Cell separation using electric fields

The present invention involves methods and devices which enable discrete objects having a conducting inner core, surrounded by a dielectric membrane to be selectively inactivated by electric fields via irreversible breakdown of their dielectric membrane. One important application of the invention is in the selection, purification, and/or purging of desired or undesired biological cells from cell suspensions. According to the invention, electric fields can be utilized to selectively inactivate and render non-viable particular subpopulations of cells in a suspension, while not adversely affecting other desired subpopulations. According to the inventive methods, the cells can be selected on the basis of intrinsic or induced differences in a characteristic electroporation threshold; which can depend, for example, on a difference in cell size and/or critical dielectric membrane breakdown voltage. The invention enables effective cell separation without the need to employ undesirable exogenous agents, such as toxins or antibodies. The inventive method also enables relatively rapid cell separation involving a relatively low degree of trauma or modification to the selected, desired cells. The inventive method has a variety of potential applications in clinical medicine, research, etc., with two of the more important foreseeable applications being stem cell enrichment/isolation, and cancer cell purging.

FIELD OF THE INVENTION 
This invention relates to methods and apparatuses for selecting specific 
cell types from cell suspensions, specifically those employing applied 
electric fields. 
BACKGROUND OF THE INVENTION 
The ability to isolate specific sub-populations of cells from cell 
suspensions is of critical importance to many applications in the 
biological sciences as well as to many therapies in clinical medicine. For 
example, the basis of many medical therapies for treating a variety of 
human diseases and for countering the effects of a variety of 
physiological injuries involves the isolation, manipulation, expansion, 
and/or alteration of specific biological cells. One particularly important 
example involves the reconstitution of the hematopoietic system via bone 
marrow or progenitor cell transplantation. More specific examples include: 
autologous, syngeneic, and allogenic stem cell transplants for immune 
system reconstitution following the myeloablative effects of severe high 
dose chemotherapy or therapeutic irradiation; severe exposure to certain 
chemical agents; or severe exposure to environmental radiation, for 
example from nuclear weapons or accidents involving nuclear power 
generators. 
Intensive chemotherapy and/or irradiation for the treatment of a variety of 
cancers, including breast cancer, has become a commonly used approach in 
cancer care centers. Such treatments are associated with severe ablation 
of the bone marrow cells required for function of the blood and immune 
systems. Such bone marrow cells are derived from a small number of 
progenitor cells known as hematopoietic stem cells in the normal bone 
marrow. Therefore, patients receiving such therapies require life-saving 
transplants of stem cells in order to survive the effects of the 
treatment. Stem cell containing tissue for transplant may be derived from 
donor marrow (allogeneic transplant) or from the patient's own bone marrow 
or peripheral blood after mobilization (autologous transplant). In both 
instances, there is a need for effective cell separation methods to enrich 
the transplant tissue in stem cells and reduce the number of undesirable 
and deleterious cells (e.g. mature T cells for allogeneic transplants and 
residual cancerous cells for autologous transplants). For example, for 
autologous adjuvant stem cell transplant therapy following myeloablative 
cancer treatments, it is believed that reinfusion of residual tumor cells 
is a major cause of post therapy relapse. Clearly, removing such cells 
from transplanted tissue would be beneficial to the patient. 
A number of cell isolation, cell separation, and cell purging strategies 
have been employed in the prior art for purifying or removing cells from a 
suspension. Prior art cell separation methods used to isolate cells or 
purge cell suspensions typically fall into one of three broad categories: 
physical separation methods typically exploit differences in a physical 
property between cell types, such as cell size or density (e.g. 
centrifugation or elutriation); chemical-based methods typically employ an 
agent that selectively kills or purges one or more undesirable cell types; 
and affinity-based methods typically exploit antibodies that bind 
selectively to marker molecules on a cell membrane surface of desired or 
undesired cell types, which antibodies may subsequently enable the cells 
to be isolated or removed from the suspension. While physical separation 
methods can be advantageous with regard to their ability to separate cells 
without causing undo damage to desired cells, current physical separation 
methods typically have relatively poor specificity and do not typically 
yield highly purified or highly purged cell suspensions. While many 
chemical and affinity methods have better selectivity than typical 
physical methods, they can often be expensive or time consuming to perform 
and can cause considerable damage to, or activation of, desired cells, for 
example stem cells, and/or can add undesirable agents to the purified or 
isolated cell suspensions (e.g. toxins, proliferation-inducing agents, 
and/or antibodies). An additional potential problem with antibody-based 
cell separation techniques typically employed for purification of stem 
cells, is that they select stem cells solely on the basis of cell surface 
markers (e.g., CD34) and will not select cells lacking such markers. 
In addition to cancer therapy, there are a number of other important 
medical therapies which exist, or are under development, that are based on 
cells derived from a variety of different types of stem cells. Examples 
include pre-exposure prophylaxis or post-exposure therapies under 
development for a variety of biological exposures that may occur naturally 
(e.g., viral exposure for example with Ebola, etc.) or be inflicted by 
mankind (i.e., biological warfare agents). A variety of gene therapies 
involving genetically manipulated stem cells, are being contemplated or 
are under development for treating a variety of blood-related diseases 
(e.g., AIDS, leukemia, other cancers, etc.). Gene therapy techniques based 
on genetically manipulated stem and/or germ cells may also be useful in 
cloning organisms, such as animals. However, genetically manipulating stem 
cells using many current technologies is difficult, typically employing 
viruses or gene carriers that can be time consuming and expensive, or may 
be dangerous to perform and may not have high yields. Current research 
findings also suggest that the practical implementation of animal organ 
transplants into human recipients also may require procedures involving 
stem cells from both the donor and recipient. Many of these promising 
therapies would require cryopreservation and storage of donor specimens 
including human stem cells, for example, as derived from the stem 
cell-rich umbilical cord blood of newborns, which can provide such donors 
with a therapeutic basis for hematopoietic reconstitution or gene therapy 
should a health emergency occur later in life. If such storage demands are 
to be realistically met, the specimens will need to have minimal volume, 
and, therefore, successful implementation of such technologies may rest on 
the development and availability of effective methods for isolating trace 
numbers of stem cells from sources such as umbilical cord blood and the 
fetal liver. In order to achieve broad implementation of the therapies 
discussed above and others, rapid and cost effective methods are needed to 
isolate, with high purity, desired target cells from suspensions having a 
diverse mix of cell types and concentrations. 
The use of applied electric fields to physically manipulate cells is known. 
Applied electric fields have been employed in the prior art for cell 
inactivation and sub-lethal cell membrane electroporation. For example, 
U.S. Pat. No. 5,048,404 to Bushnell discloses a system and method for 
sterilizing liquid foodstuffs by killing microorganisms with exposure to 
pulsed electric fields. 
Sale and Hamilton ("Effects of High Electric Fields on Microorganisms I. 
Killing of Bacteria and Yeasts," Biochim et Biophys Acta, 148:781 (1967); 
and "Effects of High Electric Fields on Microorganisms II. Mechanism of 
Action of the Lethal Effect," Biochim el Biophys A4cta, 148:789 (1967)) 
studied the effect of pulsed electric fields on suspensions of bacteria or 
suspensions of yeasts. Specifically, they investigated the effect on the 
degree of cell kill by the field as a function of field strength and 
exposure time. The effect of pulsed electric fields on the killing of 
bacteria was also studied by Huilsheger et al. ("Lethal Effects of 
High-Voltage Pulses on E. Coli K12," Radiat Environ Biophys, 18:281(1980); 
and "Killing of Bacteria with Electric Pulses of High Field Strength," 
Radiat Environ Biophys, 20:53(1981)). Hulsheger et al. studied the effects 
on bacterial cell death of a variety of experimental parameters and were 
able to demonstrate a 99.9% reduction in the number of living bacterial 
cells in suspensions after exposure to certain pulsed electric field 
parameters. 
The lysis of erythrocytes in erythrocyte suspensions by pulsed electric 
fields has also been studied both for bovine (Sale and Hamilton, "Effects 
of High Electric Fields on Microorganisms III. Lysis of Erythrocytes and 
Protoplasts," Biochim et Biophys Acta, 163:37 (1967)) and human (Kinosita 
and Tsong, "Voltage-Induced Pore Formation and Hemolysis of Human 
Erythrocytes," Biochim et Biophys Acta, 471:227 (1977); and Kinosita and 
Tsong, "Hemolysis of Human Erythrocytes by a Transient Electric Field," 
Proc Natl Acad Sci. 74:1923(1977)) erythrocytes. Knowledge derived from 
the studies above indicates that applied electric fields resulting in 
cellular transmembrane potentials on the order of 1 Volt can result in 
colloidal osmotic lysis of the erythrocytes. 
Electric fields have also been used to sublethally porate the plasma 
membrane of nucleated cells, such as leukocytes and Chinese Hamster Ovary 
(CHO) cells (Sixou and Teissie, "Specific Electropermeabilization of 
Leukocytes in a Blood Sample and Application to Large Volumes of Cells," 
Biochim et Biophys Acta, 1028:154 (1990)). Sixou and Teissie investigated 
electropermeabilization conditions to enable reversible poration of cell 
membranes, while maintaining long-term cell viability, for the purpose of 
enabling the reversibly porated cells to uptake drugs and act as 
immunocompatible drug delivery vehicles within the body. Sixou and Teissie 
studied the effect of pulsed electric field parameters on the reversible 
poration of suspensions comprising single cell types and suspensions 
comprising mixtures of two cell types (e.g. CHO cells and erythrocytes, 
and leukocytes and erythrocytes). The authors showed that reversible 
electropermeabilization is a function of the cell size and that large 
cells are reversibly porated at lower electric field strengths than small 
cells. 
While the above mentioned methods and systems for cell separation and cell 
electropermeabilization represent, in some cases, valuable and useful 
techniques for some applications, there remains a need in the art for 
simple, fast, and clean methods to selectively isolate or remove specific 
cell sub-populations from cell suspensions without causing undo damage or 
activation to the remaining cells and without employing undesirable or 
toxic agents. 
SUMMARY OF THE INVENTION 
Accordingly, the present invention can provide relatively simple, fast, and 
clean methods for cell isolation or purging based on physical differences 
between different cell types present in a suspension. Furthermore, the 
invention provides systems and methods that enable selective isolation of 
viable cells, selective cell inactivation, as well as stem cell 
electropermeabilization, using applied electric fields. 
In one aspect, the invention provides a method for creating from a 
biological sample having a given cell population, a suspension of cells 
that contain a selected viable subpopulation of the given cell population. 
The method is based on a characteristic electroporation threshold of the 
cells. The subpopulation of cells selected by the method is substantially 
limited to cells that have a characteristic electroporation threshold that 
is greater than a predetermined electroporation threshold. The selected 
suspension of cells is produced from the biological sample by first 
subjecting the sample to an electric field that has a magnitude that is 
sufficient to porate a substantial fraction of the cells in the sample 
that have a characteristic electroporation threshold less than the 
predetermined electroporation threshold. The electric field, however, does 
not porate a substantial fraction of cells that have a characteristic 
electroporation threshold greater than the predetermined electroporation 
threshold. Essentially, all of the porated cells in the sample that is 
subjected to the electric field are also inactivated. 
In another aspect, the invention provides a method for creating a selected 
subpopulation of discreet objects from a sample having a given population 
of discreet objects. A discreet object comprises an inner conductive core 
which is surrounded by a dielectric membrane. The method is based on a 
characteristic electroporation threshold of the discrete objects. The 
subpopulation of discrete objects selected by the method is substantially 
limited to discrete objects that have a characteristic electroporation 
threshold that is greater than a predetermined electroporation threshold. 
The selected suspension of discrete objects is produced from the sample by 
first subjecting the sample to an electric field that has a magnitude that 
is sufficient to cause irreversible dielectric breakdown of the dielectric 
membrane of a substantial fraction of the discrete objects in the sample 
that have a characteristic electroporation threshold less than the 
predetermined electroporation threshold. The electric field, however, does 
not cause irreversible dielectric breakdown of the dielectric membrane of 
a substantial fraction of cells that have a characteristic electroporation 
threshold greater than the predetermined electroporation threshold. 
In yet another aspect, the invention provides a method for porating cells. 
The method includes supplying a suspension of cells in a treatment volume, 
where the treatment volume includes at least two electrodes that are in 
fluid contact with the suspension. The method further involves applying a 
time varying bi-polar electrical potential across the electrodes that is 
sufficient to create an electric field that is sufficient to porate at 
least one cell in the suspension. The bi-polar electrical potential is 
varied so that the average current across the sample over the entire 
treatment time is essentially zero. 
In another aspect, the invention provides the method for reversibly 
porating stem cells. The method involves supplying in a treatment volume a 
suspension of cells including a plurality of stem cells, which stem cells 
have a characteristic size, a characteristic shape, a plasma membrane, and 
a nuclear membrane. A pulsed electric field that has a pulse duration and 
magnitude sufficient to porate the plasma membrane of a cell having a 
characteristic size and shape essentially identical to the stem cells, but 
having an effective membrane thickness substantially exceeding the average 
membrane thickness of the plasma membrane of the stem cells is then 
applied to the suspension. 
In another aspect, the invention involves a system for creating from a 
biological sample having a given cell population, a suspension containing 
a selected viable subpopulation of the given cell population. The selected 
cell population is substantially limited to cells that have a 
characteristic electroporation threshold greater than a predetermined 
electroporation threshold. The system functions by inactivating a 
substantial fraction of the cells in the sample not included in the 
selected subpopulation. The system includes a generating mechanism that 
generates an electric field of a magnitude and duration sufficient to 
irreversibly porate a substantial fraction of the cells not included in 
the selected subpopulation, while not irreversibly porating a substantial 
fraction of the cells included in the selected subpopulation. The system 
further includes a treatment cell that is electrically connected to the 
generating mechanism and is adapted to contain a cell suspension. 
In yet another aspect, the invention provides a system for selectively 
inactivating biological cells based on a difference in a characteristic 
electric poration threshold. The system includes a generating mechanism 
that generates an electric signal constructed and arranged to create 
desired electric field parameters. The system also includes a treatment 
cell that is electrically connected to the generating mechanism, includes 
at least one electrode, and includes a treatment volume adapted to contain 
a cell suspension. The electrode is in fluid contact with the cell 
suspension during operation of the system and is constructed of a porous, 
biocompatible material, which is sealed in order to reduce the release of 
gases from the electrode during operation of the system. 
In another aspect, the invention involves a cell suspension comprising a 
plurality of biological cells suspended in a liquid. The suspension 
includes one population of cells, which have a maximum of characteristic 
size not more than a predetermined value, that are substantially viable, 
and another population of cells, having a maximum characteristic size 
greater than the predetermined value, that are substantially non-viable. 
The cell suspension is obtained from a precursor suspension of 
substantially viable cells that contains as subpopulations the two cell 
populations mentioned above. The cell suspension is obtained by subjecting 
the precursor cell suspension to an electric field having a magnitude and 
duration that is sufficient to irreversibly porate a substantial fraction 
of the cells in the precursor suspension that have a maximum 
characteristic size above the predetermined value. 
In yet another aspect, the invention involves a cell suspension comprising 
a plurality of biological cells suspended in a liquid where each of the 
biological cells is enclosed by a plasma membrane. The cell suspension 
includes a subpopulation of biological cells that possess a maximum 
characteristic size in excess of a predetermined value. Furthermore, the 
cells in the subpopulation of cells having a maximum characteristic size 
in excess of the predetermined value also have a maximum transmembrane 
electrical potential that exceeds that required to cause irreversible 
dielectric breakdown of the plasma membrane of the cells. 
In another aspect, the invention provides a cell suspension comprising a 
plurality of non-cultured biological cells, including a plurality of 
viable stem cells that have a given characteristic size, suspended in a 
liquid. The cell suspension further includes a plurality of irreversibly 
porated cells, essentially all of which irreversibly porated cells have a 
characteristic size that is greater than the characteristic size of the 
stem cells. 
In another embodiment, the invention provides a cell suspension including a 
plurality of viable, reversibly electroporated stem cells. 
In yet another aspect, the invention involves a suspension comprising 
viable, human pluripotent lympho-hematopoietic stem cells, which are 
capable of differentiating into members of the lymphoid, erythroid, and 
myeloid lineages. The suspension is essentially free of mature and lineage 
committed cells and is derived from a precursor cell suspension comprising 
substantially viable cells. The suspension is derived from the precursor 
suspension by subjecting the precursor suspension to an electric field of 
sufficient duration and magnitude to inactivate a substantial fraction of 
the mature and lineage committed cells in the precursor suspension. 
Other advantages, novel features, and objects of the invention will be 
become apparent from the following detailed description of the invention 
when considered in conjunction with the accompanied drawings, which are 
schematic and which are not intended to be drawn to scale. In the figures, 
each identical or nearly identical component is illustrated in various 
figures is represented by a single numeral. For purposes of clarity, not 
every component is labeled in every figure.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention provides novel methods and systems for selectively 
inactivating biological cells, or any discrete objects having an inner 
conducting core surrounded by a dielectric layer, for example a lipid 
bilayer membrane. Specifically the invention provides methods and 
apparatus for selectively inactivating such cells or discrete objects by 
subjecting a suspension containing such cells or discrete objects to an 
applied electric field of sufficient duration and magnitude to cause 
dielectric breakdown or electroporation of the dielectric layer. The 
methods provided by the present invention can be used advantageously to 
selectively inactivate subpopulations of cells or discrete objects from a 
precursor population that contains a mixture of different cells or 
discrete objects, or mixtures of cells and non-cell discrete objects, on 
the basis of a characteristic electroporation threshold, thus providing a 
means for selectively purging or isolating cells or discrete objects from 
larger populations. One embodiment of the inventive method involves 
subjecting a sample having a given population of cells or discrete objects 
to electric field conditions sufficient to porate a substantial fraction 
of cells or discrete objects that have a characteristic electroporation 
threshold below a selected predetermined value, while not simultaneously 
porating a substantial fraction of the cells or discrete objects having a 
characteristic electroporation above the predetermined value, and 
subsequently, or simultaneously inactivating the porated cells or discrete 
objects. 
The term "biological cell" or "cell" as used herein has its commonly 
understood meaning and includes viable, potentially viable, or previously 
viable cells derived from a biological sample. Such cells include 
prokaryotic cells such as bacteria, and algae, and eukaryotic cells, such 
as yeasts, fungus, plant cells, and animal cells. Such cells typically 
have an inner, electrically conducting core comprised of cytoplasm, 
surrounded and enclosed by at least one dielectric membrane, for example 
the cytoplasmic or, equivalently, plasma membrane. Eukaryotic cells, in 
addition, typically also possess a dielectric nuclear membrane surrounding 
a conductive nucleus within the interior of the cell. 
The term "discrete objects having an inner conducting core surrounded by a 
dielectric layer" or simply "discrete object" as used herein refers to any 
object comprising a substance exhibiting a relatively low electrical 
resistivity surrounded and enclosed by a dielectric layer or dielectric 
membrane having a much higher electrical resistivity. Such discrete 
objects include biological cells as previously described, but also include 
objects such as certain viruses, sub-cellular organelles, liposomes, 
micelles, and others. Throughout the remainder of this detailed 
description, many of the methods and apparatus of the invention are 
described in relation to biological samples comprising biological cells. 
It should be understood that the invention is not so limited and that the 
invention may similarly be applied to discrete objects, as defined herein, 
other than cells. Also, whenever the term "discrete object" is used 
herein, it should be understood that the term includes, as a subset, 
biological cells. The term "dielectric layer," or "dielectric membrane," 
or "membrane" as used herein refers to a continuous layer or coating 
having a finite thickness and having an electrical resistivity 
(.OMEGA..multidot.cm) exceeding that of a conducting core which the 
membrane encloses. Typically, the electrical resistivity of the dielectric 
layer will exceed that of the inner conducting core by at least a factor 
of 10, and more typically, for example as is the case with most biological 
cells, by at least a factor of 10.sup.4 -10.sup.9. In the context of 
biological cells, the dielectric layer is defined by at least one lipid 
bilayer membrane, together with any associated structures or substances 
associated therewith which affect the effective membrane thickness or 
resistivity of the dielectric layer. "Effective," as used herein in the 
context of membrane thickness or resistivity, refers to a thickness or 
resistivity of an equivalent membrane not possessing any associated 
structures or substances affecting its dielectric properties associated 
therewith that possesses the same dielectric properties as the actual 
membrane having such associated structures or substances. 
The term "inactivating" as used herein refers to destruction of at least 
one property of a discrete object. In the context of biological cells, 
inactivating is equivalent to rendering unviable, or killing the cell. As 
applied to non-cell discrete objects, inactivate can refer to physical 
destruction of the object, or simply a destruction of the permi-selective 
diffusional barrier properties of the dielectric layer with respect to at 
least one molecular, ionic, or atomic species. In certain embodiments 
involving cells, inactivation may involve not only rendering the cells 
non-viable, but also irreparably lysing and physically disrupting and 
destroying the physical structure of the cell. 
The invention provides, in some embodiments, relatively fast and effective 
methods for purifying certain desired cells in a viable state from a 
suspension or eliminating certain undesired cells from a cell population. 
As mentioned, the methods involve exposing a suspension containing a 
population of cells to an applied electric field, which field has a 
magnitude and is applied for a duration selected to porate, and in some 
embodiments inactivate, a substantial fraction of certain subpopulations 
of cells based on their characteristic electroporation threshold. The term 
"suspension of cells" as used herein refers to a mixture of cells 
suspended in a carrier liquid. The carrier liquid may be naturally part of 
the biological sample from which the cells derive, for example blood is a 
suspension of blood cells suspended in plasma, or, for cells which are not 
normally present in a suspension, the carrier liquid can be any suitable 
diluent or medium. Preferred carrier fluids are non-toxic and 
physiologically compatible with the cells they suspend, at least for a 
time period equal to that of the electric field application procedure. 
Preferred carrier fluids are also electrically conductive. The electrical 
conductivity can be any value greater than zero, but preferably will range 
from about 10%-200% that of the conductive core of the cell. In certain 
embodiments, the conductivity or resistivity of the carrier fluid will be 
essentially equal to that of the conductive core of the cell. In other 
embodiments, in order to reduce the power consumption of the electric 
field generating apparatus and/or reduce the degree of heating of the cell 
suspension, as will discussed in greater detail herein, it can be 
preferable to utilize a carrier fluid having a resistivity that is greater 
than that of the conductive core of the cell. In order to provide a more 
uniform electrical field throughout a cell suspension being treated 
according to the invention, the cell suspension should be essentially free 
of gas bubbles. The cells in the suspension should also be individually 
suspended and as free from clumping and aggregation as possible during 
application of the electric field in order to provide the maximum 
resolution and selectivity attainable for a given set of electric field 
exposure conditions. The concentration of cells in a cell suspension, as 
will be discussed in more detail, can also affect the selection and 
performance of the applied electric field. Typically, for mammalian cells, 
the range of total cell concentrations in treated samples can range from 
about 10.sup.2 -10.sup.10 cells/ml, with preferred suspensions having from 
about 10.sup.4 -10.sup.8 cells/ml. 
The term "substantial fraction" or "substantially depleted" as used herein, 
in the context of discrete objects having a characteristic electroporation 
threshold less than a predetermined value, refers to at least 25% of such 
cells being porated by the applied electric field and inactivated, 
preferably at least 50%, more preferably at least 90%, in some embodiments 
preferably at least 99%, in some embodiments preferably at least 
99.99999%, and in some embodiments preferably essentially all of the 
discrete objects. The term "electroporation threshold" or "critical 
electroporation threshold" as used herein refers to the susceptibility of 
a particular cell or subpopulation to membrane poration by an applied 
electric field. As will be discussed in greater detail herein, the 
characteristic electroporation threshold of a cell or subpopulation of 
cells is a function of the physical and chemical properties of the cell 
that influence its interaction with an applied electric field. Differences 
between one or more of these properties between different cells or 
subpopulations may be advantageously exploited to provide a basis for 
selective isolation and inactivation according to the invention. Such 
properties include characteristic cell size, effective dielectric membrane 
thickness, cell shape, cell and/or membrane morphology, cell membrane 
capacitance, cytoplasmic electrical resistivity, etc. The term 
"predetermined electroporation threshold" as used herein refers to a 
chosen value of electroporation threshold below which a significant 
fraction of cells having such electroporation thresholds will be porated 
by the applied electric field, and above which a significant fraction of 
cells having such electroporation thresholds will not be porated. The 
predetermined electroporation threshold, as will be discussed in greater 
detail herein, can be a function of the parameters of the applied electric 
field (e.g. field strength, field duration, etc.) and the electrical 
properties of the cell suspension (e.g. fluid carrier resistivity, total 
suspension capacitance, etc). An important feature of the invention is the 
selection of such electric field parameters and suspension properties in 
order to provide conditions that selectively inactivate a significant 
fraction of one or more subpopulations of cells in a sample based on one 
or more differences in their properties that affect their characteristic 
electroporation threshold. 
The methods and apparatus provided by the present invention can be applied 
to a wide variety of discrete object separation applications, and to a 
wide variety of biological and non biological samples. One important 
embodiment of the invention provides a method of isolating or inactivating 
discrete objects based on a characteristic size. "Characteristic size" or 
"size" as used herein with respect to dimensions of cells or discrete 
objects, refers a linear dimension of a cell or discrete object as 
measured in the direction of an applied electric field to which the cells 
or objects are subjected and from an external surface of the outermost 
dielectric membrane on one side of the geometric center of the cell or 
object, through the geometric center of the cell or object, to an external 
surface of the outermost dielectric membrane on the other side of the 
geometric center of the cell or object For example, for a spherical cell 
or object, the characteristic size would be the external diameter of the 
cell or object. Specifically, the method involves porating and 
inactivating a substantial fraction of discrete objects above a certain 
predetermined threshold size, which is a function of the electric field 
and suspension properties as discussed, while leaving in an uninactivated 
state a significant fraction of discrete objects below the threshold size. 
The method can be used, for example, to inactivate cells present in viral 
preparations used in treatment, diagnosis, or research of viral diseases 
such as AIDS. It is often desirable to obtain suspensions of pure virus 
that are essentially uncontaminated with viable cells infected with such 
virus. Because virus-carrying cells will typically be substantially larger 
than viruses, the inventive method can be use to selectively inactivate 
the cells without causing undue damage to the virus. Another application 
involves isolation of certain sub-cellular organelles from cells. In this 
case, the cells can initially be porated using the inventive method in 
order to liberate the intracellular contents of the cells into the 
suspension. Subsequently, an electric field having different parameters 
can be applied to the suspension to selectively isolate one or more 
subpopulations of organelles on the basis of a difference in a 
characteristic electroporation threshold, for example due to a difference 
in characteristic size. An example of the inventive method as applied to a 
non-biological sample is the selective disruption of liposomes on the 
basis of size. Liposomes are commonly used as vehicles for drug delivery 
or for transfection of genetic material into cells. The performance of the 
liposome for its intended task and also potentially its pharmacokinetics 
within the body can be a function of the size of the liposome. Thus, the 
current invention can provide a relatively fast and easy means for 
performing a size selection of manufactured liposomes. 
Two important applications where the present invention has particular 
utility involve the purging of cancer cells from cell suspensions and the 
isolation and enrichment of stem cells or germ cells from cell 
suspensions. A "germ cell" as used herein, refers to haploid cells, or 
gametes, such as sperm or egg cells. These applications are important 
components in many clinical treatment therapies involving, for example, 
cancer treatment, organ transplant, and gene therapy. The present method 
exploits a difference in a critical electroporation threshold between the 
above mentioned cell types and the other cell types present in the cell 
suspension to effect a selection of desired cells and/or a purging of 
undesired cells. In particular, in particular embodiments, differences in 
characteristic cell size (e.g. average cell diameter) provide, at least in 
part, for the above mentioned difference in critical electroporation 
threshold. Cancer cells, for example, are often significantly larger than 
the desired population of cells, for example stem cells, in a sample and 
will generally have an electroporation threshold below that of the desired 
cells. Thus, by subjecting the suspension to a selected electric field 
having predetermined characteristics, a substantial fraction of the cancer 
cells can be inactivated without inactivating the desirable cells. 
Preferably, when a suspension initially containing such cancer cells is 
subjected to the inventive cell selection method, the concentration of 
viable cancer cells remaining is decreased by at least a factor of 10 (1 
log reduction) and most preferably by at least a factor of 100,000 (5 log 
reduction). Similarly, because for many biological samples stem cells are 
the smallest cells present, the inventive method can be used to enrich 
such a sample in viable stem cells by selectively inactivating non-stem 
cells in the sample. Preferably, the concentration of viable stem cells, 
with respect to the total number of viable cells present in the sample, is 
increased in the sample through application of the inventive method by at 
least a factor of two, more preferably by at least a factor of five, and 
in certain preferred embodiments, by a factor of 10.sup.6 or more, while, 
correspondingly, the concentration of viable non-stem cells in the sample 
is substantially depleted. One feature of the inventive method when 
applied to the purification and enrichment of stem cells, is that unlike 
typical antibody-based stem cell isolation methods, isolation of stem 
cells using applied electric fields does not rely on the presence of 
surface markers on the stem cells (e.g. CD34). The most primitive stem 
cells may not possess the cell surface markers targeted by typical 
antibody-based methods and thus, such cells will not be recovered by those 
methods. Conversely, stem cells isolated by the present methods are 
selected on the basis of their critical electroporation threshold. 
Therefore, the stem cell suspensions provided according to the inventive 
methods, will include stem cells that do not possess the surface markers 
typically used by antibody-based methods to select stem cells if such stem 
cells were initially present in the sample before application of the 
inventive methods. Specifically, one embodiment of the present invention 
can provide a suspension enriched in stem cells, which is essentially free 
of mature and lineage committed cells, and which includes as a 
subpopulation, stem cells not expressing CD34 on their surface. 
The stem cell and/or cancer cell containing suspensions can be derived from 
a variety of sources including, but not limited to, bone marrow, mobilized 
or unmobilized peripheral blood, umbilical cord blood, fetal liver tissue, 
other organ tissue, skin, nerve tissue, etc. A variety of stem cells may 
advantageously be isolated and enriched according to the invention 
including, but not limited to, hematopoietic stem cells, embryonic stem 
cells, mesenchymal stem cells, epithelial stem cells, gut stem cells, skin 
stem cells, neural stem cells, liver progenitor cells, and endocrine 
progenitor cells. One embodiment of the invention involves the isolation 
of lympho-hematopoietic stem cells, which are capable of differentiating 
into members of the lymphoid, erythroid, and myeloid lineages, from cell 
suspensions including mature and lineage committed cells to provide a 
suspension of lympho-hematopoietic stem cells that is essentially free of 
mature and lineage committed cells. The enriched stem cell suspensions 
according to the present method will also be advantageously enriched in 
pluripotent stem cells, which have the ability to differentiate into the 
full complement of mature cells derived from a particular type of stem 
cell. Also, in some embodiments, the enriched stem cell suspensions 
produced according to the invention will contain, in addition to 
pluripotent stem cells, stem cells which are committed colony forming 
cells. For example, for samples including hematopoietic stem cells, the 
enriched suspensions can advantageously include viable colony forming 
cells for granulocytes and macrophages (CFC-GM), colony forming cells for 
erythrocytes (BFU-E), colony forming cells for eosinophils (CFC-Eo), 
multipotent colony forming cells (CFC-GEMM), and immature lymphoid 
precursor cells. 
Thus, it is apparent that the present invention provides a novel method for 
cancer purging and stem cell isolation useful for a variety of medical 
therapies, an important one of which is stem cell transplantation for 
hematopoietic reconstitution after myleoablative therapies. A particularly 
attractive application for the teachings of this invention is the 
isolation of hematopoietic stem cells from bone marrow, mobilized 
peripheral blood, umbilical cord blood or fetal liver tissue, which is a 
crucial first step in an overall protocol for delivering genetically 
manipulated, stem-cell-based, pathogen countermeasures that have the 
potential to provide pre-exposure prophylaxes or post-exposure therapies, 
and immune system reconstitution. Isolation of stem cells to high purity 
prior to their genetic manipulation is essential for eliminating the 
interference and complications that occur should other leukocytes be 
present/viable during gene transfection and expansion. Cryostorage of 
large numbers of stem cell specimens will be required for large scale 
implementation of such stem cell-based-countermeasures. Efficient, 
practical cryostorage of large numbers of specimens demands small specimen 
volumes. Since the relative concentrations of stem cells in bone marrow 
mononuclear cell (BMMC) and mobilized peripheral blood mononuclear cells 
(MPBMC) specimens are approximately 1:10.sup.5, stem cell isolation and 
enrichment will-be important for achieving small specimen volumes 
advantageous for efficient cryostorage. To eliminate interference from 
non-stem cells during expansion and gene transfection, and to reduce 
volume requirements for cryostorage, isolation strategies should be 
capable of enriching stem cell concentrations by up to 10.sup.6 for BMMC 
and MPBMC specimens. 
Many presently available methods typically in use for stem cell isolation 
or cancer cell purging depend on antibody binding to cell surface 
structures or toxin-based cell inactivation strategies. These strategies 
can be sub-optimal for stem cell-enrichment because they provide in some 
cases relatively low degrees of enrichment and can add detrimental 
substances to the suspension, such as exogenous antibodies or toxins, that 
damage or can activate the isolated stem cells or be detrimental to a 
patient upon reinfusion of such cells. Other currently available stem cell 
isolation strategies involve a culture-based protocol requiring a long 
processing time, for example up to one week. In addition, many currently 
available cell isolation methods do not easily scale, and, therefore, are 
not optimal for handling the large throughput required for a widespread 
implementation of many stem-cell-based therapies. Thus, in the prior art, 
there exists a need for stem cell isolation strategies for effective 
implementation of the stem-cell-based countermeasures, which the present 
invention, in some embodiments, potentially can fill. 
The methods and apparatus provided according to the present invention can 
provide significant improvements and advantages over many prior art 
methods for performing cell separations and isolations. The present method 
is based on intrinsic differences between cell types, for example 
characteristic size and/or characteristics that effect the membrane 
breakdown voltage, such as the dielectric strength of the membrane or the 
effective membrane thickness, and does not require, in many embodiments, 
the addition or use of exogenous agents, such as antibodies or toxins, 
which can adversely affect the viability or state of activation of the 
isolated cell fraction. "Dielectric membrane breakdown voltage" or 
"membrane breakdown voltage" refers the voltage across the dielectric 
membrane layer of a cell or discrete object at the onset of poration of 
the membrane. In addition, the present method is substantially faster than 
many prior art cell separation techniques. Cell inactivation and isolation 
using the inventive method can be performed in times ranging from 
milliseconds to minutes. Finally, with appropriate selection of operating 
parameters, as discussed herein, and routine optimization of the selected 
parameters and method, the inventive method can potentially provide high 
degrees of enrichment or purging of selected cell subpopulations. 
As discussed in more detail later herein, cell selection with electric 
fields according to the invention can be used as a stand alone method or 
may be combined with one or more other cell separation methods, such other 
methods being a pre-treatment or post-treatment step. Also the method 
according to the invention can be applied to a cell suspension so that the 
applied electric field both porates and inactivates one or more 
subpopulations of cells in a single step, or, alternatively, the applied 
field may porate some or all of the cells to be inactivated without 
inactivating all of such cells, with the inactivation step performed in a 
subsequent step. In the former case, the applied electric field is 
sufficient, under the conditions of its application, to cause irreversible 
breakdown or irreversible poration of the dielectric membrane of the cell. 
"Irreversible breakdown" or "irreversible poration" refers to poration 
that is sufficient to cause death, inactivation, and/or physical 
disruption of a discrete object without a need for a secondary 
inactivating step. Inactivation and cell death due to poration are 
believed to be caused by a loss of the permi-selective nature of the 
membrane leading to cell death and/or membrane disruption, or a direct 
physical disruption of the membrane caused by extensive poration. In the 
case of a loss of the permi-selective nature of the membrane, the 
inactivation or cell death is ultimately caused by diffusion of previously 
excluded molecular species, especially small ionic species such as 
Na.sup.+, K.sup.+, and Ca.sup.++, across the membrane followed by an 
uptake of water across the membrane into the cell in an attempt to achieve 
osmotic equilibrium with the suspending fluid medium, which can lead to 
colloidal osmotic lysis and irreparable (fatal) cell lysis, or to a lethal 
disruption of cellular metabolism. For embodiments involving a method 
where the applied field porates some or all of the cells to be inactivated 
without inactivating all of such cells, where the inactivation step is 
performed in a subsequent step, the poration induced by the applied field 
is typically less extensive and not irreversible, at least for a certain 
portion of the porated cells. Given sufficient time, the reversibly 
porated cells in such samples could seal their pores and retain long-term 
viability if left in the same fluid carrier or suspending media in which 
they were subjected to the electric field. The reversibly porated cells 
may, however, be effectively inactivated by resuspending them in a 
different post-poration media, adjusting the temperature of the poration 
media, and/or adding a supplemental agent to the poration media which 
accelerates cell death, colloidal osmotic lysis, or prevents the resealing 
of membrane pores. More specific techniques and conditions are discussed 
later herein. 
The electric field is preferably applied to the cell suspension within a 
spatially defined treatment cell. The treatment cell can be designed as a 
static non-flow volumetric container in which the cell suspension to be 
treated is placed, or more preferably, the treatment cell will include an 
inlet and an outlet constructed and arranged to enable a cell suspension 
to continuously flow through the treatment volume. Systems including 
flow-through treatment cells may be arranged so that the cell suspension 
passes through the treatment cell only once (one pass) or a plurality of 
times (recirculating). In addition, either the flow or static systems may 
include multiple treatment cells. For flow systems, multiple treatment 
cells can be arranged in a series or parallel configuration. 
The treatment cell will include at least one electrode in electrical 
communication with the cell suspension to be treated. Preferably the 
treatment cell will include two electrodes placed on either side of and in 
electrical communication with the cell suspension during operation to 
which an electric potential is applied to produce an electric field within 
the treatment volume. In preferred embodiments, the treatment cell and 
electrodes are constructed and arranged to impose an electric field that 
is substantially spatially uniform within the treatment volume so that all 
cells in the suspension are exposed to similar electric field conditions. 
In some embodiments, the electric field applied to the cell suspension is 
created by an electric signal applied to the electrodes; however, it is 
also contemplated that the electric field can be induced in the sample 
cell via induction by a magnetic field. 
In order to reduce the tendency for the electrical potential applied to the 
electrodes to discharge by arcing, and in order to reduce the degree of 
electrical heating that occurs in the cell suspension, in certain 
preferred embodiments, the applied electric field is pulsed for short 
durations, such durations, except as otherwise described herein, being 
shorter than the residence time of the treated cell suspension in the 
treatment volume during the step of subjecting the suspension to the 
applied electric field. Such electric fields are hereinafter referred to 
as "pulsed electric fields" or PEFs. The shape of the electric field pulse 
is preferably substantially rectangular in shape, thus providing very 
short voltage rise and fall times and a substantially constant magnitude 
over the entire pulse length. Such rectangular pulse shapes yield the best 
performance and poration threshold resolution obtainable with the 
inventive method. While rectangular pulses are preferred, any pulse shape 
known in the art may be employed in performing the methods of the 
invention, especially when high resolution is not required, as, for 
example, when inactivating a cell type that is substantially larger than 
the desired cell type. 
As described previously, the electric field parameters required to effect a 
desired cell inactivation or isolation depend upon the nature of the 
cells, the suspension and suspending fluid, and the characteristics of the 
electric field application apparatus. The exact parameters for any given 
sample that will yield desired results must be found in practice via 
routine experimentation. What follows herein is a theoretical development 
and description of the inventive method, apparatus for performing the 
method, and important parameters affecting the performance and selectivity 
of the method to provide guidance to those of skill in the art in 
selecting parameters to develop successful cell or discrete object 
isolation and inactivation strategies. 
The Fundamental Basis of Electric Field Cell Isolation 
The mechanism by which electric fields, and particularly pulsed electric 
fields (PEFs), isolate cells can be best understood by examining the 
response of a single discrete object, as exemplified by a biological cell, 
to an externally applied electric field. A schematic illustration of such 
a system 50 is shown in FIGS. 1a-e. The externally applied electric field 
57 can be established by applying a constant voltage or voltage pulse 
across a pair of electrodes 55 and 56 that are in electrical communication 
with, and preferably in physical contact with, a cellular suspension 
containing a plurality of cells, one of which 51 is shown in FIG. 1a. 
Alternatively, the electric field 57 can be applied inductively by 
creating a time-varying magnetic field throughout the cellular suspension. 
To preserve the viability of the desired target cells, the carrying fluids 
in which the biological cells are suspended are typically buffered saline 
solutions having, in some embodiments, a standard physiological osmolality 
(e.g. 275-300 mOs/kg-water for most mammalian cells), and a pH in the 
physiological range (e.g. about 7.0-7.6 for most mammalian cells). The 
ionic strength of the solutions in certain embodiments is essentially the 
same as the ionic strength of the intracellular fluid 53 (e.g. about 0.15 
M NaCl equivalent for most mammalian cells). As such, these are conducting 
solutions. Electric field effects on cells can be estimated from the 
potential theory developed by Coulson (Coulson C A: Electricity, Oliver 
and Boyd, London, Chapter 9, 1951) incorporated herein by reference. This 
theory implies that induced transmembrane potentials depend on cell size 
and shape. Formally, the external electric field induces a potential 
across the cell 51, V.sub.cell, given by 
##EQU1## 
and where E is the field strength of the imposed electric field; d is the 
cell diameter 54, l is the projected length of the cell in the electric 
field direction 57; and f is a form factor, which is equal to 1.5 for a 
spherical cell (where l is equal to d) and is approximately unity for 
large aspect ratio cylindrically shaped cells (where l&gt;&gt;d). In the 
development to follow, the cells of interest will be assumed to be 
spherical so that d will be used for l in the following equations and f 
will be set equal to 1.5. For a more detailed discussion on the effects of 
non-spherical cell shape and angular orientation with the applied electric 
field, the reader is referred to Kinosita and Tsong (Kinosita K and Tsong 
T Y, Voltage-induced pore fonnation and hemolysis of human erythrocytes, 
Biochim et Biophys Acta. 471:227-242, 1977). 
Biological cells have an outer, semipermeable plasma membrane 52 that 
allows the cell to control its internal environment by its selective 
permeability. The proper function of this membrane is crucial to the 
viability of the cell. If the function of this membrane is altered or 
destroyed, cell death often follows. Plasma membranes are typically lipid 
bilayers which behave electrically as dielectrics, i.e., they behave as 
electrical insulators. For eukaryotic cells, as shown in FIG. 1a, the cell 
nucleus 68 and accompanying nuclear membrane 69 reside within the outer 
membrane 52, with cytoplasm 53 filling the gap between the nuclear and 
outer membranes. For prokaryotic cells, there is no nucleus or nuclear 
membrane, so the cytoplasm, which supports the cell's genetic information 
(one or more DNA molecules in the form of nucleoids) fills the entire 
intracellular volume. Cytoplasm, which refers collectively to the 
substance filling the gap 53 between the outer and nuclear membrane for 
eukaryotic cells, or the entire intracellular volume for prokaryotic 
cells, is mainly composed of cytosol, which is a semifluid concentrate 
having an electrical resistivity that is similar to that of aqueous 
solutions having a standard physiological ionic strength. As such, the 
cytosol is electrically conductive, which dictates that the intracellular 
volume of both eukaryotic and prokaryotic cells is electrically 
conductive. Thus, biological cells can be viewed as a conducting 
intracellular region surrounded by a dielectric (insulating) membrane 52. 
With this conceptual view of biological cells, application of an external 
electric field 57 causes charge separation to occur inside the biological 
cell 51 resulting in a nearly constant intracellular potential that has a 
value corresponding to the boundary average of the potential established 
on the outer surface of the cell's dielectric membrane 52. If the poles of 
the cell 51, of which there are two, are defined as the two points formed 
on the surface of the cell 51 by the intersection of a ray parallel to the 
electric field direction passing through the center of the cell, then 
application of an external electric field causes one half of the 
pole-to-pole potential drop outside of the biological cell 51 to develop 
across the membrane 52 at each pole of the cell. That is, the externally 
applied electric field 57 produces a maximum transmembrane potential, 
V.sub.m, at each pole of the cell 51 that scales as 
EQU V.sub.m =V.sub.cell /2 (2) 
or equivalently 
EQU V.sub.m =3dE/4 (3) 
for a spherical cell. 
Since, in response to an externally applied electric field 57, the 
potential drop, V.sub.cell, that develops over a cell's diameter 54 or 
projected length is transferred approximately equally across the two poles 
of the cell's membrane 52, the maximum electric field thereby imparted to 
a cell's membrane 52 is 
EQU E.sub.m =V.sub.cell /2t.sub.m, (4) 
or, for spherical cells 
EQU E.sub.m =3Ed/4t.sub.m, (5) 
or equivalently 
EQU E.sub.m =V.sub.m /t.sub.m (6) 
where E.sub.m is the electric field imparted to the membrane 52 for an 
externally applied electric field 57 of strength E; d is the diameter 54 
of the biological cell 51; and t.sub.m is the thickness of the membrane. 
Thus it is apparent from equation 5, that the imposed electric field 
within the cell membrane is directly proportional to cell size and applied 
electric field strength and inversely proportional to the thickness of the 
cell membrane. Since the size of many typical biological cells falls 
within a range of 1&lt;d&lt;50 .mu.and a typical thickness of cell membrane 52 
lipid bilayer is approximately 5 nm, the electric field strength imparted 
to the membrane 52 can be two to three orders of magnitude greater than 
the strength of the externally applied electric field 57. More 
specifically, for a typical lipid bilayer membrane thickness of about 5 
nm, a transmembrane potential, V.sub.m, of approximately one Volt will 
impart a 2 MV/cm electric field, E.sub.m, to the lipid bilayer membrane 
52. So for a 10 .mu.m diameter spherical cell, which, for example, is 
about the mean size of peripheral blood cells, a 2 kV/cm externally 
applied electric field E would generate the 2 MV/cm electric field, 
E.sub.m, in the lipid bilayer membrane. Since the dielectric strength of 
many polymers, in response to electric fields, is in the range 0.1-0.5 
MV/cm, it is reasonable to expect that a 2 MV/cm electric field imparted 
to the membrane 52 of a 10 gm diameter cell by an externally applied 2 
kV/cm electric field 57 would produce membrane pores by dielectric 
breakdown. Thus, the electric field magnification provided by the 
electrical behavior of biological cells can lead to the dielectric 
breakdown of a cell's membrane 52 when the externally applied electric 
field has sufficient strength, thereby forming irreversible pores in the 
membrane which lead to cell death. From equations 1 and 5, it is apparent 
that the susceptibility of a given cell to poration by an applied electric 
field 57 is proportional to the magnitude of the applied electric field, 
E, that is required to produce a given electric field, E.sub.m, in the 
lipid bilayer membrane (which is directly related to membrane poration) 
and is related to the size of the cells, the thickness of the dielectric 
membrane, the dielectric strength of the membrane (V/m), the shape of the 
cells and the orientation of non-spherical cells in the applied electric 
field. Thus, a difference in one or more of these properties between 
different cell types can lead to a difference in their characteristic 
electroporation threshold and can potentially be exploited to effect a 
selective cell isolation or inactivation using an applied electric field, 
as described in greater detail to follow. These relations are rigorously 
valid when the cell is immersed in a conducting fluid, but such fluid may 
not be required in some embodiments for the inventive method to be 
functional. 
For lipid bilayer membranes, which are typical of many mammalian cells and 
bacterial cells, the onset of membrane dielectric breakdown in response to 
an externally applied electric field has been relatively consistently 
observed in the prior art when the transmembrane potential, V.sub.m, 
reaches a particular critical value, namely V.sub.m =V.sub.mc, where 
V.sub.mc is the critical transmembrane potential for dielectric breakdown, 
or, equivalently, the dielectric membrane breakdown voltage. Table 1 
summarizes data from cell poration experiments assembled by Castro, et al. 
(Castro A J, et al: Microbial Inactivation of Foods by Pulsed Electric 
Fields. Washington State University, Department of Food Science and Human 
Nutrition, Pullman, Wash., 99164-6376,1993 hereinafter "Castro") showing 
the critical dimensions of various viable cells and their critical 
membrane potentials for cell membrane poration. The critical dielectric 
membrane breakdown voltage V.sub.mc for these cells were obtained by using 
Eq. 3 together with results from cell inactivation experiments which 
measured the critical threshold applied electric field, E.sub.c. 
TABLE 1 
______________________________________ 
Cell size and induced membrane potential 
for several microorganisms. 
d l .nu. V.sub.mc 
Microorganism (.mu.m) (.mu.m) (.mu.m.sup.3) f (V) 
______________________________________ 
E. coli (4 hr) 
1.15 6.9 7.2 1.06 0.26 
E. coli (30 hr) 0.88 2.2 1.4 1.15 1.05 
K. pneumoniae 0.83 3.2 1.7 1.09 1.26 
P. aeruginosa 0.73 3.9 1.6 1.07 1.26 
S. aureus 1.08 n/a 0.6 1.50 1.00 
L. monocytogenes I 0.76 1.7 0.8 1.17 0.99 
C. albicans 4.18 n/a 38.0 1.50 2.63 
______________________________________ 
In the table, v, is the volume of the cells. With the important exception 
of young cells (e.g., E. coli, 4 hr culture, in the logarithmic growth 
phase), the critical membrane potentials V.sub.mc of cells in their 
stationary phase is approximately 1 Volt. A variety of parameters can 
effect dielectric membrane breakdown voltage, V.sub.mc. Such parameters 
include, but are not limited to, the membrane thickness, the dielectric 
strength of the membrane, the dimensional and chemical uniformity of the 
membrane, etc. Since, for a given dielectric material, the threshold 
transmembrane electric field strength, E.sub.mc for dielectric breakdown 
is often similar, and since most of the cells in Table 1 have similar 
dielectric membranes (lipid bilayers together with any associated protein 
and/or carbohydrate components), Eq. 6 would suggest that the electrical 
properties, specifically the membrane thickness, of the membranes having 
similar V.sub.mc are similar. In addition to rapidly growing cells, 
another important exception to the general rule of 1 Volt being a critical 
transmembrane potential for cell poration and inactivation are spores in 
their quiescent state which have been shown to be much more insensitive to 
electric fields and appear to be sensitive only during germination and 
outgrowth when the cortex disappears and the spore coat layers dissolve as 
the cell swells (Huilsheger H., Potel J., and Niemann E -G. Electric Field 
Effects on Bacteria and Yeast Cells, Radiat. Environ. Biophys. 22:149-162, 
1983 (hereinafter "Hulsheger 19983"); and Grahl T., Sitzman W., Marki H. 
Killing of Microorganisms in Fluid Media by High-Voltage Pulses. Presented 
at 10th Dechema Annual Meeting of Biotechnologists. Karlsruhe, Germany, 
Jun. 1-3, 1992.). This behavior may be due to the quiescent spores having 
a much thicker effective dielectric membrane thickness due to the presence 
of the coat layers. Thus for a given critical transmembrane electric field 
strength, E.sub.mc, which is a function of, for example the resistivity 
and material properties of the dielectric layer, a larger effective 
membrane thickness would, according to Eq. 6, yield a larger V.sub.mc, and 
would thus require a larger critical applied electric field, E.sub.c, for 
cell inactivation (see Eq. 3). 
As shown in FIGS. 1b-e, when V.sub.m achieves the critical value V.sub.mc, 
membrane dielectric breakdown results in the formation of pores in the 
cell membrane 52, some of which may reseal upon removal of the externally 
imposed electric field. FIG. 1b illustrates the condition of a section of 
cell membrane 52 from near one pole of the cell with no external applied 
electric field (E=0). When an external field is applied that is below the 
critical field strength required for poration (E&lt;E.sub.c FIG. 1c) there is 
a separation of charge across the membrane 52 and a resulting 
transmembrane potential V.sub.m but no pore formation. This situation for 
the condition E=E.sub.c, is shown in FIG. 1d, where E is the strength of 
the externally applied electric field and, from Eq. 3: 
EQU E.sub.c =4V.sub.mc /3 d (7) 
which is the critical electric field strength for spherical cells that 
defines the onset of membrane pore 58 formation for a specific cell size 
and critical transmembrane potential. When V.sub.m is less than V.sub.mc 
(i.e., when E&lt;E.sub.c in FIG. 1c), pore formation, or at least 
irreversible pore formation, does not occur. As V.sub.m is increased 
beyond V.sub.mc (i.e., when E&gt;&gt;E.sub.c in FIG. 1e), membrane pores 59 
become more numerous, larger, and irreversible. Thus, application of 
sufficiently strong electric fields, or PEFs to cellular suspensions can 
result in the inactivation of cells by the formation of irreversible pores 
which can destroy the function of the semipermeable cell membrane 52. As 
noted earlier, the proper function of a cell's semipermeable membrane is 
required to control a cell's intracellular environment and, therefore, to 
maintain its viability. The critical applied electric field, E.sub.c, 
necessary to form irreversible pores is thus, as is apparent from Eq. 6, 
directly proportional to the critical transmembrane potential V.sub.mc, 
and thus membrane thickness, and inversely proportional to cell diameter d 
for spherical cells. 
PEF Parameters for Cell Purging and Cell Isolation 
As noted above, the lethal effect of pulsed electric fields on biological 
cells is caused by irreversible electroporation of their semipermeable 
membranes or reversible poration followed by a subsequent treatment to 
prevent membrane repair or accelerate colloidal osmotic lysis and thereby 
cause cell inactivation. An electric field applied across the cell 
electrically polarizes the cell membrane causing charge separation and 
build up of a transmembrane potential. The critical transmembrane 
potential required for membrane poration will be a function of the nature 
and thickness of the membrane, as previously mentioned, and must be 
determined experimentally for any given system; however, as previously 
discussed and illustrated by the data in Table 1, for a wide variety of 
biological cells the critical transmembrane potential associated with an 
externally applied field is approximately V.sub.mc =1 Volt. The pores 
resulting in the membrane structure of the cell due to exposure to field 
strengths above the critical value can, in certain cases, irreversibly 
increase cell membrane permeability leading to cell death. The dielectric 
membrane breakdown concept of cell inactivation is illustrated in FIGS. 
1b-e. 
Electric field strength, total exposure time, and pulse duration, for PEFs, 
can be selected to preferentially inactivate biological cells in a 
suspension which are more susceptible to electric fields due, for example, 
to their having one or more or a combination of the following properties 
with respect to other cells in the suspension: a larger average size; a 
thinner effective dielectric membrane thickness; a more spherical shape, 
etc. Of particular importance for many biological samples, especially 
those having cells with similar shapes, such as roughly spherical, and 
similar dielectric membrane thickness, is selective inactivation of cells 
based on a difference in characteristic size. Typically, the threshold 
electric field required for cell inactivation is inversely proportional to 
the characteristic size of the cell, i.e., from Eq. 7, E.sub.c 
(d)=4V.sub.mc /3d, where V.sub.mc .congruent.1 Volt is the critical 
transmembrane potential for the onset of irreversible pore formation for a 
wide variety of cell types and d is the diameter or characteristic size of 
the cell. If the undesirable cells are larger in diameter than the 
desirable cells, then the pulsed electric field method can be used to 
selectively inactivate the larger cells. By operating at electric field 
strengths just below the characteristic electroporation threshold for 
inactivation of the desirable cells, yet above the characteristic 
electroporation threshold for the undesirable cells, a substantial 
fraction of the undesirable cells can be preferentially inactivated while 
leaving a substantial fraction of the desirable cells (primitive stem 
cells for example) essentially unaltered and still viable. To further 
illustrate the utility of the concept, a specific example related to cell 
isolation from hematopoietic cell suspensions will be illustrated. Table 2 
lists the types of blood cells that typically will be present in bone 
marrow specimens during tumor cell purging and stem cell isolation 
processing. The cell diameters, relative abundance, and projected 
threshold electric field strengths (E.sub.c as calculated from Eq. 7 
assuming V.sub.mc .apprxeq.1 volt) for the onset of membrane damage are 
also provided in the table. Similar cell sizes as those listed would be 
expected for hematopoietic cells derived from mobilized peripheral blood, 
umbilical cord blood and fetal liver tissue, although the relative 
abundance of each may differ. Table 2 clearly shows that the electric 
field damage threshold for stem cells can be significantly greater than 
for the other leukocytes present in bone marrow specimens. Furthermore, 
the electric field threshold for stem cells can be more than a factor of 
two greater than for breast cancer cells. Since, as will be discussed 
below, the fraction of cells inactivated by an applied electric field 
scales exponentially with electric field strength, 1983) the factor of two 
difference in the critical electroporation threshold should allow 
essentially complete inactivation of breast cancer cells with preservation 
of the viability of the cells crucial for autologous transplantation (stem 
cells). 
TABLE 2 
______________________________________ 
Electric field damage thresholds for leukocytes and stem cells. 
Projected Electric 
Characteristic Relative Field 
Size Abundance Damage Thres- 
Cell Type (.mu.m) (%) hold (kV/cm) 
______________________________________ 
Stem 6.sup.a,b 0.001.sup.a 
2.2 
Lymphocyte (resting) 7.sup.b 21.sup.c 1.9 
Lymphocyte (active) 12.sup.d n/a 1.1 
Neutrophil 12.sup.d 73.sup.c 1.1 
Eosinophil 13.sup.d 4.sup.c 1.0 
Basophil 15.sup.d 0.1.sup.c 0.9 
Monocyte 15.sup.d 2.sup.c 0.9 
Breast Cancer &gt;15.sup.e n/a &lt;0.9 
______________________________________ 
.sup.a Berardi AC, et al: Functional isolation and characterization of 
human hematopoietic stem cells. Science, 267: 104-108, 1995. 
.sup.b Zipori D, et al: Introduction of Interleukin3 gene into stromal 
cells from the bone marrow alters hematopoietic differentiation but does 
not modify stem cell renewal. Blood 71: 586, 1988. 
.sup.c Jandl JH, Blood: Textbook of Hematology, Little, Brown and Company 
Boston/Toronto, 1987. 
.sup.d Henry JB: Clinical Diagnosis and Management by Laboratory Methods, 
16th Ed., W. B. Saunders Company, Philadelphia, PA, Vol. 1, 1979. 
.sup.e from observations by inventors 
Another feature which, in the present example, further can enhance the 
ability to perform preferential electric field isolation of stem cells 
and/or to purge relatively large tumor cells, such as breast cancer cells, 
involves the quiescent nature of stem cells. As discussed in Berardi, et 
al., stem cells are quiescent and are unaffected by an anti-metabolite 
treatment, whereas rapidly proliferating cells are inactivated by an 
anti-metabolite treatment. A similar phenomenon has been observed 
(Hulsheger 1983) with PEF inactivation of Escherichia coli (E. coli). The 
observations of Htilsheger H, et al. indicate that the stationary growth 
phase E. coli cells (quiescent cells) are much less vulnerable to the 
lethal effects of PEF's than are the larger, rapidly dividing E. coli 
cells that are in the logarithmic growth phase. Based on these 
considerations, it is expected that stem cells, due to their quiescent 
nature and smaller size, will be much less vulnerable to the lethal 
effects of electric fields and PEFs and that electric field strength can 
be used to preferentially inactivate a substantial fraction of non-stem 
cell leukocytes and tumor cells while leaving a substantial fraction of 
the stem cells unharmed. Thus, it is expected that the inventive methods 
will be an effective approach for purging tumor cells from autologous 
transplant tissue. Similarly, the inventive methods may be applied to 
other cell suspensions or suspensions on non-cell discrete objects having 
differences in characteristic size between subpopulations in order to 
selectively isolate or inactivate selected subpopulations. 
In addition to performing a selective cell isolation or inactivation on the 
basis of a difference in characteristic cell size by selecting an 
appropriate applied electric field strength, the method can also be 
employed to select cells that can be similar in size based on a difference 
in dielectric membrane breakdown voltage for example, due to a difference 
in effective membrane thickness. For example, a variety of cells, such as 
some epithelial cells and cancer cells, can have a layer of 
mucopolysaccharide coating associated with their plasma membrane which may 
increase the effective thickness of the membrane and make the cells less 
susceptible to an applied electric field than would be predicted by Eq. 7 
with V.sub.mc, assumed to be 1 Volt. In fact, assuming that the critical 
electric field imparted to the membrane required for poration, E.sub.mc, 
is similar for the cells present in the suspension, Eq. 6 indicates that 
the critical transmembrane potential V.sub.mc will be directly 
proportional to the effective thickness of the dielectric layer, and, 
therefore, from Eq. 7, the critical applied electric field strength, 
E.sub.c, for poration will also be directly proportional to the effective 
membrane thickness. Thus, an applied electric field strength may be chosen 
that is sufficient to inactivate a substantial fraction of cells having an 
effective membrane thickness below a certain predetermined threshold 
without inactivating a substantial fraction of the cells having an 
effective membrane thickness above the threshold. 
Although the threshold electric fields for the cells comprising harvested 
human bone marrow, as exemplified above, were theoretically estimated 
based on their size (see Table 1), the critical threshold electric fields 
for the cells listed in Table 1 have been previously measured. In addition 
to the importance of the magnitude of the applied electric field strength, 
total exposure time of the cells to the electric field is also an 
important parameter in determining the degree of inactivation of a given 
population of cells. In general, for cells that are selectively 
inactivated by electric fields on the basis of cell size, the electric 
field strength determines the size below which cells are preserved, and 
total electric field exposure time determines the relative reduction in 
cells having sizes above the critical size. Experiments in the prior art 
have been conducted over a wide range of pulsed electric field strengths 
and number of applied pulses and have led to an empirical model developed 
by Hulsheger 1983, herein incorporated by reference, for the surviving 
fraction of cells, s, following electric field treatment, as a function of 
the peak applied electric field strength, E, and the total time the cells 
are exposed to the electric field, t. The time t in the following model 
sums the on-time of the electric field over the total number of pulses, so 
that t=N.sub.p .tau..sub.p, where N.sub.p is the number of applied pulses 
and .tau..sub.p is the time duration of each pulse over which 
E.gtoreq.E.sub.c. Hulsheger 1983. demonstrated that bacterial cell 
surviving fraction can be roughly modeled by an empirical expression that 
is a power law function of time and an exponential function of electric 
field strength. Equation 8 provides a variant of Hulsheger's rough model 
that behaves correctly as the exposure time approaches zero for E&gt;E.sub.c. 
##EQU2## 
where, s is surviving fraction (ranging from 0.fwdarw.1), E.sub.c is the 
threshold value of the electric field strength for membrane breakdown, 
t.sub.c is an exposure time normalization constant, and k is an electric 
field normalization constant. E.sub.c, t.sub.c, and k can be empirically 
determined for a given cell suspension by fitting Eq. 8, to data taken 
relating fractional inactivation as a function of exposure time and 
applied electric field strength using any suitable regression analysis 
apparent to one of skill in the art. 
The equation for surviving fraction s can be utilized as a tool to analyze 
data collected for the surviving fraction of a particular cell population 
vs. applied electric field strength and exposure time, generated for a 
given cell suspension, and as a guide for selecting the field strength and 
duration required to achieve a desired survival fraction for cells which 
are to be porated and inactivated. By prescribing appropriate values for 
the electric field strength and total exposure time, required reductions 
in populations of cells of electroporation threshold below a critical 
value, for example having sizes larger than a critical diameter, can be 
achieved. Eq 8 indicates that s is a strong exponential function of 
electric field strength E and a weaker power law function of total 
electric field exposure time t. 
Thus, isolation and inactivation of cells or discrete objects by size 
differences according to the present invention proceeds by selecting an 
appropriate applied electric field strength E.sub.c so that cells of size 
greater than (from Eq. 7), d=4V.sub.mc /3E.sub.c will be inactivated and 
then applying an appropriate number of electric field pulses and/or total 
electric field exposure time to reduce the viable fraction of cells above 
the critical size to the desired level. 
Table 3 presents the results of cell inactivation experiments assembled by 
Castro A J, et al. on a variety of microorganisms using Hulsheger's 
original form of Eq. 8 (Hulsheger, which is obtained by removing the 
factor "+1" in Eq. 8. The threshold electric field E.sub.c derived from 
lethality measurements for E. coli in the logarithmic growth phase is 
shown in Table 3 to be 0.7 kV/cm, while the threshold field for E. coli in 
the stationary phase is more than 10 times higher, i.e., 8.3 kV/cm. The 
lower threshold electric fields required to irreparably porate the 
membranes of growing cells are related to the fact that growing cells are 
larger and must take in nutrients from the external environment, making 
them more susceptible to electric fields. 
TABLE 3 
______________________________________ 
Experimental conditions, values, and 
confidence limits for model parameters. 
E t E.sub.c 
t.sub.c k 
Microorganism (kV/cm) (ms) (kV/cm) (ms) (kV/cm) 
______________________________________ 
E. coli (4 hr) 
4-20 0.7-1.1 0.7 .+-. 3.1 
11 .+-. 9.6 
8.1 .+-. 1.8 
E. coli (30 hr) 10-20 0.7-1.1 8.3 .+-. 0.3 18 .+-. 5.7 6.3 .+-. 1.0 
K. pneumoniae 8-20 0.7-1.1 7.2 
.+-. 2.0 29 .+-. 16 6.6 .+-. 1.4 
P. aeruginosa 8-20 0.7-1.1 6.0 .+-. 0.4 35 .+-. 6.1 6.3 .+-. 1.1 
S. aureus 14-20 0.7-1.1 13 .+-. 
0.9 58 .+-. 17 2.6 .+-. 0.7 
L. mono- 12-20 0.7-1.1 10 .+-. 
2.6 63 .+-. 12 6.5 .+-. 2.5 
cytogenes I 
C. albicans 10-20 0.7-1.1 8.4 .+-. 7.5 110 .+-. 33 2.2 .+-. 0.9 
______________________________________ 
One embodiment of the invention, involving cancer cell purging and stem 
cell isolation by applied electric fields such as pulsed electric fields, 
is based on the observation that non-stem-cells are typically larger in 
size than stem cells; therefore, Eq. 7 and the observation that V.sub.mc 
is typically about 1 Volt for a wide variety of cell types implies that an 
electric field strength, E.sub.c, can be selected that will inactivate a 
substantial fraction of cells larger than the stem cell, including 
contaminating tumor cells. After selecting an appropriate predetermined 
critical electric field strength (which can be approximately E=2-2.2 kV/cm 
to preserve stem cell viability), total electric field exposure time, t, 
can then be selected with guidance from Eq. 8, and routine experimental 
optimization, in order to achieve the desired reduction in the unwanted, 
non-stem-cell populations, most importantly, tumor cell populations. 
Therefore, electric field conditions can be determined that lead to 
effective tumor cell inactivation and stem cell preservation, which is 
crucial for effective transplant tissue purging. 
Unlike stem cell isolation and purging strategies based on 
anti-metabolites, mechanical cell sorting, or antibody binding strategies, 
the present invention has the potential to isolate stem cells without 
damage and without mutation of the basic genetic molecules (DNA/RNA) 
within the cell. Most importantly, the genetic material is shielded from 
the pulsed electric fields by the conductivity of the cell nucleus and 
cytoplasm. Furthermore, at the electric field strengths of interest for 
isolating stem cells (about 1-3 kV/cm), the potential developed across 
critical bonds in these complex RNA/DNA molecules is generally not 
sufficient to break these bonds. Hence, stem cell isolation with pulsed 
electric fields should not cause undo damage to the genetic material 
within the cells. An additional advantage of using the 
electric-field-based tumor cell purging, stem cell isolation strategy is 
centered on the fact that toxic or potentially activating agents, such as 
anti-metabolites or exogenous antibodies, are typically not placed in 
physical contact with the stem cells. 
As previously mentioned, because of difficulties in applying a continuous 
potential across electrodes without arcing or discharge, electrochemical 
reactions, and excessive heat generation, the applied electric field is 
preferably supplied to the suspension as a series of short pulses, i.e. as 
a PEF. The maximum electric field pulse duration is typically limited by 
electric breakdown due to arcing between electrodes in the treatment 
volume and by single pulse heating effects. The pulse repetition rate is 
limited by the maximum temperature rise that can be sustained without 
causing undo damage to the sample. The minimum allowed pulse duration 
should be greater than the time constant at which the dielectric membrane 
charges in response to the electric field, as will be discussed in greater 
detail herein. 
Thus far, the effect of applied electric field strength and exposure time 
on the performance of the inventive methods have been discussed in detail. 
In addition, as mentioned earlier, a variety of other parameters related 
to the PEF, suspension, pulsing medium (fluid carrier), and other 
processing steps can affect the performance of the inventive method and 
should be considered when developing an effective isolation or 
inactivation protocol. What follows is a description of a number of what 
are believed are important factors related to performance. Throughout the 
description, reference will be made to one embodiment of tumor cell 
purging and stem cell isolation from suspensions of hematopoietic cells in 
order to illustrate the concepts with a concrete example. It should be 
understood that the particular example chosen is purely exemplary, and the 
methods may be practiced on a wide variety of samples for a wide variety 
of desired applications. Table 4 below summarizes some of the more 
important parameters (column 2) that can influence PEF performance, 
particularly as related to tumor cell purging and stem cell isolation, 
along with contemplated preferred ranges (column 3) of some chosen 
parameters for tumor cell purging and stem cell isolation. The table will 
serve as an outline for the discussion to follow. The "PEF" group includes 
parameters related to the nature of the applied electric field. The 
"Pulsing Medium" group includes parameters related to the properties of 
the suspension. The "Post Processing" group discusses optional treatments 
subsequent to electric field exposure that can, in some cases, enhance 
performance, and the "Heat Transfer" group includes parameters related to 
the heating effects of the applied PEFs. 
TABLE 4 
______________________________________ 
Parameters influencing PEF tumor cell purging 
and stem cell isolation efficacy. 
Group Parameter Description 
Range 
______________________________________ 
PEF -- Electric field pulse shape 
"Rectangular" 
E Electric field strength 0.5-5 kV/cm 
t Total electric field exposure time &lt;10 ms 
.tau..sub.p Electric field pulse duration 2-20 .mu.s 
Pulsing .eta..sub.ps Initial leukocyte concentration 10.sup.6 -10.sup.8 
cells/ml 
Medium .mu..sub.ps Pulsing medium ionic strength 0.015-0.15 M KCl 
equivalent 
.gamma..sub.ps Pulsing medium osmolality .ltoreq.300 mOsm/kg-water 
-- Agents for cell size modification 
-- Agents for dielectric membrane 
breakdown voltage modification 
Post .mu..sub.ls Inactivation medium ionic 0.15 M KCl equivalent, 
Proces- strength and composition Ca++ 
ing .tau..sub.ls Inactivation medium 
residence 
time 
-- Collection protocol Gradient density 
centrifugation 
Heat F.sub.p Electric field pulse rate Apparatus & pulsing 
Transfer medium dependent 
T.sub.ps Pulsing medium temperature 5-41.degree. C. 
______________________________________ 
For embodiments involving PEFs, a substantially rectangular electric field 
pulse shape is preferred for achieving optimum size selectivity. 
Rectangular pulses are those that have rise and fall times that are short 
compared to the pulse duration, and preferably shorter than the charging 
time scale of the dielectric membrane of the cells or objects to be 
inactivated (typically rise and fall times are .ltoreq.0.5 .mu.s for most 
applications of interest in the present invention), have essentially no 
overshoot during the rise- and fall-time transients, and have a 
substantially constant electric field strength between the rise and fall 
transients; preferably the difference in the maximum and minimum values in 
the substantially constant field strength region is less than 3%. 
Non-rectangular-shaped pulses, such as half-sinewave shaped or 
exponentially decaying pulses may be employed for some embodiments but 
will not provide as clearly defined an electric field strength, which can 
broaden the electroporation threshold demarcation line between 
uninactivated and PEF inactivated cells, thereby degrading the 
selectivity, resolution, and efficiency of the PEF cell inactivation 
method. 
The range of electric field strengths given in Table 4 is based on the 
critical electric field strengths given in Table 2 and represents a 
reasonable range to employ for optimization trials involving the 
inactivation of specific cell types listed in Table 2 as a function of 
electric field strength and total exposure time. With this electric field 
range, the range of inactivation that can be expected would range from no 
lethal effects on any cells to essentially total inactivation of all cell 
types. Based on Eq. 8, total electric field exposure time t can be used to 
achieve a desired reduction in the number of viable unwanted cells. The 
maximum total electric field exposure time typically will be less than 
about 10 ms for essentially complete inactivation of tumor cell 
populations. 
For embodiments involving PEFs, the inactivation of biological cells 
involves multiple steps which are dependent on the duration of the 
individual electric field pulses .notident..sub.p. Early in the pulse, the 
membrane of the cell charges, thereby producing an elevated transmembrane 
potential. The membrane charging time scale .tau..sub.m for spherical 
cells is given by (Lynch PT and Davey MR, Electrical Manipulation of 
Cells, Chapman and Hall, pp. 18-20, 1996.; and Tessie J and Tsong TY, 
Electric field induced transient pores in phospholipid bilayer vesicles, 
Biochemistry 20:(6)1548-1554, 1981.): 
EQU .tau..sub.m =1/2c.sub.m d(.rho..sub.c +.rho..sub.ps /2), (9) 
where c.sub.m is the membrane specific capacitance (typically .about.1 
.mu.f/cm.sup.2 for biological cell membranes (see Schanne O F and P. 
Cerreti E R, Impedance Measurements in Biological Cells, John Wiley and 
Sons, New York, p. 331, 1978.)), d is cell diameter, .rho..sub.c is the 
resistivity of the intracellular fluid (typically, for cytosol, .about.100 
.OMEGA.-cm), and .rho..sub.ps is the resistivity of the medium supporting 
the cells during PEF treatment (typical range of about 70-500 .OMEGA.-cm 
depending on the ionic strength of the solution). For a typical cell as 
exemplified by a hematopoietic cell, the maximum charging time constant 
will be on the order of about 0.5 .mu.s. In preferred embodiments, in 
order to achieve thorough membrane charging, the duration of the portion 
of the electric field pulse supplying an electric field strength greater 
than the critical field strength, E.sub.c, to porate the cell, for example 
the flat-topped portion of a rectangular field pulse, should be at least 
three to four times the membrane charging time constant .tau..sub.m, 
(i.e., for .tau..sub.m .about.0.4 .mu.s, .tau..sub.p &gt;1.6 .mu.s). 
After membrane charging is complete, pore formation begins and transport of 
ionic species between the inside of the cell and the pulsing medium takes 
place. It is known (Kinosita K and Tsong T Y, Voltage-induced pore 
formation and hemolysis of human erythrocytes, Biochim et Biophys Acta. 
471:227-242, 1977 (hereinafter "Kinosita 1977".) that ion transport 
processes can enhance PEF cell lysis by disrupting the osmotic balance 
across the cell membrane, which, in turn, can lead to cell swelling and 
irreversible lysis due to the uptake of water. It is also known (Kinosita 
1977) that the efficacy of inducing irreversible cell lysis by PEFs 
decreases for human erythrocytes when the electric field pulse length is 
decreased below about 10 .mu.s and improves as the pulse length is 
increased above 10 .mu.s. This phenomenon probably reflects the need for 
an electric field pulse duration that is sufficient to allow extensive 
pore development and time for ion species transport. Based on these 
observations, reasonable pulse durations for cells having a size range of 
between about 6 .mu.m and 20 .mu.m, typical for hematopoietic cells, can 
range from about .tau..sub.p =2 .mu.s to about .tau..sub.p =20 .mu.s. 
PEF cell inactivation and isolation efficacy can also be affected by the 
total concentration of cells in the PEF treatment volume. The effect of 
total concentration can be understood by comparing the total electric 
charge (Q.sub.m) required to charge the membranes of all of the cells in 
the treatment volume to a transmembrane potential of V.sub.mc =1 Volt, 
with the charge (Q.sub.p) actually supplied to the test volume by the 
electric field pulse. Q.sub.m can be expressed as: 
EQU Q.sub.m =.pi.v.sub.TV .eta..sub.c d.sup.2 c.sub.m V.sub.mc /4,(10) 
where v.sub.TV is the volume of the PEF treatment cell (cm.sup.3), 
.eta..sub.c is the concentration of cells in the PEF treatment volume 
(cells/cm.sup.3), d is the average diameter of the cells in the PEF 
treatment volume (assume for purposes of illustration .about.10 .mu.m), 
c.sub.m is membrane specific capacitance 30 (assume for purposes of 
illustration.about.1 .mu.f/cm.sup.2), and V.sub.mc is the critical 
transmembrane potential for the onset of irreversible pore formation 
(assume for purposes of illustration that V.sub.mc .apprxeq.1 Volt). Note 
that Q.sub.m is proportional to the cell concentration, .eta..sub.c. 
Q.sub.p can be expressed as: 
EQU Q.sub.p =.tau..sub.v v.sub.TV E/.rho..sub.ps w, (11) 
where .tau..sub.p is the electric field pulse length (sec), v.sub.TV is the 
PEF treatment volume (cm.sup.3), E is the electric field strength (V/cm), 
.tau..sub.ps is the resistivity of the medium supporting the cells 
(typically about 70-500 .OMEGA.-cm) in the PEF treatment volume, and w is 
the separation distance between the electrodes in the PEF treatment. Note 
that Q.sub.p is proportional to the electric field pulse length, 
.tau..sub.p, and inversely proportional to the pulsing medium resistivity, 
.tau..sub.ps. If the ratio Q.sub.m /(Q.sub.m +Q.sub.p) is not small, then 
cell concentration effects can degrade PEF cell inactivation efficacy by 
significantly increasing the effective charging time scale, .tau..sub.m, 
of the cells in the treatment volume, i.e., a significant amount of the 
total charge supplied to the treatment volume is required to simply charge 
the cells. In fact, as this ratio approaches unity, the cell membranes 
approach the situation where they have just achieved complete charging by 
the end of the electric field pulse, so there would be essentially no 
additional time for pore development and transport of ionic species 
between the cell and the pulsing medium to take place. For best 
performance, the ratio Q.sub.m /(Q.sub.m +Q.sub.p) is preferably within 
the range of about 0.0004.ltoreq.Q.sub.m /(Q.sub.m +Q.sub.p).ltoreq.0.74. 
The ratio Q.sub.m /(Q.sub.m +Q.sub.p) can be kept constant as the cell 
concentration (.rho..sub.c) is increased by increasing the pulse length 
(.tau..sub.p) proportionally and/or by appropriately reducing the pulsing 
medium resistivity (.rho..sub.ps), for example by increasing the ionic 
strength. The upper limit of cell concentration given in Table 4 
(.rho..sub.c =10.sup.8 cells/ml) has significance relative to a clinical 
PEF tumor cell purging system embodiment. For example, one liter of bone 
marrow, which is the approximate volume harvested for autologous 
transplants, contains approximately 10.sup.10 mononuclear cells, which 
must be purged before transplantation. If PEF conditions can be defined 
that provide high PEF tumor cell inactivation efficacy for a treatment 
volume cell concentration of 10.sup.8 cells/ml, then a 100 ml PEF 
treatment volume, or a flow-through system able to process 100 ml of cell 
suspension, can be used to process the entire bone marrow specimen, such a 
system is reasonable both in terms of system size and electric field pulse 
energy requirements. 
In addition to the nature of the applied PEF, the medium within which cells 
are suspended can be a significant factor in the performance and efficacy 
of the invention. The pulsing medium, in which biological cells are 
suspended, in some embodiments can have an osmolality that preserves the 
osmotic balance between the intracellular and intercellular fluids, in 
other words isotonic, where isotonic defines a solution with an osmotic 
strength (osmolality) similar to that of the suspended cells so that the 
cells do not undergo substantial osmotic pressure-driven cell volume 
regulation (.about.300 mOsm/kg-water for many mammalian cells, such as 
hematopoietic cells). If the osmotic strength of the pulsing medium 
differs substantially from that of the cytosol of the cell, the cell can 
undergo changes in cell volume, such as shrinkage or swelling that can be 
detrimental to cell viability and the performance of the inventive method, 
especially for isolations based on characteristic cell size. In some 
embodiments however, it can be desirable to suspend a cell suspension in a 
somewhat hypotonic medium either prior to pulsing, with subsequent pulsing 
performed in isotonic medium, or during the PEF treatment itself. In these 
embodiments, the suspending medium should have an ionic strength selected 
to enable at least one cell type in the suspension to undergo osmotic 
swelling, while not being low enough to cause rupture or a substantial 
loss of viability to target cells within the time frame of the treatment. 
Pre-treatment, or PEF treatment using a hypotonic suspending fluid can 
potentially improve performance for certain cell isolations by causing 
cells to become larger and more spherical in shape, and thus potentially 
more sensitive to the effects of an applied electric field. Treating cell 
suspensions that are characterized by cells having a more uniform 
spherical shape can also improve performance by reducing the effects of 
cell orientation within the electric field on poration. 
The ionic strength of the pulsing medium can be altered, while preserving 
the desired osmolality, by combining a solution comprising one or more 
solubilized electrolytes having a desired osmolality (e.g. an isotonic 
saline solution) with a solution comprising one or more solubilized 
non-electrolytes having a desired osmolality (e.g. an isotonic sucrose 
solution). There are several reasons why, for some embodiments, it can be 
desirable to reduce the ionic strength of the pulsing medium from standard 
physiological conditions (e.g. equivalent to a 0.15 M NaCl aqueous 
solution for many mammalian cells, such as hematopoietic cells). One 
reason is that it has been shown (Kinosita 1977) that PEF treatment using 
a pulsing solution having a below-physiological ionic strength followed by 
resuspension of PEF-treated cells in a standard physiological ionic 
strength solution can enhance cell destruction by colloidal osmotic lysis 
and result in a more rapid and extensive irreparable lysis of erythrocytes 
(red blood cells). This phenomenon has also been observed in the context 
of the present invention with leukocytes (white blood cells), where the 
PEF porated cells were subsequently reduced to small cell fragments by PEF 
treatment in low ionic strength medium (e.g. 10 v % PBS, 90 v % isotonic 
sucrose) followed by exposure to a physiological strength medium (e.g., 
isotonic PBS or Iscove's Modified Dulbecco's Medium (IMDM)). For 
embodiments of the inventive method utilizing a relatively lower ionic 
strength pulsing medium followed by exposure to a relatively higher ionic 
strength medium, the particular ionic strengths chosen for the pulse and 
post-treatment medium, and the exposure time of the PEF-treated cells in 
the post-treatment buffer will be selected based on routine 
experimentation with the particular cell suspension of interest to 
determine the conditions that yield the highest levels of inactivation of 
undesired cells with the best preservation of the viability of desired 
cells. Typically, the post-treatment medium will have an ionic strength 
similar to the physiological ionic strength of the cells in the treated 
suspension and the pulse medium will have an ionic strength ranging 
between 10% and 90% that of the post-treatment medium. The exposure time 
of the cells to the post-treatment medium can vary from a few seconds to 
an essentially indefinite period. It is important, however, that 
sufficient time be allowed for adequate diffusion and colloidal osmotic 
lysis to take place. In addition to, or instead of, having an ionic 
strength that is higher than that of the pulsing medium, the 
post-treatment medium may for some embodiments, have a higher osmolality 
than the pulsing medium and/or contain an agent that causes or enhances 
irreparable lysis of porated cells, such as, in some preferred 
embodiments, calcium ions. For some embodiments including a post-treatment 
step after PEF treatment designed to enhance or cause irreparable cell 
lysis, the cells porated by PEF exposure undergo irreversible poration of 
the cell membrane during PEF treatment, and post-treatment, as described, 
functions to irreparably lyse the cells that have already been inactivated 
by the PEF exposure. In other embodiments, exposure to the PEF porates, 
but does not necessarily irreversibly porate, the membranes of the cells 
to be inactivated, with inactivation and/or irreparable lysis occurring 
during a post-treatment inactivation step. 
For some embodiments, it may be desirable to remove any inactivated cells, 
lysed cells, and cellular debris from PEF treated specimens. Small cell 
fragments and debris generated by cell rupture during irreparable cell 
lysis can be separated from the viable cells using a variety of techniques 
known in the art, for example single- or multi-gradient centrifugation 
techniques. For embodiments where PEFs or post-treatment can fragment 
affected cells, it is possible to separate viable cells from cellular 
debris using standard gradient density centrifugation techniques. For 
example, Ficoll-Paque gradient density centrifugation, which is a single 
gradient separation scheme can be used. Multi-gradient centrifugation can 
also be used for other applications as apparent to one of skill in the 
art. For embodiments where inactivated cells also undergo irreparable cell 
lysis, characterized by cell rupture, it can also be advantageous to add 
to the suspending medium an agent that is able to degrade cellular debris. 
A variety of such agents apparent to one of skill in the art can be 
employed to reduce the cellular debris to its molecular components. 
Particularly preferred are enzymatic agents, and especially preferred is 
DNase to breakdown DNA dispersed in the pulsing media, and trypsin 
digestion to breakdown cell membrane and proteinaceous material. Such 
agents may be present during the PEF treatment, or alternatively, may be 
added subsequent to PEF treatment. 
Other advantages to reducing the ionic strength of the pulsing medium for 
some embodiments are related to the increased resistivity and reduced 
conductivity of pulsing solutions having a reduced ionic strength. The 
power density W.sub.p (J/cm.sup.3) and total charge density Q 
(Coulomb/cm.sup.3) input into the PEF treatment volume are both functions 
of the resistivity of the pulsing medium: 
EQU W.sub.p =E.sup.2 .tau..sub.p /.rho..sub.ps (11) 
EQU Q=E.tau..sub.p /.rho..sub.ps (12) 
where E is the magnitude of the applied electric field, .tau..sub.p is the 
pulse duration, and .rho..sub.ps is the resistivity of the pulsing medium. 
The resistivity of the pulsing medium varies inversely with ionic 
strength. Thus, according to Eq. 11, energy and power requirements of a 
PEF treatment cell can be reduced by a factor of ten, for example, by 
using a mixture of 10% by volume aqueous isotonic phosphate buffered 
saline and 90% by volume aqueous isotonic buffered sucrose as a pulsing 
medium instead of a standard physiological ionic strength medium. 
Additionally, reducing the ionic strength of the pulsing medium increases 
the resistance of the PEF treatment volume, which, for a given electric 
field strength and pulse duration, reduces the electron charge driven 
through the PEF treatment volume as shown by Eq. 12. Undesirable 
electrochemical reactions, such as free radical production, are typically 
proportional to the charge driven through the PEF treatment volume. Thus, 
reducing the ionic strength of the pulsing medium can proportionally 
decrease the production of free radicals, or other undesirable by-products 
of electrochemical reactions. Reduction of electrochemical reactions 
occurring in the PEF treatment volume during treatment can also reduce the 
pH swing of the pulsing medium during pulsing and also reduce the 
production of chlorine and hydrogen bubbles. Formation of bubbles during 
treatment is very undesirable, since the presence of bubbles can lead to 
non-uniform field distribution within the treatment volume and locally 
elevated electric field intensities, which can significantly reduce the 
size selectivity of the inventive cell isolation methods. In general, it 
is desirable to maintain the pH of the pulsing medium relatively constant 
during PEF treatment and within a range that is not detrimental to the 
viability of the cells being treated (typically about pH 7-7.6). A variety 
of buffer systems apparent to one of skill in the art for use in this 
range that are sufficiently non-toxic to cells can be employed including, 
but not limited to phosphate buffers, BES, MOPS, TES, HEPES, DIPSO, and 
TAPSO. Preferred for embodiments where precise pH control is especially 
important, are buffer systems including one or more strong organic buffers 
such as those referred to as "Good" buffers (Good, N. E., et al. , 
Biochemistry, 5:467(1966); Good, N. E., and Izawa, S., Meth. Enzymol., 
24(Part B):53(1972); Ferguson, W. J. and Good, N. E., Anal. Biochem., 
104:300(1980)), and especially 
N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES). 
One important consideration in designing and implementing a PEF isolation 
or inactivation strategy is heat effects and temperature rise due to heat 
generated within the PEF treatment cell due to the energy deposited into 
the treatment cell by the electric field. One issue that depends heavily 
on heat transfer effects is whether the total exposure time of the treated 
suspension should be achieved with a single pulse or with a series of 
pulses. Since many biological cells can be non-selectively inactivated by 
overheating, which is not a function of electroporation threshold, and 
since a single long electric field pulse can, without sufficient heat 
removal, cause a greater amount of heating of the cells for a given total 
exposure time, it is preferable, for many embodiments, to apply the total 
electric field exposure time as a series of pulses, rather than as a 
single pulse of longer duration. Since cyclic heating and cooling is 
particularly destructive for blood cells, pulse duration should be kept 
short enough to minimize cell and cell suspension temperature excursions 
beyond the physiological range. The rate, or frequency, at which electric 
field pulses may be applied is related to the energy deposited in the 
pulsing medium per electric field pulse (Joule heating), the geometric 
shape and heat transfer characteristics of the particular PEF treatment 
volume and the density and thermal conductivity of the pulsing medium 
(which collectively dictate heat removal rates), and the heat capacity of 
the pulsing medium. There are two components to the total temperature rise 
in the PEF treatment volume as a function of the electrical energy input: 
the temperature jump .DELTA.T.sub.j (.degree.C.) that occurs for each 
individual electrical pulse; and the steady pulsing temperature rise 
.DELTA.T.sub.s (.degree.C.), which is a function of the volume and heat 
transfer characteristics of the PEF treatment cell. Each time a pulse is 
applied to the pulsing medium, the temperature will jump. The magnitude of 
the temperature jump is proportional to the power density W.sub.p given by 
Eq. 11, and can be expressed as: 
EQU .DELTA.T.sub.J (.degree.C.)=.tau..sub.p E.sup.2 /.phi..sub.pm .rho..sub.ps 
c.sub.p, (13) 
where .DELTA.T.sub.J is the temperature jump (.degree.C.), .tau..sub.p is 
the electric field pulse length (with a typical range for cell isolations 
involving hematopoietic cells of about for example 2-20 .mu.s), E is the 
electric field strength (with a typical range for cell isolations 
involving hematopoietic cells of about for example 0.5-5 kV/cm), 
.phi..sub.pm is the density of the pulsing medium (typically .about.1 
g/cm.sup.3), .rho..sub.ps is the resistivity of the pulsing suspension 
(with a typical range for cell isolations involving hematopoietic cells of 
about for example 70-500 .OMEGA.-cm), and c.sub.p is the specific heat of 
the pulsing medium (typically .about.4.19 J/g.degree. C.). Under the most 
extreme conditions presented in Table 4, and listed in parenthesis 
directly above, the maximum temperature jump per electric field pulse will 
be .DELTA.T.sub.J =0.86.degree. C. The steady temperature rise is a 
function of the heat input per pulse, the number of pulses per unit time 
(frequency), and the heat transfer rate of the PEF treatment cell tending 
to remove heat from the treatment volume. One dimensional conduction heat 
transfer considerations may be applied for the static PEF treatment cell 
embodiment to determine the steady pulsing, time average temperature rise 
at the midplane between the electrodes imparting the electric field to the 
treatment volume relative to the temperature of the bounding electrodes. 
This formulation assumes that convective transport driven by temperature 
induced density gradients are of negligible importance. Equation 13a below 
(see Holman J P. Heat Transfer, 5th Ed., McGraw-Hill, Inc., New York, 
1981, p. 35) describes the average temperature rise for one dimensional 
steady pulsing volumetric heat deposition by PEFs into a treatment volume 
bounded by two plane electrodes. 
EQU .DELTA.T.sub.Cl,ave =qw.sup.2 /8.kappa. (13a) 
Where q is the volumetric heat deposition rate given by: 
EQU q=F.tau..sub.p E.sup.2 /.rho..sub.ps (13b) 
And where w is the separation distance between the electrodes, .kappa. is 
the thermal conductivity of the pulsing medium, F is the pulse repetition 
frequency, .tau..sub.p, is the electric field pulse length, E is the 
strength of the imposed electric field, and .rho..sub.ps is the 
resistivity of the pulsing medium. Equations 13a and 13b indicate that the 
mid-plane temperature increases as the square of the electrode separation 
distance, linearly with pulse repetition frequency, the square of electric 
field strength, and inversely proportional to the resistivity of the 
pulsing medium. Since the average midplane temperature rise given by Eq.'s 
13a and 13b, which constitutes the maximum average temperature rise in the 
PEF treatment volume, is inversely proportional to the resistivity of the 
pulsing medium, an increase in pulse repetition frequency by a factor of 
ten can be realized while maintaining the same midplane temperature rise 
by increasing the pulsing medium resistivity by a factor of ten. This can 
be accomplished by decreasing the ionic strength of the pulsing medium to 
10% of standard physiological ionic strength by the methods described 
previously. The temperature jump given by Eq. 13 in response to the 
application of each electric field pulse can be imagined to be 
superimposed on the average steady pulsing temperature rise 
.DELTA.T.sub.Cl,ave as a periodic temperature spike with an exponential 
decay and a frequency corresponding to electric field pulse repetition 
frequency. 
For illustrative purposes, if the electrode spacing is 0.318 cm and using 
the most extreme conditions presented in Table 4, which gave a maximum 
temperature jump per electric field pulse of .DELTA.T.sub.J =0.86.degree. 
C., the steady pulsing temperature rise at the midplane between the two 
electrodes will be .DELTA.T.sub.Cl,ave (.degree.C.)=5.16 F(Hz). Thus, if 
the electrodes are maintained at a temperature of 25.degree. C. and one 
wishes not to exceed a midplane peak temperature no greater than 
37.degree. C., then the average midplane temperature cannot exceed 
.DELTA.T.sub.Cl,ave =37-25-.DELTA.T.sub.J, which is .DELTA.T.sub.Cl,ave 
=11.14.degree. C., which constrains the pulse repetition frequency to be 
no greater than F=11.14/5.16=2.16 Hz. If the ionic strength of the pulsing 
medium was reduced by a factor of ten, the pulsing medium resistivity 
would increase by a factor of ten, which would allow operating at a pulse 
repetition frequency of 21.6 Hz for which .DELTA.T.sub.Clave ape would 
remain at 11.14.degree. C. 
For illustrative purposes, assuming a worst-case heating situation by 
neglecting any PEF treatment volume heat removal effects, the total 
electric field exposure time (t=N.sub.p .tau..sub.p, where N.sub.p is the 
number of applied electric field pulses) that may be applied without 
exceeding a predefined pulsing medium temperature rise can be expressed 
as: 
EQU t=.phi..sub.pm .rho..sub.ps c.sub.p .DELTA.T.sub.max /E.sup.2,(14) 
where .DELTA.T.sub.max is the maximum predefined allowable temperature rise 
(.degree.C.). For example, if pulsing is initiated with a PEF treatment 
cell pulsing medium temperature of 25.degree. C. and we wish not to exceed 
a final pulsing medium temperature of 37.degree. C., then the predefined 
maximum allowable temperature rise is .DELTA.T.sub.max =12.degree. C. 
Based on .DELTA.T.sub.max, and the strength of the applied electric field 
the allowable total electric field exposure time for a given cell 
suspension in a PEF treatment cell can be determined by Eq. 14. Eq. 14 
also shows that if a low ionic strength pulsing medium is used, the 
allowable electric field exposure time calculated for a given 
.DELTA.T.sub.max is directly proportional to the decrease in ionic 
strength; for example, reducing the ionic strength by a factor of ten 
would increase the allowable exposure time by about a factor of ten. 
Furthermore, since under low ionic strength conditions, the energy 
transferred to the PEF treatment volume is reduced, for a given desired 
total exposure time , the pulse frequency may be increased without 
exceeding the predefined maximum temperature rise, thus allowing for a 
more rapid isolation or inactivation. Accordingly, the inventive PEF 
strategy can be a very rapid approach to cell purging and cell isolation. 
For a single- or multi-pass flow-through PEF treatment cell embodiment of 
the invention, Eq. 14 can be manipulated to a form that describes the 
temperature rise of a fluid element of the pulsing medium as it traverses 
from the inlet to the exit of the PEF treatment volume. This formulation 
represents an upper bound on the temperature rise since it neglects heat 
transfer to the bounding electrodes as the fluid element passes through 
the PEF treatment volume. If N.sub.p is the number of electric field 
pulses applied during the residence time of the pulsing medium in the PEF 
treatment volume and .tau..sub.p is the duration of each of the electric 
field pulses, then the total electric field exposure time is .tau.=N.sub.p 
.tau..sub.p and the resulting temperature rise is given by Eq. 14a below: 
EQU .DELTA.T.sub.res =.tau.E.sup.2 /.phi..sub.pm .rho..sub.ps c.sub.p(14a) 
As illustrated above for the static PEF treatment cell embodiment, 
reduction of the ionic strength of the pulsing medium, which results in an 
increase in the resistivity of the pulsing medium, can allow a 
corresponding increase in the number of pulses that can be applied during 
the residence time of the pulsing medium in the PEF treatment volume. 
Thus, for the flow-through PEF treatment cell embodiment, it can be 
beneficial to use the lowest ionic strength pulsing medium allowable in 
order to maximize the number of pulses that may be applied in a single 
pass without exceeding the temperature rise limitations beyond which 
thermal effects impact cell viability. The relationships given above 
provide guidance to the skilled practitioner for selecting reasonable 
pulse repetition frequencies that are appropriate, for a specific pulsing 
medium and electric field pulse intensity and duration, for inactivating 
selected cells from a cell suspension having a maximum allowable 
temperature rise. 
Another previously mentioned factor that can influence the way in which an 
applied electric field interacts with a cell suspension, and the 
selectivity of an applied electric field at inactivating cells based on a 
critical electroporation threshold, is the shape and orientation of cells 
within the field. This factor is important for any cell inactivation 
involving non-spherical shaped cells. A problem arises with such samples 
because non-spherical cells, in a given sample, are typically randomly 
aligned with respect to the electric field direction. While such a random 
alignment is not a problem for hematopoietic stem cells or other 
essentially spherical cells, random orientation can reduce the 
effectiveness, especially for cells with large aspect ratios, of cell 
inactivation with applied electric fields. Eq. 1 shows that the 
transmembrane voltage, V.sub.m, that results from an applied electric 
field E is directly proportional to the projected length, l, of the cell 
in the electric field direction. Thus, for cells with large aspect ratios, 
l can be highly variable depending on the orientation of the cell. Since 
it is typically desirable to apply an essentially time-invariant 
transmembrane voltage for a predetermined length of time in order to 
obtain more easily predictable and controllable poration results, it is 
therefore desirable to align cells that are not substantially spherical so 
that they have a more consistent and predictable orientation with respect 
to the electric field direction. For embodiments of the invention where it 
is desired to align the axes of cylindrical or oval shaped cells to 
achieve maximum PEF inactivation efficiency, an AC field can be applied 
across the sample to accomplish this function. The AC field is preferably 
selected to provide an essentially uniform oscillating electric field 
during the PEF treatment period, and has a magnitude selected to be 
sufficient to align the cells with their long dimensions parallel to the 
PEF field direction for optimum size selectivity by the PEF field, without 
porating the cells or unduly overheating the pulsing medium. The 
theoretical treatment of this cell alignment technique is discussed in 
detail by Lynch (Lynch P T and Davey M R. Electrical Manipulation of 
Cells, Chapman and Hall, New York, Chapter 4, 1996), herein incorporated 
by reference. 
Some preferred embodiments of the invention include the use of an applied 
electric field for cell inactivation that is a bipolar electric field. A 
"bipolar electric field" as used herein refers to an electric field that 
is pulsed or otherwise applied to a sample so that the average current 
across the sample over the total treatment time is essentially zero. The 
use of bipolar electric fields in the context of the present invention 
provides several advantages over non-bipolar fields. When an electric 
field is applied across a sample, particularly a blood sample, 
electrochemical reactions can occur which can produce free radicals, other 
deleterious compounds, and species that can shift suspension pH and/or 
generate bubbles. Such electrochemical effects are, as previously 
indicated, undesirable. Within the context of the present invention, the 
inventors have found that undesirable electrochemical effects can be 
reduced or eliminated by utilizing a bipolar electric field pulsed so that 
the average current across the sample over the treatment time is 
essentially zero. Because the application of the bipolar electric field 
involves essentially equal current flows across the sample for each 
applied polarity, the reversible electrochemical reactions induced by the 
applied electric field component having a first polarity, can be 
substantially reversed by the applied electric field component having the 
opposite polarity, thus yielding a situation characterized by no net 
electrochemical reaction over the treatment time. 
There are a variety of ways to apply a bipolar electric field to the sample 
as apparent to one skilled in the electrical engineering arts. For some 
embodiments, the pulses across the sample are of essentially equal 
magnitude, duration, and number, but alternate pulses are of opposite 
polarity, while for other embodiments, the pulse having a first polarity 
may be of greater magnitude but shorter duration while the pulse of the 
reverse polarity is of lower magnitude and longer duration, so that the 
total average current flow is essentially zero. In another embodiment, an 
electric field pulse having a first polarity is utilized together with a 
DC current having an opposite polarity selected so that the magnitude and 
duration of each is selected to yield an essentially zero net current in 
order to achieve no net electrochemical reaction within the sample. In yet 
another embodiment for creating a desired bipolar electric field, the 
pulse used to create the PEF field may also be utilized to charge a 
suitable capacitor. When the original PEF pulse terminates, the capacitor 
then discharges back through the solution containing the cells at a rate 
determined in part by a resistance in the discharge path to produce the 
desired bipolar field. 
In addition to reducing undesired electrochemical reactions, bipolar PEFs 
can provide an additional advantage in the selective inactivation/lysing 
of larger cells. The additional advantage lies in that the first pulse 
component having a first polarity results in a charge across the membrane 
of the cell which remains for some period of time after the first pulse 
component terminates. If a second pulse component having an opposite 
polarity is applied across the cell during this time, the voltage across 
the cell can be effectively doubled for a short period of time. This 
doubling effect is greater for larger cells than for smaller cells because 
of the larger membrane charging time scale for larger cells (see Eq. 9). 
This effect can potentially enhance size-selective destruction of the 
larger cells, thereby enhancing the cell selectivity of the invention for 
certain applications. 
Temperature may also be utilized to enhance cell inactivation by the 
inventive methods. In general, biological cells are less capable of 
repairing membrane damage at sub-physiological temperatures. This behavior 
can be utilized to increase inactivation of cells in response to an 
applied electric field. For example, in one embodiment the cells are 
subjected to a PEF in a solution that is maintained within a physiological 
range of temperatures (for most mammalian cells, approximately 
33-39.degree. C.), the porated cells are then resuspended in a solution at 
a lower temperature, for example room temperature (approximately 
25.degree. C.) or lower, but above the freezing temperature of the 
suspension. The lower temperature solution delays repair of porated 
membranes and thus can increase the degree of cell inactivation by 
colloidal osmotic lysis. 
Alternatively, since cell repair (i.e. the closing of pores in a porated 
cell) will not take place when the cell temperature is dropped much below 
body temperature, the PEF exposure itself can be performed at a lower 
temperature, thereby permitting irreversible poration to occur at an 
electric field strength that can be lower than that which would otherwise 
be required. In addition, utilizing a lower PEF subjecting temperature for 
any given applied electric field strength can improve the kill rate (i.e. 
reduce the surviving fraction) from that suggested by Eq. 8 as determined 
for the same given field strength but a higher PEF subjecting temperature. 
For embodiments of the invention involving applications where it is desired 
to selectively inactivate one or more cell types having a characteristic 
size greater than a predetermined value while simultaneously leaving 
substantially viable another desired cell type, or group of cell types, 
having a characteristic size below the predetermined value, the greater 
the difference between the predetermined value of size and the 
characteristic size of the desired cells, in general, the easier and more 
selective the isolation. "Substantially viable" as used herein indicates 
that at least about 10% of the cells in the population are viable, 
preferably at least 50%, more preferably at least 90%, and most preferably 
at least 95%, while conversely, "substantially non-viable" as used herein 
indicates that at least about 25% of the cells in the population are 
non-viable, preferably at least 75%, more preferably a least 90%, more 
preferably at least 95%, and most preferably essentially all of the cells 
in the population are non-viable. For applications where the difference in 
characteristic size of the desired cell type and the undesired cell type 
is small, a variety of strategies can be utilized to improve the 
performance of the PEF isolation protocol. One method is to remove the 
cells that are close in size to the desired cells by performing a 
preliminary cell separation step that separates cells on some basis other 
than a difference in size. Depending upon the particular application, 
suitable cell separation methods include but are not limited to: flow 
cytometry; affinity cell chromatography; and centrifugation. 
For example, in an application involving the isolation of hematopoietic 
stem cells from other blood or bone marrow cells, both red blood cells 
(about 7 1m diameter) and resting lymphocytes (about 7 .mu.m diameter) are 
dimensionally the closest to the stem cell (about 6 .mu.m diameter). 
Because the number of red blood cells is much greater than the number of 
all of the nucleated cells combined, and because the red cells have a 
higher density than the nucleated cells, they are typically separated from 
a sample by density gradient centrifugation, using for example a 
Ficoll-Paque single gradient having a density of 1.07 g/cm.sup.3 (which 
will also remove polymorphonuclear leukocytes such as neutrophils), before 
PEF application. If the size selectivity of the PEF isolation for a 
particular embodiment is not sufficient to select stem cells over the 
resting lymphocytes with the desired level of purity, the resting 
lymphocytes, or other small cells, can be removed from the pulsing medium 
prior to PEF treatment. This can be achieved, by a variety of cell 
separation methods known in the art including CD34 targeted antibody 
affinity bin mg techniques that are selective for CD34.sup.+ cells, such 
as hematopoietic precursor cells including some stem cells. An alternative 
is to add an agent to the cell suspension that can preferentially modify 
(increase or decrease) the critical electroporation threshold of a subset 
of the cells to make them more or less susceptible to inactivation by 
electric fields. For example in one embodiment, the small resting cells, 
such as resting lymphocytes, may be eliminated by activating the resting 
lymphocytes using an agent that forces the resting lymphocytes into their 
active state so as to achieve their mature size (typically about 12 .mu.m) 
where hey can be more easily inactivated without adversely affecting the 
stem cell population. A variety of suitable activating agents are known in 
the art including protein agents, such as cytokines, lymphokines, 
chemokines, anti-cell surface marker antibodies, and cell receptor 
antagonists. Other suitable stimulants include lectins, such as Phaseolus 
vulgaris lectin (PHA), and concanavalin A. As specific examples, 
monoclonal anti-CD40 antibody, CD40 ligand, or interleukin-4 (IL-4) can 
efficiently activate resting B-cells (see Valle A, Zuber CE, Deffrance T. 
Djsou, et al., Activation of human B lymphocytes through CD40 and 
Interleukin 4. Eur J Immuno 19:1463-1467, 1991. Banchereau J, de Paoli P, 
Valle A, Garcia E, et al., Long term human B cell lines dependent on 
Interleukin 4 and anti-CD40. Science 251:70-72, 1991. both incorporated 
herein by reference), while interleukin-2 (IL-2) and concanavalin A can 
ef:ciently activate T-cells (Berger SL, Lymphocytes as resting cells. 
Methods in Enzymology (eds) Jakoby W and Pastan I. Academic Press. Vol 
LVIII:486-494, 1979. Waxman J and alkwill. (eds) Interleukin-2. Blackwell 
Science Publications, Oxford, 1992. both incorporate in by reference). 
Another factor that can affect the performance of selective cell 
inactivation by electric fields on the the basis of a difference in a 
characteristic electroporation threshold based on cell size is a 
concurrent difference in the dielectric membrane breakdown voltage, for 
example, due to a difference in the effective membrane thickness, between 
cell types. In such cases, it can be advantageous to add an agent to the 
cell suspension, prior to or concurrently with electric field application, 
that can modify (increase or decrease) the dielectric membrane breakdown 
voltage of one or more cell types. For example, for applications where a 
larger, undesirable cell type possesses a thicker effective membrane 
thickness than a smaller desired cell type, as previously discussed, the 
larger cell having the thicker effective membrane thickness will typically 
have a higher critical dielectric membrane breakdown voltage and thus 
require a larger electric field strength for inactivation than a 
comparably sized cell having a thinner membrane. Depending on the 
difference in characteristic size and effective membrane thickness between 
the two cell types, the ability of the electric field to selectively 
porate the larger cells while not affecting the smaller cells can be 
diminished or eliminated. A specific example of an application where this 
phenomenon may arise is in the use of PEFs for selectively inactivating 
certain tumor cells from tissue samples or hematopoietic cell suspensions. 
A variety of tumor cells, for example many epithelial tumor cells, have 
associated with their plasma membrane a relatively thick layer of 
mucopolysaccharide, known as the glycocalyx, that can increase the 
effective thickness of the membrane dielectric layer making the cells less 
susceptible to the PEF. For application, where this phenomenon is an 
important consideration, e.g. when cell kills are substantially less than 
would be predicted based on cell size alone, the performance of the PEF 
method can be improved by removing the glycocalyx layer prior to 
subjecting the cell suspension to the PEF treatment. Agents that can be 
used for effectively reducing or eliminating the glycocalyx layer on cell 
membranes include enzymes such as hyaluronidase, collagenase, pronase, 
elastase, and trypsin (Gruenert D C, Basbaum C B, and Widdicombe J H. 
Long-term culture of normal and cystic fibrosis cells grown under 
serum-free conditions, In Vitro Cell. Dev. Biol 26, 411-418, 1990. Lechner 
J F, Babcock M S, Marnell M, Narayan K S, and Kaighn M E. Normal human 
prostate epithelial cell cultures, Methods in Cell Biology, 21B, 195-225, 
1980. Stamfer M R. Isolation and growth of human mammary epithelial cells, 
Journal of Tissue Culture Methods, 9, 107-115, 1985.). Alternatively, an 
agent that preferentially increases the dielectric membrane breakdown 
voltage of a desired cell type could be used, in addition to or instead of 
the above mentioned agents, in some embodiments to make the cells less 
susceptible to inactivation by electric fields. 
In some embodiments, where it is desired to alter the apparent membrane 
breakdown voltage or characteristic size of one or more subpopulations of 
cells in a heterogeneous population of cells, for example when the 
electroporation thresholds of desirable and undesirable cellular subsets 
are comparable, such alterations can be effected by attaching material to 
cellular subsets using antibodies that have immunospecificity for the 
cells, especially monoclonal antibodies. For example, metallic beads 
coated with a monoclonal antibody that can bind the bead to a specific 
cell surface antigen can produce two distinctly different effects 
depending upon the surface density of the beads attached to the cell. If 
the surface density of the attached beads is very high, such that an 
almost continuous layer of metallic beads exists on the surface of the 
cell, then the resulting metallic structure will behave as a Faraday cage, 
which will shield the cell from the effects of the imposed electric 
fields. If the surface density of the beads on the cells is low, however, 
then the each bead will behave as an antenna, which can make the effective 
size of the cell larger, thereby making the cells more susceptible to the 
lethal effects of the imposed electric fields than implied by the original 
size of the cell. Thus, agents, such as antibody coated metallic beads, 
can be used to alter the electroporation thresholds of specific cells, 
thereby enhancing PEF selection or inactivation characteristics. 
A typical stem cell, shown in FIG. 2a, also possess a unique morphology 
that can make them less susceptible to poration by electric fields than 
their size would suggest. 
Morphologically, stem cells are typically small in size (.about.6 .mu.m in 
diameter for hematopoietic stem cells) with a faint halo of cytoplasm 72 
between the nuclear sack 74 surrounded by nuclear membrane 73, and outer 
(plasma) membrane 71. The next larger nucleated hematopoietic cells, 
resting lymphocytes (.about.7-8 .mu.m in diameter), typically have a much 
larger gap between their nuclear and outer membranes. The arrangement of 
the stem cell's nuclear 73 and outer membranes 71 being separated by a 
very small distance can cause the nuclear 73 and outer membrane 71 to 
become electrically coupled and to charge together as one effective 
dielectric layer of thickness approximately equal to the sum of the 
thicknesses of the nuclear and outer membranes. Provided the electric 
field pulse length is small compared to the discharge time scale of the 
nuclear membrane, electric field strengths considerably greater than the 
value implied by the diameter of the stem cell and the critical 
transmembrane voltage, V.sub.mc, for the outer membrane 71 will be needed 
to form temporary or irreversible pores in stem cells. Specifically, for 
PEF pulse durations less than the characteristic discharge time scale of 
the nuclear membrane 73, the critical electric field required for the 
onset of poration would be: 
EQU E.sub.comp .apprxeq.E.sub.c (t.sub.om +t.sub.nm /t.sub.om (15) 
where E.sub.coup is the required electric field strength to porate the cell 
for electrically coupled nuclear 73 and outer 71 membranes, E.sub.c is the 
critical field strength as calculated from Eq. 7, t.sub.om is the 
effective thickness of the outer membrane 71, and t.sub.nm is the 
effective thickness of the nuclear membrane 73. 
FIG. 2b illustrates electrically the reasons the nuclear and outer 
membranes of the stem cell can charge as one membrane when the electric 
field pulse duration is small compared to the discharge time scale of the 
nuclear membrane. The poles 81, 82 of the cell are defined as those two 
points on the surface of the cell which are closest to the electrodes 
imposing the electric field. The membrane gap is defined as the smallest 
distance between the nuclear and outer membranes. The pole-to-pole 
membrane gap resistance 80 is the resistance in the membrane gap between 
opposite poles of the cell. The membrane gap resistance 77 is the gap 
resistance between the inner and outer membranes. When the membrane gap is 
very small, the membrane gap resistance 77 will be much less than 
pole-to-pole membrane gap resistance 80. This is typically the case for 
the stem cell. Under these conditions the nuclear and outer membranes will 
charge together as one membrane of thickness approximately equal to the 
sum of the thicknesses of the nuclear and outer membranes. When the gap is 
significant, which will be the case for most other cells in the blood and 
immune system, the pole-to-pole membrane gap resistance 80 is less than 
the membrane gap resistance 77, and the nuclear membrane does not 
participate significantly in electric field effects, so that just the 
outer membrane charges. If R.sub.pp is taken as the pole-to-pole membrane 
gap resistance 80 and C.sub.pp is the capacitance of the dual membrane 
system, then the discharge time scale of the nuclear membrane will be no 
greater than .tau..sub.nm &lt;R.sub.pp C.sub.pp. Provided the electric field 
pulse is significantly less than .tau..sub.nm, the nuclear and outer 
membranes will behave electrically as one membrane of thickness equal to 
the sum of the nuclear and outer membrane thicknesses (see Eq. 15) and 
during this time, electric field strengths much greater than implied by 
the diameter of the stem cell (see Eq. 7) will be required to form 
irreversible pores. This effect can enhance the ability to isolate stem 
cells utilizing the PEF methods of this invention and can reduce the need 
to activate cells that are close in size to the stem cells in order to 
increase the difference in characteristic size. 
While the discussion above has been with respect to the poration of larger 
cells to isolate or segregate stem cells which typically have a smaller 
size, there are also applications where it may be desirable to porate the 
stem cells. Heretofore, an effective technique for electroporating stem 
cells has not existed. Poration of stem cells can be achieved according to 
the present invention by initially using the PEF method to isolate stem 
cells, and subjecting the isolated stem cells to an electric field of 
appropriate magnitude for porating the stem cells. For example, if the 
electric field pulse duration, .tau..sub.p, being utilized is less than 
.tau..sub.nno, then the appropriate magnitude would be determined by 
employing Eq. 15 in combination with Eq. 7; however, if the pulse duration 
is longer than .tau..sub.nm, then the appropriate magnitude would be 
determined by employing Eq. 7. The objective in porating stem cells can be 
reversible, non-lethal poration or irreversible poration to lyse or kill 
the cells. One embodiment of the present inventive methods can enable the 
temporary, reversible poration of the stem cells to allow, for example, 
genetic material to enter the stem cells, producing genetic mutations or 
recombinations for gene therapy. The inventive stem cell transfection 
technique may be preferable to many currently employed techniques, such as 
using viral transfection, since the PEF poration technique can enable the 
genetic material to enter a larger percentage of the stem cells and can 
result in a higher survival rate for the mutated stem cells. Such mutated 
stem cells can, for example, be utilized in a variety of gene therapy 
techniques, for cloning, or to provide immunity against specific adverse 
biological agents such as viruses, bacteria, and various toxins. 
Since a variety of known methods exist for extracting viable cells from 
mixtures containing viable/dead cells and cellular debris, selective 
inactivation of cells by the methods provided by the present invention 
represents a potentially important step toward achieving high purity 
isolation. PEF cell inactivation can, via post-PEF spontaneous cell lysis 
(colloidal osmotic lysis), transform the PEF inactivated cells into ghost 
membranes and free nuclei dispersed in the cellular suspension. Single or 
multiple gradient centrifugation techniques can then be used to separate 
the viable cells, and, if desired, any residual intact non-viable cells, 
from the cellular debris composed primarily of ghost membranes and free 
nuclei. Viable cells can also be extracted from the PEF-treated mixture of 
viable/dead cells and cellular debris by using cell sorting, e.g. FACS or 
flow cytometry, or antibody binding strategies that are commercially 
available for a variety of cell types. Alternatively, if the population of 
selected viable cells are to be expanded, there may be no need to remove 
the post-PEF non-viable cells and debris, since the expansion process can 
produce a post-expansion population of viable cells that will render 
insignificant the relatively small numbers of inactivated cells and 
residual cellular debris, or the debris will simply decompose during the 
expansion culture. 
FIG. 3 presents a flow chart summarizing the steps of a typical PEF cell 
isolation strategy according to the invention. It should be reemphasized 
that for any given cell suspension and desired cell isolation or 
inactivation, the PEF parameter values for optimal performance must be 
selected based on routine experimentation and optimization with guidance 
from the theoretical development presented previously. The general 
procedures described below may be employed both during screening tests to 
determine optimal PEF parameters for cell isolation, and during actual 
cell selections with predetermined optimal parameters. 
Initially, before the beginning of the procedure, PEF operating parameters 
(e.g. electric field strength, total exposure time, pulse duration and 
frequency, etc.) are selected as described previously based, in part, upon 
a difference in a characteristic electroporation threshold difference 
between desired and undesired cells (determines choice of electric field 
strength) and a desired degree of cell inactivation (determines choice of 
exposure time). The first step 61 of the procedure 60 is to prepare the 
cell suspension. The cell suspension is prepared by uniformly dispersing 
and suspending cells in a physiologically compatible, conductive medium. 
In some cases, e.g. blood, the sample to be treated may already be 
suspended in a suitable medium, in other cases, e.g. cells from solid 
tissue or organs, the cells may need to be dispersed and resuspended in a 
suitable medium. The viability, concentration and identity of the cells 
present in the pre-PEF treated suspension can be determined by a variety 
of methods known in the art. Viability, for example, for many cells, such 
as mammalian cells, can be determined by trypan blue dye exclusion. 
Concentration may be determined by manual or automated cell counting 
techniques, for example manual counting with a hemacytometer, or automated 
counting by light scattering techniques. Individual cell types can be 
enumerated and marked for further tracking by standard immunophenotyping 
techniques known in the art, such as by using cell-specific dyes or 
dye-labeled antibodies (e.g. fluorescently labeled antibodies) that have 
specificity for certain cell surface antigens specific to certain cell 
types. The labeled cells can then be quantified by standard techniques, 
for example, fluorescence microscopy or flow cytometry. 
The second step 62 of the procedure 60 includes various optional 
pre-treatment methods, or pre-PEF cell separations, used to enhance the 
performance of the PEF treatment. A variety of such methods were discussed 
previously and include: isolation of a subpopulation of cells by cell 
separation methods such as centrifugation, for example, density gradient 
centrifugation; osmotic cell lysis of red blood cells; cell affinity 
chromatography; fluorescence activated cell sorting (FACS); etc. Other 
treatments that can be employed at this step include treatments designed 
to enhance a difference in characteristic electroporation threshold, such 
as: hypotonic swelling of the cells; treatment with an agent to remove a 
glycocalyx layer from one or more cell types to reduce the effective 
membrane thickness; treating the cell suspension with an activating agent 
to increase the characteristic size of one or more cell types, etc. For 
applications involving the isolation of hematopoietic stem cells from 
blood or bone marrow, typically during this step, mononuclear cells are 
purified from the sample from step 1 by using a standard Ficoll-Paque 
density gradient centrifugation technique, followed by ACK (product 
10-548, BioWhittaker, Walkersville, Md.) lysis of residual erythrocytes. 
The mononuclear cells are then washed and resuspended in an appropriate 
pulsing buffer. Optionally, an activating agent, such as those mentioned 
previously, may be added to the suspension to activate resting 
lymphocytes, or, if tumor cells having a thick glycocalyx layer are 
present, an enzyme may be added to at least partially remove this layer. 
The third step 63 of the method 60 involves subjecting the cell suspension 
to the pulsed electric field. Electric field strengths and durations may 
be selected for this step that are sufficient to cause irreversible 
poration and inactivation of the porated cells, or alternatively, that are 
sufficient to reversibly porate but not inactivate porated cells. For some 
purposes, the PEF treated cell suspension produced during this step is in 
a final, usable form; however, for many applications, additional post-PEF 
steps are required or desirable for attaining a final cell suspension. 
The fourth step 64 of the method 60 is an optional step performed to 
inactivate cells that are porated but not inactivated in the previous step 
63. This step 64 is typically employed for embodiments where the PEF 
parameters chosen in step 63 are sufficient to porate but not inactivate 
the undesired cells; however, the step may also be employed for 
embodiments utilizing PEF conditions selected for inactivation in order 
to, for example, increase the total fraction of cells inactivated, speed 
up the inactivation process, or physically disrupt the structure of the 
inactivated cells by irreversible lysis. As previously discussed, the 
methods of this step typically involve resuspending the PEF treated cells 
in an inactivation medium, or adding one or more supplemental agents to 
the PEF medium to create an inactivation medium in situ, designed to 
accelerate colloidal osmotic lysis, prevent cell membrane repair, or both. 
As previously discussed, the inactivation medium for use in this step 
typically will have one or more of the following properties: a higher 
ionic strength than the pulsing medium; a higher osmolality than the 
pulsing medium; a lower temperature than the pulsing medium; or an agent 
(e.g. Ca.sup.++) that specifically promotes colloidal osmotic lysis of 
porated cells. 
The fifth step 65 is an optional step performed to remove inactivated cells 
and cellular debris from the suspension containing viable PEF isolated 
cells. This step can be performed by a variety of techniques apparent to 
the skilled artisan, including, but not limited to centrifugation, 
filtration, and adsorption. In some embodiments, suitable agents may be 
added in order to break up or reduce cellular debris, such agents 
including, for example DNase, trypsin, or other enzymes. For applications 
involving hematopoietic cell isolations, after PEF treatment, the stem 
cell enriched suspension is typically subjected to a Ficoll-Paque gradient 
density centrifugation to remove inactivated cells and cellular debris. 
For selected applications, it may be desirable to further purify, isolate 
or treat a subpopulation of cells from the PEF treated cell suspension. 
Such secondary, or supplemental cell separation or other treatments 
comprise an optional sixth step 66. Any of the previously mentioned cell 
separation techniques can potentially be employed for this step. In some 
cases, the secondary purification may be used to further enrich or purify 
the subpopulation initially isolated by PEF treatment, while in other 
cases, the secondary isolation may involve separating a sub-subpopulation 
of cells from the PEF isolated subpopulation, which sub--subpopulation may 
not be distinct with respect to a critical electroporation threshold. Such 
a secondary separation is potentially useful, for example, for 
applications where it is desirable to separate a particular type of 
progenitor cell from a PEF isolated stem cell suspension. As previously 
discussed, preferred stem cell suspensions isolated by PEF can contain a 
variety of different lineage committed colony forming cells in addition to 
the pluripotent cells. One or more of these cell sub-types may be isolated 
by, for example, FACS by utilizing one or more labeled antibodies with 
immunospecificity to cell surface markers present on particular cell 
sub-types. 
The final optional seventh step 67 comprises any treatments that are 
performed on or using the PEF treated cell suspensions. Such treatments 
can include, for example, expansion and/or differentiation of the selected 
cells by in vitro cell culture techniques, transfection or other genetic 
modification of selected cells, etc. For example for applications 
involving the isolation of stem cells, since the fraction of stem cells 
present in a typical pre-treatment sample is very small (about 1:10.sup.6 
in bone marrow aspirate), depending on the volume of sample processed, the 
final quantity of isolated stem cells may not be sufficient to engraft a 
patient (typically about 10.sup.6 CD34.sup.+ cells/kg body weight are 
required). In such cases, the quantity of stem cells can be amplified 
through standard stem cell amplification and expansion techniques 
(Zandstra, A. J., et al. "Advances in hematopoietic stem cell culture," 
Curr Opin Biotechnol 9:146-151(1998)). Cell culture techniques can also be 
employed to activate the isolated stem cells to differentiate along 
desired lineage paths, thus increasing the number of committed progenitor 
cells reinfused into a patient. An additional post-PEF isolation treatment 
that can optionally be performed on the isolated stem cells, for example 
for gene therapy applications, is reversible poration and transfection of 
the cells with genetic material using PEFs to electroporate the stem 
cells. 
In most applications, and especially when screening to determine optimal 
PEF parameters, it is desirable to characterize the post-PEF treatment 
cell suspension with regard to cell quantity, cell viability, and cell 
identity in order to evaluate performance. The techniques described for 
characterizing the pre-treatment suspensions can also be employed to 
characterize the post-treatment populations. From comparison of the pre- 
and post-treatment cell suspensions, yield, selectivity, enrichment, and 
depletion determinations can be inferred. The inventors have found that 
for analyzing post-PEF suspensions of hematopoietic cells, an effective 
and convenient method to simultaneously determine cell viability, cell 
apoptosis, and cell concentration, and cell identity, is to stain the 
post-PEF cells obtained after the third step 63 or the fifth step 65 above 
with a combination of propidium iodide, Annexin-V, and fluorescently 
labeled antibodies specific to CD3, CD14, CD19, CD45, CD34, and CD38 cell 
surface markers. These cell specific fluorescently labeled markers allow 
enumeration of lymphocytes (CD3+, CD45+), monocytes (CD14+, CD45+), 
primitive progenitor cells (CD34+), and certain hematopoietic stem cells 
(CD34+, CD38-). The viability stain propidium iodide is a DNA stain. Thus 
for cells with disrupted membranes, yet still containing a nucleus, this 
dye will stain non-viable cells. Annexin-V, however, stains 
phosphatidylserine (PS) which migrates from the inside to the outside of 
the plasma membrane during normal apoptosis. Thus it is normally used as 
an indicator of apoptotic, not yet non-viable cells. Whether or not the PS 
migrates to the external surface of the cell as a result of PEF treatment 
is unimportant. What is important is that should PEFs result in the 
discharge of the nucleus from the cell, Annexin-V will stain the PS on the 
inside of the plasma membrane, since this stain has access to the inside 
of the cell due to membrane disruption. Thus, ghost cells should have a 
bright Annexin-V fluorescence signature. Therefore, gating based on both 
PI and Annexin-V has been found by the investigators to give the best 
screening for viable cells. Additional markers specific to cancer cells 
can also be included for applications involving cancer cell purging. 
While the above description in association with FIG. 3 is intended to 
illustrate some representative methods and strategies for performing the 
inventive cell isolations, it should be understood that the above 
description is only illustrative and exemplary, and that the invention can 
be performed otherwise than described above without departing from the 
spirit and scope of the invention as presented in the appended claims. 
Also, the above description presents and describes various methods and 
techniques that can for certain embodiments and applications be associated 
with the invention; however, additional or substitute methods may be used 
as apparent to the skilled artisan, and details and descriptions that, in 
some cases, may be necessary to perform the invention but that are known 
or available to those skilled in the art are not necessarily included or 
described herein. 
The inventive electric field cell/discrete object isolation/inactivation 
methods can potentially be performed using a wide variety of 
electroporation equipment known in the art (see for example: Gene Pulser 
II, BioRad, Calif.; ECM-2001, BTX, Calif.; Multiporator, Eppendorf, N.Y.; 
Electroporator II, Invitrogen, Calif.; PA-4000, Cytopulse, Md.). Although 
there are numerous electroporation systems available, they are not ideally 
suited for cell selection/inactivation discussed herein because they have 
been designed for electroporation or electrofusion applications for which 
cell preservation is key, not cell inactivation. They are also typically 
not capable of processing the number of cells appropriate for either 
research or clinical implementations of the cell selection/inactivation 
strategies discussed herein, since they are limited by the electric field 
pulse energies they can deliver. FIG. 4 shows one embodiment of a batch 
system 85 including elements useful for performing the inventive PEF 
methods. The main components of the illustrated system are: an electric 
field pulse driver 101, which applies voltage pulses to the PEF electrode 
enclosure assembly 95 (which includes the PEF treatment cell); a power 
supply 102, which can be external or internal to the pulse driver; an 
optional oscilloscope 103 for electric field waveform monitoring; a 
trigger generator 104; and a control/data acquisition system 105, 
preferably including a computer, for controlling the system and gathering 
and processing data. The control/data acquisition system 105, trigger 
generator 104, oscilloscope 103, power supply 102, and pulse generator 101 
are electrically coupled via appropriate electrical connections 106, and 
together comprise an electric field generating mechanism 90. The pulse 
driver 101 of the generating mechanism 90 is electrically coupled to the 
PEF electrode enclosure assembly 95 via cathode connecting line 100 and 
anode connecting line 99. Also included in the overall system 85 is an 
optional forced circulation cooling system 98 which forces cooling fluid 
through the PEF electrode enclosure assembly 95 through lines 96 and 97 to 
remove heat from the treatment cell that is generated by the electric 
field and for controlling the temperature of the treatment cell. 
The PEF electrode enclosure assembly 95 of system 85 is shown in greater 
detail in FIG. 5. The electrode enclosure assembly 95 includes a PEF 
treatment cell 110 that is designed to physically mate with cathode 114 
and anode 112. FIG. 5 is a cross-sectional view of the PEF electrode 
enclosure assembly 95 taken by slicing the PEF electrode enclosure 
assembly 95, as oriented in FIG. 4, into the plane of the drawing. The PEF 
electrode enclosure assembly 95 also includes an annular enclosure 122 
that is sealingly mated to the top plate 113 and bottom plate 111. Top 
plate 113 and bottom plate 111 are connected to each other at spaced 
intervals using a plurality of spacer rods 120 which are transversed by a 
threaded rod 126 with threadingly attached nuts 121 which may be tightened 
to securely connect the top 113 and bottom 111 plates, or loosened and 
removed for disassembly of the PEF electrode enclosure assembly 95. 
Annular enclosure 122 circumscribes and defines an enclosed space 123 that 
may be evacuated via evacuation port 125 or pressurized with a gas via 
pressurization port 124 in order to more thoroughly insulate the 
electrical components of the assembly. The treatment cell 110 and cathode 
114 is supported by a fluid-cooled cathode support rod 115 that includes 
cooling fluid channels 116 and 117. The anode 112 also preferably includes 
fluid cooling channels 118 and 119 to provide additional heat removal 
capacity from the treatment cell 110. The fluid cooling channels are in 
fluid communication with forced circulation cooling system 98 shown in 
FIG. 4. Forced circulation cooling system 98 is preferably sized and 
designed to maintain the cathode 114 and anode 112 at a selected 
temperature controlled to +/-0.1 degrees C over a temperature range of at 
least 4-50 degrees C. Heat removal and temperature control of the pulsing 
medium is effected by conductive heat transfer from the medium to the 
temperature controlled anode 112 and cathode 114. The PEF electrode 
enclosure assembly 95 should be constructed with appropriate seals so that 
enclosed space 123 can be maintained under high vacuum without significant 
leakage. The PEF electrode enclosure assembly 95 can be constructed from a 
variety of materials apparent to one of skill in the art. The anode 
assembly 112, and cathode 114, should be constructed from conducting 
materials such as metals. In one particular embodiment, the cathode 114 
and anode 112 are constructed of copper. In some preferred embodiments, 
anode assembly 112 is removable from bottom plate 111 to allow easy access 
and removal of test cell 110 without the need to disassemble the entire 
PEF electrode enclosure assembly 95. The top 113 and bottom 111 plates can 
be constructed of any strong stiff material. Preferred plates are 
constructed from an insulating material such as a strong plastic, for 
example Lexan. Annular enclosure 122 is preferably constructed from a 
transparent material such as plexiglass. 
The static test cell 110 is shown in exploded view in FIG. 6. The test cell 
comprises an anode end block 131, which mates to anode 112 via element 
138, a cathode end block 132, which mates to cathode 114 via element 144. 
Each end block includes a plate 137, which is circular in the illustrated 
embodiment, and a plurality of holes 136 through which connecting elements 
pass in order to assemble and seal the treatment cell 110. The end blocks 
are in contact with plate electrodes 133 and 134 which are in turn 
separated by the fluid containing annular spacer 135. Annular spacer 135 
includes a channel 139 that, when the treatment cell 110 is assembled, 
provides a passage in fluid communication with an internal volume 140 
defined by the annular wall 141 of the spacer 135 and the planar walls 142 
of plate electrodes 133 and 134. In operation, the cell suspension to be 
treated is inserted and removed from the treatment volume 140 via channel 
139. The treatment cell 110 is sized to provide a treatment volume having 
a desired total volume. Small scale experimental systems typically have a 
treatment volume of about 1-5 ml, while large scale clinical devices 
preferably have a treatment of 0.1-1 liter. 
End blocks 131 and 132 can be constructed of any suitable conducting 
material. Preferred end blocks are constructed from metal. Particularly 
preferred end blocks are constructed from copper and subsequently gold 
plated, or are constructed from tungsten. Annular spacer 135 is 
constructed from an insulating material that is preferably biocompatible. 
A preferred material for constructing the annular spacer 135 is tempered 
glass (e.g. Pyrex.RTM. glass). Electrodes 133 and 134 can be constructed 
from any suitable conducting material, with preferred electrodes being 
constructed from conducting materials that are biocompatible and which do 
not release toxic amounts of electro-catalyzed reaction products during 
application of the electric field to the sample. Particularly preferred 
electrodes 133 and 134 are constructed from graphite carbon. For 
embodiments utilizing porous graphite electrodes 133 and 134, the 
electrodes are preferably degassed after introduction of a pulsing medium 
or cell suspension into test cell 110 so that bubbles are not released 
from the electrodes into the medium during application of the PEFs. 
Degassing can be accomplished by a variety of methods apparent to the 
skilled practitioner, for example the pulsing medium can be added to the 
assembled test cell 110 at sub-ambient temperature and -subsequently 
heated to ambient or physiological temperature to release gas bubbles, or 
the pulsing medium can be added to an assembled treatment cell 110 
maintained under vacuum during the adding step. A particularly preferred 
electrode arrangement that can reduce or eliminate the release of bubbles 
trapped in the porous matrix is constructed from graphite which is 
subsequently sealed with a sealing agent so that the surfaces 142 in 
contact with the pulsed suspensions are rendered essentially non-porous. 
In preferred embodiments, the porous matrix is sealed with a thin layer of 
pyrolytic carbon. The test cell 110 is also preferably constructed and 
arranged so that an essentially spatially uniform electric field is 
applied to the treatment volume 140. During use, it is important that the 
suspension undergoing treatment completely fill treatment volume 140 so 
that there is no meniscus that can distort the electric field 
distribution. Also, preferred treatment cells 110 are constructed and 
lapped to have very smooth mating surfaces on components 131, 133, 135, 
134, and 132, so that when assembled, the treatment cell 110 is fluid 
tight without the need for supplemental seals, such as washers or O-rings. 
A schematic diagram of a preferred continuous flow PEF system 150 is 
illustrated in FIG. 7. The system includes a flow-through treatment cell 
151 having two electrodes 152 and 153 in fluid contact with a flowing 
suspension being treated that enters the treatment cell 151 through line 
168 and exits through line 169. The electrodes 152 and 153 are 
electrically coupled to a generating mechanism 90, which can be 
essentially identical in arrangement as that previously described. System 
150 also includes pump 160 for pumping cell-free pulsing medium through 
the system, and pump 161 for pumping a cell suspension through the system. 
Pumps 160 and 161 pump fluid via lines 162 and 163 respectively, into line 
164, which is in fluid communication with three-way valve 165. Three-way 
valve 165 can be set to direct the pumped fluid to the treatment cell 151 
via line 168 or to a waste container 167 via line 166. Pulsing medium or 
treated suspension exiting the treatment cell 151 via lines 169 and 172 
can be controllably directed to a sample container 177 via line 176 or a 
waste container 175 through adjustment of three-way valve 173. 
Particularly preferred embodiments of system 150 also include a 
supplemental pump 170 which controllably pumps a desired substance into 
the stream of PEF treated cells via line 171. The desired substance can be 
a variety of materials useful in the fourth step 64 through the sixth step 
66 of the exemplary procedure 60 discussed previously in reference to FIG. 
3. For example, the substance supplied by pump 171 can be DNase or be 
Trypsin, added to inactivate released DNA and reduce cellular debris, or a 
medium added to raise the ionic strength and/or osmolality of the 
suspension in order to accelerate or bring about inactivation through 
colloidal osmotic lysis. The system as arranged allows the treatment cell 
151 to be initially primed with pulsing medium before addition of cell 
suspension, and also allows the treated suspension to be diverted to waste 
until optimal PEF conditions are established. Also optionally included in 
the system 150 but not shown are mechanisms downstream of the treatment 
cell 151 for removing inactivated cells or cellular debris, such as 
filters or flow cytometers. In addition, for some embodiments, it may be 
advantageous to provide a cooling system, such as system 98 in FIG. 4, to 
remove heat from the flow-through treatment cell 151. 
The preferred flow-through treatment cell 151 includes graphite electrodes 
constructed from similar material as discussed for electrodes 133 and 134 
previously. The electrodes can be enclosed in a flow chamber constructed 
of an insulating material such as a plastic, or preferably, Pyrex glass. 
The electrodes 152 and 153 are elongated in shape and are positioned so 
that their long axis is parallel to the direction of fluid flow and have 
extended planar surfaces 154 and 155 in contact with the fluid in 
treatment volume 178. The electrodes are preferably constructed and 
arranged so that the electric field applied to the fluid in treatment 
volume 178 is substantially spatially uniform and so that the electric 
field strength upstream and downstream of the main electric field 
treatment region (the region bounded by the portion of the electrodes 152 
and 153 that have surfaces, which are in contact with the cell suspension 
during operation, that are essentially parallel to one another) 
essentially never exceeds the strength in the main electric field 
treatment region. In order to accomplish this, preferred electrodes 
include contoured regions 156 and 157 (scale exaggerated in the FIG.) 
adjacent the inlet and outlet regions of the test cell 151. Well 
established electrode profiles exist (e.g. Rogowski, Ernst, or Chang 
profiles) that can be implemented to obtain the purpose of contours 156 
and 157. The treatment volume 178 of the test cell 151 is defined, for 
rectangularly shaped test cells, as the product of the length 179, the gap 
width 195, and the height h (into the plane of the figure and not shown) 
so that v.sub.TV .apprxeq.lwh, where v.sub.TV is the total volumetric 
capacity of treatment volume 178, l is the length of the test cell 151, 
and w is smallest gap width 195 between electrodes 152 and 153. These 
dimensions are chosen for a particular application to yield a desired 
volumetric throughput having a desired total treatment residence time in 
the treatment volume 178 and a shear rate below the value that can cause 
damage to the cells. For a test cell having an essentially uniform 
rectangular cross-section for flow, the average residence time 
.tau..sub.res of fluid in the treatment volume v.sub.TV is: 
EQU .tau..sub.res =v.sub.TV /u=lwh/u (16) 
where u is the volumetric flow rate of the fluid. The maximum laminar shear 
rate .GAMMA. can be approximated by: 
EQU .tau.=2u(w+h)/(wh).sup.2 (17) 
The treatment cell dimensions and throughput flow rate should be selected 
to provide a desired total exposure time t to the electric field for a 
given applied pulse duration, .tau..sub.p, and pulse frequency F. For 
example, if the desired total treatment time is t, then the necessary 
pulse frequency would be: 
EQU F=(ut)/(lwh.tau..sub.p) (18) 
For one exemplary embodiment of treatment cell 151, length 179 is 37.3 mm, 
the gap width 195 is 8 mm, and the height (into the plane of the figure) 
is 4 mm. 
Startup and operation of the flow-through system 150 can proceed as 
follows. The system, in this exemplary embodiment, employs syringe pumps 
for pumps 160, 161, and 170. With pump 160 loaded with cell-free pulsing 
medium, pump 161 loaded with the cell suspension to be treated, and pump 
170 loaded with pulsing medium supplemented with DNase, three-way valve 
165 is positioned to direct fluid to the treatment cell 151, and three-way 
valve 173 is positioned to direct fluid to waste container 175, and the 
lines and treatment cell 151 is flooded with pulsing buffer by activating 
pump 160 in order to remove bubbles from the system. Next, pump 160 is 
stopped, three-way valve 165 is repositioned to direct fluid to waste 
container 167, and pump 161 is activated to pump cell suspension to waste 
container 167 in order to remove any bubbles from line 163. Three-way 
valve 165 is then switched to direct the cell suspension to the treatment 
cell 151 and the generating mechanism 90 is activated to apply PEFs to the 
treatment volume 178. As soon as it is determined that the system is 
functioning properly, three-way valve 173 is positioned to direct the 
treated cell suspension into cell collection container 177. When a desired 
quantity of cell suspension has been processed, pump 161 is shut off and 
the generating mechanism 90 is switched off. Pump 170 may be operated as 
desired during PEF treatment to add DNase to the treated cell suspension 
to prevent coagulation of any cellular debris. The sequence of events just 
described is preferably computer controlled and pertinent PEF system data 
is automatically collected and stored by a data acquisition system. In 
alternate embodiments of flow through system 150, instead of passing 
through the treatment cell 151 only once, the cell suspension can be 
recycled back to the inlet of the treatment cell for a plurality of PEF 
treatments. Also in some embodiments, instead of supplying a pulsed 
electric field to the treatment volume 178, the field is maintained at an 
essentially constant value during the PEF subjecting step, and the average 
exposure time of the cells in the suspension is simply the average 
residence time of the cells in the treatment volume 178 determined by the 
suspension flow rate. 
Pulse driver 101 (see FIG. 4) for use in systems 85 and 150 can be any 
suitable pulse driver known in the art that is capable of producing pulsed 
electric fields within the PEF treatment volume of sufficient magnitude 
and duration for inactivating discrete objects of interest. Preferred 
pulse generators are able to induce an electric field magnitude of at 
least about 5 kV/cm, more preferably at least about 10 kV/cm, and most 
preferably at least 20 kV/cm in the treatment volume. Preferred pulse 
generators are able to supply an electric field pulse duration above a set 
point electric field strength of between about 2-20 .mu.s at pulse 
frequencies between about 0 to 10 kHz. Preferred pulse drivers are able to 
produce a substantially rectangular pulse shape, as previously discussed, 
with rise and fall times not exceeding about 0.5 .mu.s. Preferred pulse 
driver mechanisms also include control circuitry to allow the maximum 
pulse voltage to be controllable by the user and to terminate pulsing upon 
detection of an electrical short circuit or arc. While pulse drivers based 
on gas switches, such as thyratrons and spark gaps, can be employed for 
use in the invention, because of their typically superior durability and 
reliability, pulse drivers based on all-solid-state switch technology are 
preferred. 
Design specifications for the pulse driver are determined from the required 
maximum applied electric field strength, maximum required electric field 
pulse duration, the size of the treatment volume, and the electrical 
resistivity of the suspensions being treated. The pulse driver for use in 
a particular application must be designed to supply a maximum pulse 
energy, e.sub.p (Joules/pulse), determined by: 
EQU e.sub.p =.tau..sub.p v.sub.TV E.sup.2 /.rho..sub.ps (19) 
where .tau..sub.p is the maximum pulse duration, v.sub.TV is the treatment 
volume, E is the maximum required electric field strength, and 
.tau..sub.ps is the resistivity of the sample being treated. For impedance 
matched conditions, the load voltage, V.sub.l, is one-half the maximum 
charge voltage of the pulse driver. The load voltage requirements of the 
pulse driver can be determined by: 
EQU V.sub.l =Ew (20) 
where E is the maximum required electric field strength, and w is the 
distance between the electrodes (see FIG. 7). The resistance load of the 
treatment cell which the pulse driver must be able to handle is determined 
by: 
EQU R=.rho..sub.ps w.sup.2 /v.sub.TV (21) 
where R is the impedance load (.OMEGA.), .rho..sub.ps is the resistivity of 
the sample being treated, w is the distance between the electrodes, and 
v.sub.TV is the treatment region volume. 
FIG. 8 is a block diagram of one embodiment of a pulse driver system 180 
for use in the inventive PEF systems. The pulse driver 180 produces the 
substantially rectangular bipolar pulse shown in FIG. 9. The bipolar 
rectangular pulse shown in FIG. 9 has short rise 201 and fall 202 times 
separated by flat regions 203 of substantially constant voltage. The 
trailing opposite polarity segment 204 of the waveform serves to reduce 
and/or reverse electrochemical reactions that otherwise can produce 
hydrogen and chlorine bubbles, which can degrade performance and affect 
suspension pH. In other embodiments, instead of supplying a bipolar pulse 
as shown in FIG. 9, the pulse generator may instead supply a unipolar 
pulse, as shown in FIG. 10, while simultaneously supplying a reverse 
polarity DC current properly matched to essentially equal the 
time-averaged current of the electrical field pulses. The pulse shape 
shown in FIG. 10 is not substantially rectangular in shape but is instead 
in the shape of a half sine-wave. As previously discussed, such pulse 
shapes will, in general, yield poorer electroporation threshold 
selectivity than more rectangularly shaped pulses. 
The pulse driver system in FIG. 8 that produces the waveform shape shown in 
FIG. 9 includes two pulse driver circuits 205 and 206, one 205 for a 
positive polarity voltage pulse, the other 206 for a trailing, negative 
polarity pulse. Each of the pulse drivers includes a DC power supply (182 
and 183), a storage capacitor 184, and a stack of integrated bipolar 
transistors (IGBTs) 185 and 187, which are solid state switches that apply 
the electrical energy stored in the storage capacitors 184 to the PEF 
treatment cell 191. The IGBTs each include a switch stack and gate trigger 
186, 188, and a diode 189, 190 on the output line. The IGBTs are stacked 
in series and parallel combinations to provide enhanced voltage and 
current capabilities. Power is supplied to the pulse driver system from a 
source 181 of facility line power. Preferred pulse driver systems also 
include circuitry 193 for process control and circuitry 192 for system 
diagnostics and data acquisition as known in the art. In preferred 
embodiments, the electric field strength developed in the PEF treatment 
cell 191 is determined by circuitry 192 for system diagnostics and data 
acquisition by use of a calibrated high voltage probe/oscilloscope system. 
The current waveform can be measured using a Pearson coil and is, in 
preferred embodiments, recorded on the same oscilloscope as the voltage 
waveform. The time integrated product of the current and voltage waveforms 
can then be used to determine pulse energy. Pulse energy thus determined, 
together with measurements of the pulse repetition frequency and the 
temperature of the electrodes in the treatment cell 191 can be used, 
together with a heat transfer mathematical model describing the heat 
transfer characteristics of the treatment cell 191, to calculate the 
temperature evolution of the pulsed suspension. Alternatively, the 
temperature of the pulsed suspension can be directly measured in a 
plurality of locations within the treatment volume with suitable 
temperature probes or thermocouples. Diagnostic 192 and control 193 
systems also, in preferred embodiments, include one or more computer 
elements, for example integrated personal computers, to control system 
operation and perform data reduction and analysis. 
The function and advantage of these and other embodiments of the present 
invention will be more fully understood from the examples below. The 
following examples are intended to illustrate the operation of the present 
invention, but not to exemplify the full scope of the invention. 
EXAMPLES 
Introduction 
The experimental examples that follow, presented as five separate cases, 
demonstrate pulsed electric field cell-size selectivity, hematopoietic 
primitive progenitor cell enrichment, and tumor cell purging. Two PEF 
cell-size selection examples are presented (Cases 1 and 2). In Case 1 PEFs 
were applied to a suspension of peripheral blood mononuclear cells 
resulting in a step-wise reduction in size distribution of the PEF treated 
viable cells as a function of electric field strength and total electric 
field exposure time. In Case 2 PEFs were applied to a suspension of 
peripheral blood mononuclear cells, illustrating lymphocyte enrichment by 
selective inactivation of monocytes in PEF treated specimens as a function 
of electric field strength and electric field exposure time. Case 3 
illustrates the enrichment of hematopoietic stem cells in PEF treated 
peripheral blood progenitor cell specimens. In Case 3, PEFs were applied 
to mobilized peripheral blood specimens harvested from a patient by 
leukopheresis. Case 3 illustrates the dependence of stem cell enrichment 
on electric field strength for a fixed electric field exposure time. Two 
cases are presented that illustrate PEF tumor cell purging (Cases 4 and 
5). In Case 4, PEFs were applied to a suspension of peripheral blood 
mononuclear cells seeded with a megakaryocyte tumor cell line (CMK). This 
case illustrates the selective inactivation of the tumor cells with 
simultaneous preservation of lymphocyte and monocyte cells as a function 
of electric field strength and fixed total electric field exposure time. 
In Case 5 PEFs were applied to a suspension containing only mammary gland 
breast tumor cells (MCF-7). This case illustrates the PEF inactivation 
characteristics of this breast tumor line as a function of electric field 
strength, pulse duration, and total electric field exposure time. The 
examples given by Cases 1-5 were performed under non-optimized PEF 
conditions. Thus, these results represent a lower end demonstration of the 
capability of the PEF cell selection strategy. 
Methods and Materials 
The methods and materials for the five example cases are given together 
below and are broken into three categories: cell preparations, cell 
assays, and PEF apparatus. 
Cell Preparations 
Four cellular systems were used in the example cases: peripheral blood 
mononuclear cells (PBMCs), mobilized peripheral blood mononuclear cells 
(hereafter referred to as peripheral blood progenitor cells, (PBPCs)), 
megakaryocyte tumor cells (CMKs), and mammary gland breast tumor cells 
(MCF-7s). 
PBMCs were used in Cases 1, 2, and 4. The PBMCs were obtained from healthy 
donors by harvesting approximately 60 ml of peripheral blood per test day 
by venipuncture through a 21G, 1 inch needle (Beckton Dickinson, 305175) 
into a syringe (Beckton Dickinson, 309663) containing 1 ml Heparin (5000U, 
Elkins-Sinn, Inc., A-0400H). The PBMCs cells were separated from the whole 
blood using standard density gradient centrifugation techniques. Under 
sterile conditions, the blood was diluted with twice its volume with 
1.times. phosphate buffered saline (PBS, Mediatech, 21-031-CV) and then 
aliquoted (30-40 ml) into 50 ml conical centrifuge tubes. Ficoll-Paque 
(Pharmacia Biotech, 17-0840-03) was slowly dispensed into the bottom of 
each centrifuge tube, and the tubes were centrifuged (IEC, Centra GP8R, 
rotor no. 228) at 2000 RPM for 30 minutes with the brake off. The PBMCs at 
the density interface were collected by aspiration with a 5 ml pipet. The 
recovered PBMC layers were combined into a single 50 ml centrifuge tube. 
The resulting PBMC suspension was then diluted 10.times. using PBS, 
aliquotting into as many 50 ml centrifuge tubes as required, followed by 
centrifugation at 1500 RPM for 5 minutes. The supernatant was aspirated, 
and one pellet was resuspended in 10 ml PBS. This 10 ml suspension was 
then used to resuspend the pellets of all remaining tubes. PBS was then 
added to the resulting suspension, bringing the total volume to 50 ml. 
This 50 ml suspension was centrifuged at 1500 RPM for 5 minutes; the 
supernatant was aspirated; the pellet was then resuspended in 20 ml IMDM 
(Sigma, I 2762) with 10% fetal calf serum (FCS, Sigma, F 2442), and then 
transferred to a flask and placed in an incubator at 37.degree. C. 
overnight in preparation for PEF treatment the next day. The next morning 
the contents of the flask were transferred to a 50 ml centrifuge tube. The 
flask was washed three times with 10 ml PBS: these PBS wash volumes were 
added to the 50 ml tube containing the bulk of the PBMC specimen. The 
resulting suspension was then centrifuged at 1500 RPM for 5 minutes; the 
supernatant was aspirated, and the cells were suspended in the desired 
pulsing buffer. Pulsing and post-PEF treatment buffers are discussed in 
more detail in a subsequent section. 
Cell preparation for Case 3 was identical to that for Cases 1 and 2 with 
the following exceptions. The cells for Case 3 were obtained by 
leukopheresis from a cancer patient that had been administered granulocyte 
colony stimulating factor (G-CSF). More specifically, mobilization of the 
patient's peripheral blood was effected by administering G-CSF (10 .mu.g 
per kg body weight per day) subcutaneously for 4-6 days, with apheresis 
collections beginning on day 4 until 2.5.times.10.sup.6 CD34+ cells per kg 
body weight were obtained. The resulting cell suspension contained 
approximately 2.times.10.sup.8 leukocytes in approximately 2 ml PBS. This 
cell preparation was used, instead of the whole blood in the protocol 
described above for obtaining PBMC preparations. Before suspending the 
PBPCs in the pulsing buffer, the resting lymphocyte population was 
activated to move these cells to their larger, active state in order to 
improve for stem cell enrichment. To activate the lymphocytes, the PBPC 
suspension, interleukin-2 (IL-2, 50 IU/ml, R n' D Systems) and PHA (0.25 
.mu.g/ml, Sigma) were added to a culture medium (20 ml, IMDM, 10% FCS) and 
incubated at 37.degree. C. for 36 hours. After activation (incubation), 
the suspension was centrifuged at 1500 RPM for 5 minutes, the supernatant 
was aspirated, and the resulting pellet was resuspended in the desired 
pulsing buffer. 
For Case 4, PBMCs, prepared as described above for Cases 1,2 and 4, were 
seeded with a megakaryocyte tumor cell line (CMK, Sato T. et al., Br. J 
Haematol. 1989 Jun; 72(2): 184-90). For Case 5, a suspension containing 
only mammary gland tumor cells (MCF-7, ATCC no. HTB-22) was exposed to 
PEFs. Preparation of the MCF-7s for PEF treatment is described below with 
modifications to the procedure for the CMK line noted. MCF-7 (ATCC no. 
HTB-22) cells were thawed under the following conditions. A vial of cells 
was thawed at 37.degree. C., then transferred to a 50 ml tube (Fisher, 
14-959-49A) containing 30 ml of 1.times. Iscove's medium without glutamine 
(IMDM, Fisher, MT15 016LV) and centrifuged at 1500 RPM for five minutes at 
room temperature. The supernatant was discarded and the pellet was 
resuspended in 20 ml of MCF-7 culture-medium and cultured at 37.degree. 
C., 5% CO.sub.2. The MCF-7 culture medium used was 1.times. Dulbecco's 
modification of Eagle's medium, without glutamine, including 4.5 g/L 
glucose, and supplemented with 2 mM L-glutamine (Fisher/Cellgro, 
MT-25-005-LI), 50 I.U./ml of penicillin, and 50 .mu.g/ml streptomycin 
(Fisher/Cellgro, MT-300-01-LI) and 20% FCS. The cells were split upon 
reaching confluence, typically every three to four days, as follows. 
Culture supernatant was aspirated and replaced with 5-6 ml of trypsin (2.5 
g/L 1:250 in HBSS without calcium or magnesium, Fisher/Cellgro, 
25-050-11). After incubation at room temperature for 5 minutes, the flask 
was rinsed using 10 ml of 1.times. Dulbecco's Phosphate buffered saline 
without calcium or magnesium (PBS, Fisher/Cellgro, MT-21-031-CV) 
supplemented with 1% fetal calf serum (FCS, Sigma, T-2442). The rinse 
solution was then transferred to a 50 ml tube, and the volume was made up 
to 50 ml with PBS, supplemented with 1% FCS, before centrifuging the tube, 
as described above. After centrifugation, the supernatant was discarded 
and the pellet was resuspended in 10 ml of MCF-7 culture medium. Five 
milliliters of this suspension was transferred to one of two 75 cm.sup.2 
flasks, each containing 15 ml of MCF-7 culture-medium, prior to reculture 
under the above described conditions. If there were more than two flasks 
to be split, then the excess cells were frozen as follows. The cells were 
trypsinized and washed as previously described, and then resuspended in 1 
ml of freezing medium (10% DMSO, Fisher, D128-500, 90% FCS), per flask and 
transferred to labeled cryovials (Fisher, 5000-1020) prior to transfer to 
a -70.degree. C. freezer. MCF-7 PEF treatment commenced the morning after 
these cells achieved half-confluency in the culture flasks. On the morning 
of a PEF experiment, the MCF-7s were trypsinized using the above protocol, 
the recovered cells were then washed using PBS, and the centrifuge pellet 
was then suspended in pulsing buffer in preparation for PEF treatment. 
For the CMK line, the cell suspensions were thawed as previously described 
for the MCF-7 line and cultured in CMK culture medium at 37.degree. C., 5% 
CO.sub.2. The CMK culture medium comprised 1.times. RPMI 1640 medium, 
without glutamine, supplemented with 2 mM L-glutamine (Fisher/Cellgro, 
MT-25-005-LI), 50 I.U./ml of penicillin, and 50 .mu.g/ml streptomycin 
(Fisher/Cellgro, MT-300-01-LI) and 20% FCS. The CMKs were split 1:10 every 
3-4 days by transferring about 2 ml of the cell suspension to a new 75 
cm.sup.2 flask containing 18 ml of CMK culture medium. Excess cells were 
frozen as previously described. On the morning of a PEF treatment 
experiment, the CMKs were transferred from the culture flasks to 50 ml 
Falcon tubes (Becton Dickinson, 2098). As part of this transfer, the cells 
were passed through a separation filter (Miltenyi Biotec, Inc., 414-07) to 
remove large-scale debris. The cells were then washed in PBS and 
resuspended in the pulsing buffer in preparation for PEF treatment. 
Pulsing Medium 
For Cases 1 and 2, IMDM was used as the medium in which the PBMCs were 
suspended for PEF treatment. After PEF treatment, the treated specimens 
were combined with an equal volume of IMDM. The resulting specimen 
remained at room temperature until preparation for flow cytometry 
analysis. 
For Cases 3,4, and 5, the cells were suspended in a low ionic strength 
medium (10%v/v PBS, 90%v/v isotonic sucrose solution). This low ionic 
strength pulsing medium was formulated to be isotonic. After PEF 
treatment, the treated specimen was combined with an approximately equal 
volume of IMDM. The inactivation protocol using a low ionic strength 
pulsing medium, followed by resuspension in a higher ionic strength 
medium, was used in these cases to investigate whether the combination 
would result in more extensive post-PEF fragmentation of PEF porated cells 
by colloidal osmotic lysis. The low ionic strength pulsing buffer was 
formulated as follows. Twenty milliliters of 1.times. sterile PBS was 
combined with 180 ml of distilled/deionized water. To this solution, 16.6 
g of powdered sucrose was added (Fisher, BP220-212). This solution was 
then sterilized by passing it through a 0.2 .mu.m filter (Nalgene, 
291-3320). The pH of the resulting solution was checked using a Beckman 
.PHI.40 pH meter and was found typically to lie in the range 7.4-7.6. 
Prior to final preparation of the cellular suspensions for pulsing, trypan 
blue exclusion using phase contrast microscopy was employed to enumerate 
the number of viable cells present in PBS suspensions of the various 
cells. Given the number of cells desired in the cellular suspensions for 
pulsing, the trypan blue results were used to determine the volume of PBS 
cell suspension required to provide the desired number of cells, which 
volume was then centrifuiged to pellet the cells. The pellet was then 
suspended in the required amount of pulsing buffer. 
Cell Assays 
Pre- and post-PEF cell enumerations were performed using both trypan blue 
exclusion, using a hemacytometer (Fisher, 02-671-10) under phase contrast 
optical microscopy, and a wide variety of well established flow cytometry 
protocols. Trypan blue exclusion was used primarily for determining the 
number of viable cells to suspend in the pulsing medium for each 
experiment. Flow cytometry, using a variety of viability and conjugate 
antibody fluorescent stains, was used to enumerate pre- and post-PEF 
viable cell numbers, including lymphocytes (B- and T-cells), monocytes, 
primitive progenitor cells (including stem cells), and tumor cells (CMK 
and MCF-7). A Becton Dickinson FACScan flow cytometer was used to perform 
the analytical assays. For Cases 1, 2, 3, and 4, cell viability was 
determined by flow cytometry using either propidium iodide (PI, Molecular 
Probes, P-3566) or TO-PRO-3 (Molecular Probes, T-3605) DNA staining 
combined with light scatter characteristics. Viable lymphocytes were 
identified as those cells scoring low for the particular DNA viability 
stain used (either PI or TO-PRO-3) while staining brightly for CD3 
(T-cells, Fisher, OB9515-02 or -09) or CD19 (B-cells, Fisher, Becton 
Dickinson, 340409 or 340364). Viable monocytes were identified as those 
cells scoring low for the DNA viability stain used while staining brightly 
for either CD11b (Becton Dickinson, 347557), CD13 (Fisher, OB9555-02 or 
-09), or CD14 (Fisher, OB9560-02 or -09) and CD45 (leukocytes, Fisher, 
OB9625-02 or -09). Viable hematopoietic stem cells were identified as 
those cells scoring low for the DNA viability stain used while staining 
brightly for CD34 (Fisher, OB9595-02 or -09) and dimly for CD38 (Fisher, 
OB9610-02 or -09). Viable CMK tumor cells were identified as those cells 
scoring low for the DNA viability stain used, staining dimly for CD14, and 
staining brightly for CD45, while also being outside of the light scatter 
compartments for lymphocytes and monocytes (light scatter gating). 
For Case 5, involving the PEF inactivation characteristics of MCF-7 tumor 
cells, viable MCF-7s were identified as those cells scoring low for both 
the apoptotic membrane stain Annexin-V (Caltag, Annexin VV01-3) and the 
DNA stain PI. Light scatter gating was also used to discriminate cells 
from cell debris that was smaller than the smallest of the MCF-7 cells. 
Both PI and Annexin-V viability stains were used for MCF-7 analysis based 
on the following considerations. Propidium iodide is a DNA stain. Thus, 
for cells with disrupted membranes, yet still containing a nucleus, this 
dye will stain non-viable cells. Annexin-V, however, stains 
phosphatidylserine (PS) which migrates from the inside to the outside of 
the plasma membrane during normal apoptosis. Thus, it is normally used as 
an indicator of apoptotic, but still viable cells. Whether or not the PS 
migrates to the external surface of the cell as a result of PEF treatment 
is unimportant. What is important is that should PEFs result in the 
discharge of the nucleus from the cell, Annexin-V will stain the PS on the 
inside of the plasma membrane, since this stain has access to the inside 
of the cell due to membrane disruption. Thus, ghost cells should have a 
bright Annexin-V fluorescence signature. Therefore, gating based on both 
PI and Annexin-V has been found by the inventors to give the best 
screening for viable MCF-7 cells. 
Test and Control Specimens 
On a given test day, both control specimens and PEF treated specimens were 
prepared. Two types of controls were prepared: 1) a stock cell control 
specimen and 2) a PEF test cell control specimen. Stock cell suspension 
refers to the cellular suspension in pulsing medium from which fixed 
volume aliquots were taken for loading into the PEF treatment cells. The 
stock cell suspension controls were prepared by placing the same volume of 
stock cell suspension into a 15 ml centrifuge tube as would be loaded into 
the PEF treatment volume. An additional 5 ml of IMDM was then added to the 
same tube. The PEF treatment cell controls were prepared by loading a PEF 
treatment cell with the appropriate volume of cell suspension and allowing 
it to stand at room temperature for the period of time it would normally 
take to treat a specimen with PEFs; however, during this time, no PEFs 
were applied to the PEF treatment cell control specimens. When the 
standing period expired, the specimen was removed from the PEF treatment 
cell and placed in a 15 ml centrifuge tube to which 5 ml of IMDM was 
added. The PEF treated specimens were handled in the same way as the PEF 
treatment cell controls, except PEFs were applied to the treated 
specimens. 
The control specimens served two purposes. First, a comparison of the total 
viable cell counts, as provided by flow cytometry, between the stock cell 
controls and treatment cell controls gave an indication of the fraction of 
cells lost simply by virtue of residence in the PEF treatment cells. 
Second, the cytometry counts from the treatment cell controls for each 
cell type were used to normalize the cytometry counts for each cell type 
for the PEF treated specimens. This allowed for computation of the 
surviving percent for each cell type for each set of PEF conditions. To 
obtain consistent and meaningful cytometry counts, the following general 
procedure was followed. By virtue of the fact that the PEF treatment cells 
employed had a fixed volume, and that the stock cell suspension controls 
were prepared using a stock cell specimen having the same volume as the 
PEF treatment cells, all of the specimens prepared on a given test day had 
the same number of input cells. When preparing the control and PEF treated 
specimens for flow cytometry analysis, all cellular material present prior 
to PEF treatment was ultimately contained in the aliquot the cytometer 
drew from when performing the flow cytometry assays. Thus, if the volume 
of each cytometry aliquot was the same for all specimens on a given test 
day, then acquiring counts for a fixed acquisition time for all specimens 
would provide viable counts that could be compared across the specimens 
prepared on a given test day. Most importantly, the viable counts for the 
treatment cell controls could then be used to normalize the viable counts 
for each PEF treated specimen, thereby providing the information needed to 
compute surviving percent for each PEF-exposed specimen. 
Flow Cytometry 
The following procedures were used during preparation of the control and 
PEF-treated specimens for flow cytometry analysis. 100 .mu.l of DNase 
I(100 mg; 500 Kunitz-units per mg solid, Sigma, DN-25) was added to each 
specimen to minimize coagulation by released DNA. The specimens were then 
vortexed and centrifuged for 6 minutes at 1500 RPM. The supernatant was 
aspirated and the pellet was washed with 5 ml PBS. When preparing for PI 
and Annexin-V staining, the specimens were centrifuged again for 6 minutes 
at 1500 RPM; the supernatant was aspirated, and 100 .mu.l of 1.times. 
calcium buffer was added (Bender MedSystems, BMS306BB), followed by 
addition of 150 .mu.l of propidium iodide (PE conjugate) and 5 .mu.l of 
Annexin-V (FITC conjugate). These specimens were incubated at room 
temperature for 15 minutes. Next, 300 .mu.l of 1.times. calcium buffer was 
added, the sample vortexed, and then the specimens analyzed under flow 
cytometry. PI/Annexin-V staining was the only staining used for Case 5 
where the PEF inactivation characteristics of MCF-7s (with no other 
cellular species present) is presented. PI staining, in conjunction with 
other conjugate monoclonal antibody stain, was used for Cases 1 and 2, 
where the inactivation of PBMCs is presented: Annexin-V viability staining 
was not used for these cases (Cases 1 and 2). For Case 1, the specimens 
were also stained using the conjugate monoclonal antibodies CD3 and CD11b 
in order to enumerate lymphocyte (T-cells) and monocyte/granulocyte cells, 
respectively. For Case 2, the specimens were also stained with the 
conjugate monoclonal antibodies CD3 and CD13 in order to enumerate 
lymphocyte (T-cells) and monocyte cells, respectively. For Case 3, the 
washed pellets were resuspended in 200 .mu.l PBS, which was then aliquoted 
to two 100 .mu.l specimens. The conjugate monoclonal antibody stains CD14 
and CD45 were added to one of these aliquots for enumeration of lymphocyte 
and monocyte cells, respectively. The conjugate monoclonal antibody stains 
CD34 and CD38 were added to the second aliquot for enumeration of 
primitive progenitor cells (hematopoietic stem cells). Both aliquots were 
then incubated at room temperature for 15 minutes. These aliquots were 
then washed with 2 ml PBS, centrifuged for 6 minutes at 1500 RPM; the 
supernatant was then aspirated, and 500 .mu.l of 1 .mu.g/ml TO-PRO-3 
viability stain was added to the tube. The resulting aliquots were then 
incubated for 15 minutes at room temperature, vortexed, and analyzed under 
flow cytometry. The staining for Case 4 was very similar to Case 3, except 
the specimens were not stained with the CD34/CD38 conjugate monoclonal 
antibodies. Rather, these specimens were stained with the conjugate 
monoclonal antibodies CD3 and CD19 (in addition to CD14 and CD45) in order 
to enumerate T- and B-cells, respectively. 
When analyzing Cases 1, 2, 4, and 5 under flow cytometry, approximately 
100,000 cytometer events were found to give adequate resolution of the 
cell types of interest (lymphocytes, monocytes, tumor cells). Due to their 
rare frequency, however, 500,000-1,000,000 cytometer counts were required 
to identify the CD34+/CD38- stem cell population for Case 3. 
PEF Apparatus 
The apparatus used for the examples that follow was a batch PEF treatment 
system, i.e., PEF treatment takes place in a fixed, static PEF treatment 
volume. The batch PEF system is comprised of a batch PEF treatment cell, 
an electrode enclosure assembly that holds the PEF treatment volume during 
exposure to PEFs, an electric field pulse generator that applies voltage 
pulses to the PEF treatment cell, and a computer control/data acquisition 
system. 
The essential details of the PEF treatment cell used for the examples is 
illustrated in FIG. 6. The treatment volume is formed by stacking the end 
blocks 131, 132, graphite disk electrodes 133, 134, and Pyrex annular 
spacer 135 as shown in FIG. 6. This treatment cell has a sealless design, 
i.e., no O-ring or gasket seals are employed to seal the PEF treatment 
volume 140, defined by the mating of the Pyrex annular spacer and graphite 
disks. Rather, all mating surfaces were lapped to a waviness of no more 
than 2.5 .mu.m over the diameter (8.26 cm) of the disks involved the 
assembly. Furthermore, all mating surfaces were lapped to a number 4 
surface finish, which had an RMS variation in surface deviations of +/-0.1 
.mu.m. The graphite disks were made of ISO-63 graphite, which had an 
average pore size of 1 .mu.m. The tolerances were chosen to prevent blood 
cells, which are typically greater than 5 .mu.m, from finding their way 
into the interface regions of the mating treatment cell components, or 
penetrating into the pores of the graphite disk electrodes. Nylon screws 
were employed to hold the treatment cell assembly together. Since all 
internal surfaces of the treatment cell were either normal to the electric 
field direction (e.g. conducting surfaces) or were parallel to the 
electric field direction (e.g. insulating surfaces), the utilized 
treatment cell geometry provided very uniform electric field strength over 
the entire treatment volume. The end blocks of the treatment cell were 
made of gold coated copper. The gold coating was included to minimize 
surface oxidation and to provide excellent electrical contact with the 
assembly that holds the treatment cells during PEF treatment. The pulsing 
suspension was loaded and unloaded from the PEF treatment cell through the 
radial channel 139 in the Pyrex annular spacer by using a pipetting 
syringe (pipetting needles, Fisher, 14-825-16N); 10 ml disposable 
syringes, Fisher, 14-823-2A). 
The treatment cell just described was used for Cases 1, 2, 3, and 4. It is 
referred to hereafter as the Type A test cell. For Case 5, a variant of 
the Type A test cell was used. This variant is referred to as the Type B 
test cell (not shown) and was similar to the Type A test cell previously 
described with the following differences. The type B test cell used 
tungsten electrodes, rather than graphite and the annular spacer was made 
of plexiglass that had four radial holes (1 mm I.D.), equally spaced 
around its perimeter, that penetrated from the outer surface through to 
the inner surface. A silicon washer, which had an outside diameter equal 
to the inside diameter of the plexiglass spacer and which was 
approximately 0.05 mm thicker than the plexiglass annular spacer, was 
inserted inside the plexiglass spacer during assembly of the Type B test 
cell, which silicon washer ultimately formed the circumferential bounding 
wall of the PEF treatment volume. Once the Type B test cells were 
assembled, they were loaded and unloaded with cell suspension through the 
1 mm I.d. holes using A 21G hypodermic needles mated to 1 ml syringes (21G 
needles, Becton Dickinson, 305176; 1 ml syringes, Becton Dickinson, 5602). 
The internal dimensions of the test cells that were critical for the heat 
transfer and electric field strength calculations, were as follows. For 
the Type A test cells, the diameter of the cylindrical treatment volume 
was 4.45 cm, and the gap between graphite electrodes, when the test cell 
is assembled, was 3.2 mm, which yields a volume of 4.94 ml. For the Type B 
test cells, the diameter of the cylindrical treatment volume was 1.91 cm, 
and the gap between graphite electrode was 2.5 mm, which yields a volume 
of 0.72 ml. The parts making up the test cells were fabricated using 
standard methods known in the art. 
In general, electrochemical reactions take place in the region near the 
electrode surfaces when a current is passed through the electrodes into an 
electrolyte solution. For the present application, were thousands of 
electric field pulses may be applied, gold is not a good electrode 
candidate. This is because free chlorine ions can react with the gold, 
forming a gold salt, which dissolves in the electrolyte. Tungsten is a 
reasonably inert electrode material, but it can react with free oxygen 
ions to form an oxide layer on the electrodes. Carbon electrodes 
(graphite) are the preferred electrode material since graphite is 
substantially inert with respect to the ionic species formed during PEF 
treatment. 
To demonstrate to the importance of controlling electrochemical reactions, 
separate experiments were performed where electric field pulses were 
applied to isotonic potassium chloride solutions. Plumes of bubbles were 
observed to be released from both the anode and cathode electrodes of the 
Type A test cell. Furthermore, after a thousand pulses, the pH of the 
solution shifted from 7.0 to 6.0. However, when a DC counter-current was 
driven through the test cell during pulsing, which had a current equal to 
and opposite of the time average of the primary electric field pulses, no 
bubble formation was detected and the swing in pH was undetectable. These 
experiments illustrated the ability of using a reverse polarity current to 
control electrochemical reactions in unbuffered aqueous solutions having 
high (i.e. physiological) ionic strengths. Reverse polarity currents were 
not employed in the example cases to follow since the pulsing media used 
for those cases was typically of lower ionic strength and included a 
buffer to counter the effects of low concentrations of electrochemically 
produced products. 
Once the test cells were loaded with the desired cellular suspension, the 
test cells were then mounted in an electrode/enclosure assembly that 
supports the test cell when the PEFs are applied to the test cell. The 
electrode/enclosure assembly used for the example cases following has 
essentially the same features as the assembly shown in FIG. 5. This 
assembly serves three purposes. First, it provides electrical contact 
between the electric pulse generator and the PEF treatment cell. Second, 
it provides temperature control for the test cell. Third, it encloses the 
test cell during PEF treatment, thereby protecting personnel in close 
proximity to the system from high voltage components. With reference to 
FIG. 5, the temperature of the test cells were controlled by contact with 
temperature-controlled cathode 114 and anode 111 supports of the 
electrode/enclosure assembly 95. The temperature of the cathode and anode 
supports is controlled by circulating temperature controlled water through 
these structures using a heater/chiller system (Neslab, RTE-110). This 
system is capable of setting the temperature of the test cells to 
+/-0.2.degree. C. over the range 0-50.degree. C. After mounting the test 
cells in the electrode/enclosure assembly, the test cells were allowed to 
reach thermal equilibrium with the temperature of the cathode and anode 
structures in the electrode/enclosure assembly. 
The electric pulse generator system used for the examples consisted of an 
energy storage capacitor (3 kV, 500 .mu.f, Maxwell, 38683), which was 
connected to an all-solid-state pulse driver designed and fabricated by 
the inventors for driving a CO.sub.2 laser. The pulse driver (electric 
pulse generator) is referred to as the COLD-I. This driver switches the 
energy stored in the 500 .mu.f capacitor via solid-state SCR switches to a 
20:1 saturable core transformer into an impedance matched load (.about.8 
.OMEGA.). This driver, as presently configured, can deliver fixed duration 
voltage pulses (.about.5 .mu.s duration) at voltages up to 8 kV. Under 
these conditions, the deliverable pulse energy is .about.80 J. The storage 
capacitor was charged via a Maxwell (1 kV, 5 kJ/s, CCDS 501P372-208) high 
voltage charging power supply. Normally, the voltage output of the COLD-I 
is to high for use for PEF cell selection. This was remedied by using a 
high power voltage divider circuit, which simply comprised two resistors 
in series connected to ground. This voltage divider circuit could 
attenuate the voltage pulses delivered to the PEF test cell by factors of 
4-20 simply by changing the resistance of the two resisters in the 
circuit. 
A Pearson coil current transducer was used to monitor the current delivered 
to the PEF test cell. A second Pearson coil transducer measured the 
current through a precision 100 .OMEGA. resister connected across the PEF 
test cell. The voltage applied to the test cell was derived from the 
second Pearson coil transducer signal by multiplying it by 100 .OMEGA.. 
These current and voltage signals were displayed on a LeCroy oscilloscope 
(9410). FIG. 10 presents the voltage waveform produced by the COLD-I 
driver when applied to a Type A test cell containing a room temperature 
isotonic potassium chloride solution, which corresponds to imposing a 2.2 
kV/cm electric field in this saline solution. 
An IBM personal computer clone was used for system control and data 
acquisition. During PEF treatment, a control program was run that issued 
pulse driver trigger signals. The frequency and number of the trigger 
signals was set by the operator based on the PEF conditions desired. The 
magnitude of the voltage pulses delivered to the PEF test cell, however, 
was set by a dial associated with the Maxwell charging power supply. The 
output trigger signal from the PC control computer was delivered to an 
opto-isolator circuit, which prevented any electric noise from feeding 
back to the PC. The output of the opto-isolator circuit was delivered to 
an HP 214-B trigger generator, which, in turn, sent a 100 volt, 2 .mu. 
pulse to the COLD-I driver, which triggered the COLD-I, thereby applying 
the desired electric field pulse to the PEF test cell. 
Upon completion of a PEF treatment experimental trial, the voltage and 
current waveform data was downloaded from the oscilloscope to the PC 
computer via a GPIB interface. After the data was downloaded, 
post-processing computations were performed. More specifically, the 
following quantities were computed: peak voltages and currents, the 
resistance of the test cell, the full-width at half-maximum (FWHM) 
electric field pulse length, and the energy delivered to the test cell per 
electric field pulse. For the cases that follow, the electric field 
strengths quoted were those based on the peak voltage of the pulses 
delivered to the PEF test cell. Furthermore, the electric field pulse 
lengths quoted were the FWHM values. After computing the single pulse 
energy deposited to the test cell, the average midplane temperature rise 
in the PEF test cell and the temperature jump per electric field pulse for 
a given PEF treatment trial was computed. These temperatures, when quoted 
in the cases that follow, were derived using Eqs. 13, 13a, and 13b given 
earlier in the detailed description. 
Case 1. Step-Wise PEF Inactivation of PBMCs 
The ability of PEFs to selectively inactivate cells in a step-wise, 
size-dependent manner with increasing electric field strength and exposure 
time is demonstrated in this case. The stock cell suspension contained 
PBMCs suspended in IMDM at a concentration of 1.1.times.10.sup.6 cells/ml. 
The pulsing medium was of standard physiological ionic strength. The input 
cells and stock cell suspension were prepared as previously described. 
Type A test cells were used in the Case I trials. Pulsed electric fields, 
having strengths in the range 1.4-1.8 kV/cm, were applied to the 
specimens. The total electric field exposure times were in the range of 
0.18-5.97 ms, and the electric field pulse length was about 5.75+/-0.2 
.mu.s (FWHM). The total electric field exposure time was varied over the 
noted range by varying the number of applied electric field pulses over a 
range of 30-1000 pulses. The single pulse energy deposited to the test 
cells ranged from 0.54-0.88 J/pulse. The electric field pulses were 
applied at 1 Hz. The end blocks of the test cells were maintained at 
35.degree. C.+/-0.2.degree. C. Based on Eqs. 13, 13a, and 13b, the average 
midplane temperature varied over the range from about 35.2-35.4.degree. C. 
and the temperature jump per electric field pulse varied over the range 
0.03-0.04.degree. C. One stock cell control specimen and one test cell 
control specimen were prepared before commencing PEF treatments, and one 
test cell control specimen was prepared after all PEF treatments had been 
performed for the test day in question. The control and PEF treated 
specimens (about 5 ml each) were placed in 15 ml centrifuge tubes after 
preparation, to which an approximately equal volume of IMDM was added as 
previously described. These specimens were then analyzed by flow cytometry 
for enumeration of viable cell types and numbers as also previously 
described. 
FIG. 11 presents total surviving percent of all viable cells, on the 
y-axis, as a function of total electric field exposure time, for three 
different electric field strengths. These results indicate that PEF 
lethality, expressed as surviving percent, increased with increasing 
electric field strength and total electric field exposure time as 
predicted by Eq. 8. Further, FIG. 11 indicates that the onset of 
significant cell inactivation occurred at approximately 1.8 kV/cm. This 
measured threshold strength is in reasonable agreement with the threshold 
strength for the resting lymphocytes given in Table 2, which would be 
expected since the most abundant population of cells in the PBMC specimens 
were resting lymphocytes. 
FIGS. 12a-12d show viability scatter plots that were derived from flow 
cytometry assays of the specimens for this case. The results given in 
these figures were generated for specimens exposed to a fixed electric 
field strength of 1.8 kV/cm. FIG. 12a shows the scatter plot for the test 
cell control specimen, whereas FIGS. 12b, 12c, and 12d show the scatter 
plots for 30, 100, and 300 applied electric field pulses respectively. The 
y-axes in these figures show relative propidium iodide (PI) intensity, and 
the x-axes show forward scattered (FSC-H) light angles, which are 
proportional to cell size. High PI values are indicative of non-viable 
cells. High FSC-H values are large cells. Regions represented by R1 and R2 
in these figures correspond to viable cells. In FIG. 12a, the viable cells 
in R1 and R2 form a pattern that looks like a pan with a handle. It can be 
seen that as one scans from FIG. 12a to 12d, the handle (i.e. viable cells 
in R2) disappears. These figures also show that the fraction of cells that 
scored high for PI increased with increasing total electric field exposure 
time (total number of applied pulses). Thus, for a field strength of 1.8 
kV/cm, FIGS. 12a-12d indicate that the applied PEFs were selectively 
killing the larger cells. 
FIGS. 13a-13c present CD1 lb/CD3 flow cytometry scatter plots for the 
corresponding specimens shown in FIGS. 12a-12d. FIG. 13a shows results for 
the control specimen. Relative intensity of CD11b staining is shown on the 
y-axes, and relative intensity of CD3 staining is shown on the x-axes. 
FIGS. 13b, 13c, and 13d correspond to PEF treated specimens that received 
30, 100, and 300 electric field pulses of 1.8 kV/cm strength respectively. 
Cells scoring high for CD11b are predominately monocytes. Those scoring 
high for CD3 are predominately T-cells. Based on Table 2, the T-cells (a 
subset of lymphocytes) are typically about 7 .mu.m in size, whereas the 
monocytes are typically about 15 .mu.m in size. The upper left quadrant of 
FIG. 13a displays a distinct monocyte population that diminishes 
significantly as one scans from FIG. 13a to FIG. 13b to FIG. 13c and 
finally to FIG. 13d. In contrast, however, the T-cell population (lower 
right quadrant) has not been reduced as significantly as the monocyte 
population. Thus, the 1.8 kV/cm PEFs have selectively depleted the 
monocyte cells while preserving a significant fraction of the T-cells. 
Case 2. PEF Enrichment of Lymphocytes Over Monocytes 
The ability of PEFs to enrich a PBMC specimen in lymphocytes by selective 
inactivation of the larger monocytes was demonstrated in this case. The 
stock cell suspension contained PBMCs suspended in IMDM at a concentration 
of 1.5.times.10.sup.6 cells/ml. The pulsing medium was of standard 
physiological ionic strength. The input cells and stock cell suspension 
were prepared as previously described. Type A test cells were used for the 
Case 2 trials. Pulsed electric fields, having strengths in the range 
1.2-1.6 kV/cm, were applied to the specimens. The total electric field 
exposure times were in the range 0.15-5.70 ms, and the electric field 
pulse length was about 5.17+/-0.3 .mu.s (FWHM). The total electric field 
exposure time was varied over the noted range by varying the number of 
applied electric field pulses over a range of 30-1000 pulses. The single 
pulse energy deposited to the test cells ranged from 0.36-0.77 J/pulse. 
The electric field pulses were applied at 1 Hz. The end blocks of the test 
cells were maintained at 35.degree. C.+/-0.2.degree. C. Based on Eqs. 13, 
13a, and 13b, the average midplane temperature varied over the range 
35.1-35.3.degree. C. and the temperature jump per electric field pulse 
varied over the range 0.02-0.04.degree. C. One stock cell control specimen 
and one test cell control specimen were prepared before commencing PEF 
treatments, and one test cell control specimen was prepared after all PEF 
treatments had been performed for the test day in question. The control 
and PEF treated specimens (about 5 ml EACH) were placed in 15 ml 
centrifuge tubes after preparation, to which an approximately equal volume 
of IMDM was added as previously described. These specimens were then 
analyzed by flow cytometry for enumeration of viable cell types and 
numbers as also previously described. 
The data in FIG. 14 is presented in the same format as shown previously in 
FIG. 11. The results indicate that PEF lethality, inversely proportional 
to surviving percent, increased with increasing electric field strength 
and total electric field exposure time as predicted by Eq. 8. The results 
presented in this figure exhibit the same trends as the data presented 
FIG. 11 for Case 1. 
Using light scatter, viability stain gating, and CD3 and CD13 antibody 
fluorescence, viable lymphocyte and monocyte cells populations were 
enumerated independently. FIGS. 15a-15j present the results in forward 
scatter histogram format with relative numbers of cells on the y-axes and 
size on the x-axes. FIGS. 15a and 15f show the monocyte (FIG. 15a) and 
lymphocyte (FIG. 15f) histograms for the control specimens. Similarly, 
FIGS. 15b and 15g, 15c and 15h, 15d and 15i, and 15e and 15j show the 
monocyte and lymphocyte histograms, respectively within each pair of 
FIGS., for an electric field strength of 1.4 kV/cm and total electric 
field exposure times of 0.15 ms (30 pulses, FIGS. 15b and 15g), 0.33 ms 
(65 pulses, FIGS. 15e and 15h), 0.70 ms (140 pulses, FIGS. 15d and 15i), 
1.50 ms (300 pulses, FIGS. 15e and 15i). The size difference between the 
monocytes and lymphocytes can be inferred by considering the location of 
the corresponding distributions on the forward scatter axis: the monocytes 
are farther to the right (i.e. larger) relative to the lymphocytes 
(compare FIGS. 15a and 15f for example). Progressively comparing 
corresponding pairs of FIGS. for related PEF treatment conditions and 
increasing electric field exposure times (i.e. comparing FIGS. 15a and 
15f, then FIGS. 15b and FIG. 15g, etc.), indicates that as total electric 
field exposure time increased, there was a large reduction in the number 
of monocytes, with only a minor reduction in the number of lymphocytes. 
(Note that the scale for the monocyte histograms changes from FIG. 15b to 
FIG. 15c, and again from FIG. 15d to 15e). FIGS. 15a-15j clearly 
demonstrate the size selective inactivation characteristics of PEFs for 
this cellular system. It is also noteworthy that the steepness of the tail 
on the right hand side of the lymphocyte distributions increases with 
increasing electric field exposure time, an indication that the larger 
lymphocytes were inactivated before the smaller ones. 
The results for this case have been replotted in FIG. 16 as lymphocyte 
enrichment (y-axis) as a function of total electric field exposure time 
(x-axis) for three different electric field strengths. Lymphocyte 
enrichment was defined as the ratio of lymphocytes to monocytes for the 
control specimen divided by this ratio for the PEF treated specimens. This 
figure clearly shows that lymphocyte enrichment increased with both 
electric field strength and total electric field exposure time. At a field 
strength of 1.6 kV/cm and a total electric field exposure time of .about.5 
ms, essentially no monocytes were detected by flow cytometry thereby 
yielding an essentially infinite value for lymphocyte enrichment. 
Case 3. PEF Enrichment of Stem Cells in PBPC Preparations 
The ability of PEFs to enrich PBPC specimens for hematopoietic stem cells 
by selective inactivation of the larger cells present in heterogeneous 
PBPC mixtures was demonstrated in this case. The stock cell suspension 
contained PBPCs suspended in a low ionic strength pulsing medium (10%v/v 
PBS, 90%v/v isotonic sucrose solution) at a concentration of 
6.6.times.10.sup.6 cells/ml. The PBPCs were harvested from patients, by 
leukopheresis, that had received G-CSF treatments as previously discussed. 
Prior to PEF treatment, the resting lymphocytes in the PBPC preparation 
were activated, as previously discussed, thereby stimulating these cells 
to their active state which nearly doubled their size. Type A test cells 
were used for the Case 3 trials. Pulsed electric fields, having strengths 
of 1.7, 1.8, and 1.9 kV/cm, were applied to the specimens. The total 
electric field exposure time was 5.30 ms (1000 applied pulses), and the 
electric field pulse length was 5.30 .mu.s (FWHM). The single pulse energy 
deposited in the test cells ranged from 0.11-0.15 J/pulse. The electric 
field pulses were applied at 1 Hz. The end blocks of the test cells were 
maintained at about 35.degree. C.+/-0.2.degree. C. Based on Eqs. 13, 13a, 
and 13b, the average midplane temperature varied from about 
35.0-35.1.degree. C., and the temperature jump per electric field pulse 
varied from about 0.005-0.007.degree. C. One stock cell control specimen 
and one test cell control specimen were prepared before commencing PEF 
treatments, and one test cell control specimen was prepared after all PEF 
treatments had been performed The controls and PEF treated specimens (each 
about 5 ml) were placed in 15 ml centrifuge tubes after preparation, to 
which an equal volume of IMDM was added as previously described. The 
specimens were then analyzed by flow cytometry for cell identification and 
enumeration as previously described. 
Using viability (TO-PRO-3) and CD34, and CD38 antibody staining, viable 
primitive progenitor cells were enumerated in the control and PEF treated 
specimens. Hematopoietic stem cells were identified as those viable cells 
that scored low for the viability stain, high for the CD34 fluorescence 
marker, and low for the CD38 marker (i.e., CD34+/CD38- or CD34 single 
positive cells). The results for this case are presented in FIG. 17 in bar 
chart format. The shaded bars in FIG. 17 correspond to the surviving 
percent of all cells in the suspension. The unshaded bars correspond to 
stem cell enrichment, which was defined as the ratio of viable stem cells 
to total viable cells in the PEF treated specimens normalized by the same 
ratio for the control specimens. FIG. 17 shows that the total surviving 
percent of cells decreased with increasing electric field strength. 
However, stem cell enrichment increased with increasing electric field 
strength. In fact, at 1.9 kV/cm, enrichment approached 1 log. 
Significantly, the enrichment increased inversely proportional to the 
decrease in total surviving percent. This indicates that the stem cell 
population was being preserved and demonstrates the ability of PEF's to 
enrich PBPC specimens for stem cells. 
It should be re-emphasized that these results were obtained under 
conditions that were not optimized for stem cell enrichment. More 
specifically, the electric field waveform shape used for this case (see 
FIG. 10) was Gaussian in shape, rather than rectangular, and the length of 
the pulse (.about.5 .mu.s) was very short. As discussed previously, the 
electric field waveform shape is preferably rectangular (with very short 
rise/fall times and with an essentially constant field strength between 
rise and fall ) for obtaining more optimal size selectivity. Also, as 
discussed previously, short electric field pulses, such as the 5 .mu.s 
pulses used in the present case are generally less effective for cell 
inactivation than pulse durations greater than about 10 .mu.s. 
Case 4. PEF Purging of CMK Tumor Cells in PBMCs 
This case demonstrates the ability of PEFs to purge PBMC suspensions of 
tumor cells. The stock cell suspension included PBMCs and CMK tumor cells 
suspended in a low ionic strength pulsing medium (10% v/v PBS, 90% v/v 
isotonic sucrose solution) at 2.5.times.10.sup.6 cells/ml. The CMK tumor 
cells represented about 14% of the total number of cells. The CMKs are a 
megakaryocyte line whose size approximates epithelial tumor cell types. 
The PBMCs and CMKs were prepared as previously described. Type A test 
cells were used for the Case 4 trials. Pulsed electric fields, having 
strengths in the range 1.2-1.8 kV/cm, were applied to the specimens. The 
total electric field exposure time was 3.6 ms (1000 applied pulses), and 
the electric field pulse length was 3.6 .mu.s (FWHM). The single pulse 
energy deposited to the test cells ranged from 0.04-0.10 J/pulse, and the 
electric field pulses were applied at 1 Hz. The end blocks of the test 
cells were maintained at 35.degree. C.+/-0.2.degree. C. Based on Eqs. 13, 
13a, and 13b, the average midplane temperature varied from about 
35.02-35.04.degree. C. and the temperature jump per electric field pulse 
varied from about 0.002-0.005.degree. C. One stock cell control specimen 
and one test control specimen were prepared before commencing PEF 
treatments, and one test cell control specimen was prepared after all PEF 
treatments had been performed. The controls and PEF treated specimens 
(each about 5 ml) were placed in 15 ml centrifuge tubes after preparation, 
to which an approximately equal volume of IMDM was added as previously 
described. The specimens were then analyzed by flow cytometry for cell 
identification and enumeration as previously described. FIGS. 18a-18f show 
the flow cytometry data from the analysis of the input cells for this 
case. FIGS. 18a, 18c, and 18e are the light scatter plots (no viability 
stain gating) for the CMK cells alone, the PBMCs alone, and the CMK/PBMC 
mixture, respectively. FIGS. 18b, 18d, and 18f are viability stain 
histograms for the CMKs alone, the PBMCs alone, and the CMK/PBMC mixture 
respectively. Cells staining for the viability stain (TO-PRO-3) with an 
intensity greater than 102 were considered dead. FIG. 18b indicates that 
the CMK cells had a component (i.e. that population above 102 on the 
x-axis) that was either dead or represented cell debris. These dead cells 
or debris showed up in the light scatter plot (FIG. 18a) as a band of dots 
that extend up and to the right from the origin. The viable CMK population 
appeared as a cluster of dots that is centered vertically in FIG. 18a and 
slightly to the right of center horizontally. FIG. 18c indicates that were 
three clusters of dots for the PBMCs. The cluster near the origin 
represents fine cell debris. The next cluster to the right represents the 
lymphocyte population, and the cluster furthest to the right represents 
the monocyte population. The lymphocyte, monocyte, and CMK clusters are 
evident in FIG. 18e. 
FIGS. 19a-19f qualitatively illustrate the effect of applying 1000 electric 
field pulses, each with a strength of 1.8 kV/cm, to the CMK/PBMC mixture. 
FIGS. 19a, 19b, and 19c are a light scatter plot, CD14/CD45 bivariate 
plot, and TO-PRO-3 viability histogram, respectively for the control 
specimen. FIGS. 19d, 19e, and 19f present the same corresponding 
information as FIGS. 19a, 19b, and 19c respectively, for the specimen 
treated with 1000 electric field pulses, each having a strength of 1.8 
kV/cm. As in FIG. 18e, the lymphocyte, monocyte, and CMK populations are 
evident in FIG. 19a. Circled region R1 (in FIGS. 19a and 19d) enclose the 
CMK population. Bracketed region R3 (in FIG. 19c and 19f) defines viable 
cells as judged by TO-PRO-3 viability staining intensity. Scatter plots 
shown in FIGS. 19b (control) and 19e (PEF treated specimen) were gated on 
both R1 and R3, so the cells appearing in these two figures represent 
viable cells in the R1 light scatter compartment, which is dominated by 
the CMK tumor cells. The lower right quadrant of FIG. 19b shows a well 
defined CMK population. The upper right quadrant, however, shows that the 
R1 region also contained a small number of monocytes. FIG. 19e shows that 
both the CMKs and monocytes were eliminated by application of 1000 
electric field pulses of 1.8 kV/cm strength. However, the light scatter 
plot (FIG. 19d) for the PEF treated specimen indicates that the specimen 
still contained healthy lymphocyte and monocyte populations. The change in 
the TO-PRO-3 viability histograms (FIG. 19c and FIG. 19f) also indicates 
that only the CMK population had been affected by PEF treatment (the 
central peak in FIG. 19c, which is missing in FIG. 19f corresponds to the 
CMK population, which was derived by considering FIG. 18b). 
FIGS. 20a-20f examine the effect of PEFs on the lymphocyte population. 
FIGS. 20a, 20b, and 20c are light scatter, CD3/CD19 bivariate plots, and 
TO-PRO-3 viability histograms, respectively, for the control specimen. 
FIGS. 20d, 20e, and 20f are the light scatter, CD3/CD19 bivariate plots, 
and TO-PRO-3 viability histograms, respectively, for the specimen that was 
treated with 1000 electric field pulses, each having a strength of 1.8 
kV/cm. In FIG. 20a and FIG. 20d, regions labeled R4, R6, and R7 correspond 
to the light scatter compartments that enclose the lymphocytes, monocytes, 
and CMKs, respectively. The TO-PRO-3 viability histograms in FIG. 20c and 
FIG. 20f were gated on R4, which means that only events in the lymphocyte 
compartments in the light scatter plots are displayed in FIG. 20c and FIG. 
20f. FIGS. 20b and 20e are gated on R8 (the TO-PRO-3 viability ranges in 
either FIG. 20c or FIG. 20d) and R4 (the lymphocyte compartment in either 
FIG. 20a and FIG. 20d). Thus, FIG. 20b and FIG. 20e display only the 
viable cells from the lymphocyte light scatter compartments. The conjugate 
monoclonal antibody fluorescence marker CD3 stains T-cells, a subset of 
lymphocytes. The fluorescence marker CD19 stains B-cells. Thus, the upper 
left quadrants in FIG. 20b and FIG. 20e contain viable T-cells, whereas 
the lower right quadrants contain viable B-cells. Comparison of FIG. 20b 
and FIG. 20e indicates there was little difference in the abundance of 
viable T- and B-cells for the control and PEF treated specimens. However, 
FIGS. 19b and 19e clearly show that the CMK cells had been almost entirely 
eliminated under the same PEF conditions. 
FIG. 21 presents the full set of data collected for this case. In this 
figure, the surviving percents of the relevant cell types (y-axis) are 
presented as a function of electric field strength (x-axis). Recall that 
the total electric field exposure time was constant for each field 
strength and was produced by applying 1000 electric field pulses. The 
total electric field exposure time was 3.6 ms. This figure clearly shows 
that an almost 2-log reduction in CMKs was achieved at 1.8 kV/cm without 
impacting the viability of the lymphocytes. Also, it is evident that the 
monocytes were just beginning to be affected at 1.8 kV/cm, since the 
monocyte surviving percent curve is beginning to decrease at this field 
strength. Comparing the PBMC total surviving percent curves presented in 
FIGS. 11 and 14 with the lymphocyte surviving percent curve in FIG. 21, 
shows the lethal effects of PEFs occurs at lower electric field strengths 
in FIGS. 11 and 14. As already indicated, the results presented in these 
examples have been acquired under non-optimized conditions. The 
differences in PEF efficacy found by comparing FIGS. 11 and 14 with FIG. 
21 may be due to pulse length and pulsing medium ionic strength 
differences. The pulse length for the results in FIG. 21 was approximately 
30% shorter than for the results in FIGS. 11 and 14. Further, the results 
in FIG. 21 were obtained using a low ionic strength pulsing medium, rather 
than a standard physiological ionic strength medium as used for the 
results presented in FIGS. 11 and 14. 
As noted, the pulsing medium used for this case was a low ionic strength 
pulsing buffer (10% v/v PBS, 90% v/v isotonic sucrose solution). This low 
ionic strength pulsing medium was used for two reasons. First, as 
previously discussed, application of PEFs to cells in a low ionic strength 
pulsing medium, followed by resuspension in a standard physiological 
strength buffer, can lead to more extensive fragmentation of the of the 
PEF porated cells. It was observed during PEF cell selection experiments 
that post-PEF specimens that were treated with PEFs in a low ionic 
strength pulsing medium, followed by resuspension in a higher ionic 
strength medium, included far fewer trypan blue stained cells than when 
treated with PEFs under conditions where the pulsing buffer was of 
standard physiological ionic strength, even though the reductions in 
viable cells were comparable. This result indicates that the combination 
of a low ionic strength pulsing buffer and a higher ionic strength 
post-PEF resuspension buffer led to greater fragmentation of the PEF 
porated cells by colloidal osmotic cell lysis than for conditions where 
the pulsing buffer was of standard physiological ionic strength. Secondly, 
a lower ionic strength pulsing medium requires lower energy input to 
achieve the same electric field strengths. 
Case 5. PEF Inactivation Characteristics of Breast Tumor Cells 
The efficacy of PEF inactivation of breast tumor cells was investigated in 
this case. The stock cell suspension contained only breast tumor cells 
(MCF-7), which were suspended in a low ionic strength pulsing medium 
(10%v/v PB5, 90%v/v isotonic sucrose solution) at a total concentration of 
1.2.times.10.sup.6 cells/ml. The MCF-7s were prepared as previously 
described. Type B test cells were used for the Case 5 experiments. Pulsed 
electric fields, having field strengths in the range of 1.0-2.0 kV/cm were 
applied to the specimens. Two electric field pulse lengths were used for 
this case: 3.50 and 5.25 .mu.s (FWHM). The total electric field exposure 
time was 3.5 ms (1000 applied pulses) for the shortest electric field 
pulse length, whereas the total electric field exposure time for the 
longest electric field pulse was 4.7 ms (900 pulses). The slight reduction 
in pulse number for the longer pulse length experiments was included to 
keep the total electric field exposure time for the longer pulse length 
experiments, based on the pulse length at 95% of the peak electric field 
strength, approximately the same as for the shorter pulse length 
experiments. The single pulse energy deposited to the test cells was in 
the range of 0.003-0.020 J/pulse. The electric field pulses were applied 
at 1 Hz. The end blocks of the test cells were maintained at about 
35.degree. C.+/-0.2.degree. C. Based on Eqs. 13, 13a, and 13b, the average 
midplane temperature varied over the range from 35.01-35.04.degree. C., 
and the temperature jump per electric field pulse varied over the range 
0.001-0.007.degree. C. One stock cell control specimen and test cell 
control specimen were prepared before commencing PEF treatments, and one 
test cell control specimen was prepared after all PEF treatments had been 
performed for each set of tests. The controls and PEF treated specimens 
(each about 0.72 ml) were placed in 15 ml centrifuge tubes, to which about 
5 ml of IMDM was added as previously described. These specimens were then 
analyzed by flow cytometry for enumeration of viable MCF-7s, also as 
previously described. 
For this case, viable MCF-7s were identified as those cells that fluoresced 
dimly for both propidium iodide (PI) and Annexin-V stains. FIG. 22 
presents results typical of PEF inactivation experiments using the MCF-7 
cell line. More specifically, this figure illustrates the combined effect 
of increasing electric field pulse length and exposure time, as well as 
electric field strength. The curve shown by the dashed line represents 
data obtained by applying 1000 electric field pulses, each pulse having a 
3.5 .mu.s FWHM pulse length. The curve shown by the solid line represents 
data obtained by applying 900 electric field pulses, each pulse having a 
5.3 lus FWHM pulse length. Thus, the total electric field exposure time 
for the dashed curve was 3.5 ms, whereas the total electric field exposure 
time for the solid curve was 4.7 ms. 
FIG. 22 clearly shows that increasing PEF pulse length and exposure time 
resulted in increased tumor cell purging efficacy. Further, the 5.3 
.mu.pulse length produced about a 2.3 log reduction in viable tumor cells. 
It is important to note that the 2.3 log reduction shown may significantly 
understate the efficacy of the PEF process. Optical microscopy indicated 
that the input cell population included clumps of agglomerated cells, each 
clump containing from about 3-10 cells. Thus, the number of viable MCF-7 
cells in the control specimens, which were used to normalize the data for 
viable cells contained in the PEF-treated specimens, could be low by about 
a factor of 3-10. Thus, the surviving percents reported for the 
PEF-treated specimens could be high by a factor of 3-10. In addition, 
application of PEFs to this cell line had two effects: 1) the PEFs breakup 
the clumps of aggregated cells, and 2) inactivation of the cells. The 
breakup of cells was clearly observed by the increase in total events 
recorded by the cytometer. Total events were observed to increase by at 
least a factor of two once PEFs had been applied to a specimen. It is also 
believed the inflection points in the curves of FIG. 22 could be due to an 
increase in cell numbers due to disaggregation by the PEFs, followed by 
subsequent inactivation. Significantly, some of the cells in clumps will 
be shielded from the PEFs until such clumps are completely reduced to 
monodispersed cells. Once monodispersed, they can then be inactivated by 
the PEFs. Thus the presence of clumped cells in the cell suspension before 
treatment would imply that not all of the cells in the specimens 
experienced the same effective electric field exposure time. Thus, 
increasing the electric field pulse length, may be an effective way to 
lead to more efficient PEF clump disaggregation, which, in turn, could 
allow the use of lower field strengths and possibly shorter exposure times 
to achieve inactivation of cells that tend to clump, like the MCF-7 tumor 
cells. The MCF-7 tumor cell inactivation results presented here show that 
at least a 2.3 log reduction in this breast cancer line was achieved at 
PEF conditions which led to a 1 log enrichment of stem cells, without 
significant loss of viable stem cells, in Case 3 presented above. 
Case 6: Enzymatic Removal of a Glycocalyx Membrane Layer 
A small fraction of the cells within a given epithelial cell line, such as 
the MCF-7 line, secrete a mucopolysaccharide (mucin), which can coat the 
cell plasma membrane. This mucin coat can function to increase the 
effective thickness of the membrane of these cells, which can, in turn, 
require higher electric field strengths for their inactivation. The 
inventors have experimentally determined, using standard mucin stains and 
optical microscopy, that a fraction of the MCF-7 cells have a mucin coat 
and that the coat can be removed by enzyme digestion. To achieve this, the 
MCF-7s were subjected to a hyaluronidase digestion protocol just prior to 
their suspension in the pulsing medium. The mucin digestion protocol 
involved resuspending the trypsinized cells in a digestion solution (500 
ug/ml hyaluronidase, Sigma, H4272 30 mg; 94 mM potassium phosphate 
monobasic, Sigma, P8416; 6 mM Sodium phosphate dibasic, Sigma, S-5136) and 
incubating the solution for 30 minutes at 37.degree. C. Using standard 
techniques for mucin staining under optical microscopy it was found that 
this digestion protocol essentially entirely removed the extracellular 
mucin coats. 
While the invention has been shown and described above with reference to 
various embodiments and specific examples, it is to be understood that the 
invention is not limited to the embodiments or examples described and that 
the teachings of this invention may be practiced by one skilled in the art 
in various additional ways and for various additional purposes.