Automated cell culture and testing system

A cell culture and testing system provides a completely self-contained environment in which living tissues may be placed and where living tissues may be nutrified, oxygenated and maintained within a range of temperatures within which life may be sustained. The system includes aspects permitting administering of drugs or other substances to living tissues and monitoring of results accruing from such administration. In the preferred embodiment, the system is completely self-contained and sealed and may be operated both through use of an external power supply and an internal back-up power supply. The system is maintained at a positive pressure slightly above atmospheric pressure to prevent contamination from the surrounding environment. The system includes at least three levels of containment to completely isolate living tissues from ambient surroundings and the system has been successfully tested under conditions of zero gravity.

BACKGROUND OF THE INVENTION 
Laboratory-scale culture and testing of cells and tissues derived from 
mammalian sources typically involves the use of specialized containers. 
The physical and chemical nature of the chosen vessel dictates the choice 
of handling methods and technical limitations of the experimental process. 
Static vessels such as petri dishes and tissue flasks, roller bottles 
which are rotated to provide continuous bathing of cells which grow 
attached to the walls of the vessel, or spinner flasks in which a moving 
paddle continually suspends cellular material in nutrient broth or media 
are commonly employed. In all of these approaches, the chosen vessel must 
be associated with a laminar flow hood for aseptic set-up and servicing. 
Furthermore, these vessels do not have independent means for controlling 
temperature. As such, such vessels must be placed within an incubator 
designed to regulate temperature and control atmosphere during 
maintenance. 
In a further limitation, such vessels are, by design, open systems having 
direct gaseous communication with the ambient environment. For this 
reason, accidental contamination of the contents of the vessel with 
atmospherically borne microbial elements is common. When contamination 
with such ambient elements occurs, the user must reject the contents of 
the container from the particular study at hand, thereby causing loss of 
data and time. Systems which employ vessels, such as those listed above, 
are highly labor intensive, inconvenient, expensive, unreliable for 
maintenance of sterility and may also be wasteful of laboratory space. 
While the prior art does evidence the existence of devices which, in 
limited ways, may be employed to grow and maintain living cells and 
tissues, most of these devices are incorporated into bioreactor-type 
inventions which utilize cells as living chemical fabricators to produce 
proteins, enzymes, monoclonal antibodies, hormones, drugs, pesticides and 
other substances. In such devices, the focus is on the desired end product 
and not on the cells themselves. The cells are simply the means to the end 
product meant for applications outside the system. 
Given the limitations of known systems Concerning sterility, contamination, 
reproducibility of results, size, expense and reliability, a need has 
developed for a single self-contained device able to sustain life of 
living cells and tissues, grow the tissues, facilitate performance of 
experimentation on the tissues and obtain results of such experimentation, 
free of risk of contamination, loss of sterility, and in a reproducible 
manner. It is with these thoughts in mind that the present invention was 
developed. 
U.S. Pat. No. 4,725,548 to Karrer discloses a method and fermenter for 
growing tissue cells. In the Karrer device, once the cells are grown, they 
are transferred from the device via a harvest pipe for use external to the 
Karrer device. The present invention differs from the teachings of Karrer 
as contemplating a completely self-contained device wherein cells and 
other living tissues may be grown and wherein testing and experimental 
procedures utilizing these living tissues and cells occur within the 
confines of the completely self-contained device. 
Several devices and systems are known in which cells and tissues are grown 
in gas permeable plastic bags. Those patents known to Applicant which 
employ such structure are U.S. Pat. Nos. 3,102,082 to Brewer, 3,941,662 to 
Munder et al., 4,142,940 to Modolell et al., and 4,829,002 to Pattillo et 
al. In such devices, the containers may be filled aseptically with media 
and bulk additives. System implementation is hampered by inherently 
limited cellular capacity and tedious turnover of media following 
application of hormones or drugs to the cultures. Systems such as those 
disclosed in these patents require use of an incubator to provide thermal 
and atmospheric maintenance. Passaging of cultured cells at confluence is 
done manually and the cells must be removed physically from the system. 
This transfer technique may facilitate contamination of cultured cells by 
microorganisms. Furthermore, the systems disclosed in these patents do not 
permit monitoring of the metabolic status of cells during culture. The 
present invention overcomes these deficiencies in these prior art designs. 
An alternative to the static bag culture system is the hollow fiber 
bioreactor approach described in the Knazek et al. patents, U.S. Pat. Nos. 
3,821,087, 3,883,393, 4,184,922, 4,200,689, 4,206,015 and 4,220,725. These 
systems employ membrane capillaries encased in rigid plastic housings 
sealed in such a manner that two discrete volumes are established which 
communicate via pores of molecular dimensions which traverse the 
membranes. The cells are inoculated into a static volume external to the 
capillaries. A mobile volume is contained within the membrane proper and 
the connected vessels and conduits. This volume may include a 
replenishable media reservoir and gas permeable tubing or membrane 
oxygenating devices which serve to adjust acidity in the media by allowing 
equilibration with controlled concentrations of carbon dioxide and 
facilitating uptake of oxygen. The media are circulated by means of a 
pump. While very high cell density is obtainable with the systems 
disclosed in these patents, direct observation of cells within the system 
is not feasible. In a further aspect, set-up must be accomplished under a 
laminar flow hood for aseptic set-up and servicing. Additionally, the 
systems disclosed in these patents must reside within a carbon dioxide 
incubator or equivalent cabinet. Furthermore, application of hormones or 
drugs is difficult and passaging of cells is manual, leading to potential 
for biological contamination. 
U.S. Pat. No. 4,650,766 to Harm et al. discloses a culturing apparatus 
which is compatible with hollow fiber bioreactors such as those disclosed 
in the Knazek et al. patents. Patentees Harm and Peluso describe a culture 
apparatus which provides integral gassing of media and provision of 
heating to eliminate the need for incubators. The patented apparatus is 
limited by the fact that the system is of a single pass design resulting 
in inefficient use of media. Fluid flow is very slow resulting in loss of 
water through the exchange membrane. The osmolarity of the media increases 
to the detriment of the cells or tissues under culture. Dissolved gas 
components are the limiting nutrients in the system and new media is 
constantly moved over the cells to provide these materials. Open lines run 
from the outlet of the bioreactors to a supplemental fraction collector. 
The termination of these lines is open to the environment providing ready 
access for microbial contamination of the system. Gassing and heating of 
the medium is concurrent favoring formation of gas bubbles in the flow 
path. As is well known, presence of bubbles in a flowing system is lethal 
to cells in culture. Multiple media sources are mixed and distributed via 
manifolds thereby providing the potential for a single contamination nidus 
to infect all bioreactors in the system. While parallel flow paths are 
possible in the design, to thereby provide potential for reduction in the 
impact of casual contamination, gassing is continuous resulting in waste 
of supplies. Thermal regulation is determined by external water bath 
controls allowing the potential for rapid temperature variations which may 
be lethal. The Harm et al. system provides no automated control or 
monitoring nor is any facility for the introduction of drugs or hormones 
provided. Additionally, no computer control is provided. 
U.S. Pat. No. 4,629,686 to Gruenberg discloses an apparatus for delivering 
a controlled dosage of a chemical substance to cell or tissue cultures. In 
the Gruenberg system, a series of pre-diluted media preparations are 
selectively applied to the organ or tissue of interest. A computer 
controls the selection of which concentration to apply. The system is 
single pass in design but does include a sterile dispenser for elimination 
of potential contamination via the fraction collector/emitter. Temperature 
is maintained via a circulating water bath providing potential for rapid 
temperature fluctuations which may be lethal to the tissues contained 
therein. The Gruenberg device may not be utilized for large scale testing 
of multiple drugs. The present invention differs from the teachings of 
Gruenberg as providing a multiple pass system, at least two levels of 
containment, provision for testing of multiple samples with multiple drugs 
simultaneously and as including extremely close control over nutrient 
supply, oxygenation and temperature. 
U.S. Pat. No. 4,116,778 to Belousov et al. discloses a plant for continuous 
cultivation of microorganisms. Belousov et al. fail to contemplate 
administration of drugs or other substances to living tissues nor 
monitoring of results of such administration. Furthermore, Belousov et al. 
provide no control of the temperature of the cells therein. Other 
significant differences from the present invention also exist. 
U.S. Pat. No. 4,894,342 to Guinn et al. discloses a bioreactor based 
fermenting device designed to grow cell products. Guinn et al. include a 
humidification process for gas, bubble arrestors and optical fluid level 
monitoring as well as color detectors. Temperature control is provided 
through flowing fluid leading to the potential for rapid temperature 
variations which would be lethal to living tissues. Furthermore, the Guinn 
et al. system offers no protection to the operator from accidental 
exposure to infectious or toxic materials. 
U.S. Pat. No. 4,446,229 to Indech discloses a method of tissue growth 
wherein fetuses are transplanted to a unit which circulates blood through 
an artificial vasculature lung and kidney apparatus. The unit is subjected 
to ultraviolet light from an integral source to provide sterilization. 
Such devices usually generate ozone which is toxic to the living tissues. 
However, Indech fails to disclose any provision for removal of ozone which 
is generated. A base tissue of non-immunoreactive mesentery is presented 
to the transplant to which an outgrowth of vasculature is encouraged. 
Indech claims a method for aseptic addition of materials into the system 
for testing purposes with respect to the implanted tissue or fetus. Indech 
fails to disclose methods employed to maintain a sterile barrier. The 
Indech device is only applicable concerning tissues, embryos and fetuses 
which are competent to form vasculature tissue denovo. 
U.S. Pat. No. 4,889,691 to Argentieri discloses a modular tissue 
superfusion chamber including a receptacle support having a recess for 
receiving one of a plurality of modular bath containers which hold tissue 
samples being tested for electrophysiological responses to commands. While 
the receptacle support includes a Peltier heater therein, the tissues are 
exposed to the environment and, as such, may not be isolated from 
potential contamination. 
U.S. Pat. No. 4,680,266 to Tschopp et al. discloses a cell culture chamber 
with means for automatic replenishment of nutrient. Tschopp et al. 
disclose a device which may be utilized for carrying out or implementing 
biological experiments under zero gravity conditions. While this aspect is 
in common with the teachings of the present invention, the present 
invention differs from the teachings of Tschopp et al. in many respects. 
Firstly, the Tschopp et al. device is non-mechanical in nature and relies 
on osmotically pumped fluid moving through channels drilled into the body 
of the apparatus, passing over attached cells which reside in a cavity and 
grow on removable glass windows. The spent media is conveyed into the 
general cavity of the apparatus. The unit is not self-sustaining and must 
reside in an incubator so that thermal and atmospheric conditions may be 
regulated. The device of Tschopp et al. may not be replenished or serviced 
and cannot be sampled during the course of an experiment. 
U.S. Pat. No. 3,065,148 to Ferrari, Jr. discloses a method and apparatus 
for use in conducting studies on cells. The Ferrari, Jr. device uses very 
short-term exposure of the cells to test materials and relies upon changes 
in the rate of carbon dioxide evolution by the cells as an indicator. The 
individual cells may be recovered in this test scheme and effluent may be 
fractionated to test for the release of recognized indicators of cell 
stress and toxic response. Since most cells in the mammalian body are 
anchorage-dependent, this testing scheme is of severely limited utility in 
current state of the art laboratories. The Ferrari, Jr. device may not be 
effectively used for toxicity and carcinogenicity screening. 
SUMMARY OF THE INVENTION 
The present invention relates to an automated system for culturing and 
testing of cells and tissues. The system is microgravity-adapted and 
provides a compact, full function growth and experimentation platform for 
the culture and testing of microorganisms, plant cells and cells derived 
from animal tissue sources. The system is compatible with both suspended 
cell ferments and anchorage-dependent cell production either on hollow 
fibers or microcarriers. 
The inventive system provides two aseptic containment barriers to protect 
workers from accidental escape of noxious or infectious fluids while 
affording a fully controlled thermal and gaseous environment for the 
cultures. The apparatus is attitude independent for operation in 
conditions of normal gravity and in conditions of weightlessness. 
Additionally, a prototype of the inventive device has successfully flown 
on the Space Shuttle and has successfully withstood the stress of orbital 
launch and recovery while successfully operating during the mission. 
If desired, the inventive device may be provided with as many as four 
levels of containment so that complete isolation from the ambient 
environment may be assured. The system is designed to be capable of "stand 
alone" operation for the maintenance of cell growth and production without 
monitoring, provides an intelligent interface for operator control, has an 
uninterruptable power supply for added system reliability, incorporates an 
integral fraction collection facility which eliminates the need for 
breaking of sterile barriers through sample withdrawal and features a 
precision metering system for the introduction of controlled volumes of 
media or media enriched with test materials at known concentrations. 
When the inventive device is made of a size occupying a volume of 
approximately 2 cubic feet, up to 20 culture reactors may be supported in 
self-contained fashion each containing up to 10,000,000,000 cells for a 
period of up to 28 days without servicing and replenishment. If desired, 
the system may be reconfigured to allow extended cycle life when used with 
fewer than 20 culture reactors. 
The inventive system has in-line detectors and monitoring devices allowing 
continuous assessment of the viability of metabolic state of each cell 
without the need for invasive procedures. In conjunction with these 
devices, the system includes computer control allowing adjustment of rates 
of oxygenation, nutrient feed and operation of heat control to maintain 
all life sustaining factors well within desired ranges. 
More particularly, the present invention includes the following 
interrelated objects, aspects and features: 
(A) In a first aspect of the present invention, in order to completely 
isolate all functions of the inventive device, up to four levels of 
containment may be provided. At minimum, three levels of containment are 
provided including an innermost level of containment comprising multiple 
independent fluid systems consisting of the cell containing vessels, fluid 
reservoirs, test material reservoirs, propulsion and metering devices, 
routing devices, gas exchange devices, spent media sump containers, as 
well as fraction collecting devices and receiving vessels. The innermost 
level of containment also includes a single gas environment system 
consisting of a pressure vessel containing ultra-high purity gas mix, a 
pressure reduction device or regulator, high efficiency filtering 
appliances, static routing system which interfaces with the individual 
fluid systems and individual flow control devices. Outside this innermost 
level of containment, the next level of containment comprises an outer 
chamber which receives gas released from the inner level of containment 
and is fluidly connected with the inner level of containment with a 
one-way valve serially connected with a filter unit to prevent moisture, 
infectious agents and aerosols from exiting the inner containment. A 
third, outer level of containment may be provided to completely isolate 
the two inner levels of containment from the surrounding environment. 
(B) The various components contained within the inner level of containment 
were broadly listed above. Therewithin, a fluid pathway is provided which 
provides parallel supply of media, nutrients and chemical agents to the 
cells contained therein. In one important aspect, the reservoirs of fluid 
are flexible in nature and are pressurized by surrounding elastic sleeves. 
Plural pumps are provided to allow the various functions of the fluid 
pathway to occur including a circulating peristaltic pump having rollers 
sufficiently wide enough to simultaneously pump fluid through a plurality 
of parallel arranged flexible tubes. In the fluid pathway are a plurality 
of parallel arranged bioreactors which receive cells and other living 
tissues which are to be maintained therein. Fluids which are administered 
to the bioreactors are oxygenated in a way limiting the possibility of 
toxic bubbling. The device includes integral computer control for the 
various valves and pumps in the fluid pathway allowing control of 
administration of fluids as well as removal thereof. 
(C) The innermost level of containment also includes a gas pathway 
permitting oxygenation of living tissues via the fluid pathway as will be 
described in greater detail hereinafter. A bottle of pressurized gas 
including a life sustaining percentage of oxygen is incorporated within 
the device and various pressure regulators, filters and valves are 
provided to convey the life sustaining gas within a desired range of 
pressure and humidity to the inboard oxygenators. As briefly described 
above, spent gasses from the bioreactors are expelled from the innermost 
level of containment to the next level of containment via filters and 
one-way valves. Appropriate sensors are provided within the gas pathway to 
sense dissolved oxygen and/or acidity so that, responsive to receipt of 
this data, life sustaining gas may be refreshed. 
(D) The bioreactors are mounted within recesses in an aluminum heat sink 
having a Peltier-type heating/cooling mechanism incorporated therewith. 
Embedded within the heat sink is a temperature sensor which senses the 
heat sink temperature and conveys this information to the onboard computer 
control so that operation of the Peltier heating/cooling device may be 
controlled responsive to sensing of heat sink temperature. Use of a metal 
heat sink is an advance in systems of this type because (1) it prevents 
rapid temperature changes which are potentially life threatening and (2) 
it allows accurate, consistent temperature control even under zero gravity 
conditions. The Peltier device may heat or cool depending upon the 
polarity of electrical connection. Under computer control, this polarity 
may be adjusted and the unit itself may be pulsed on and off at a desired 
frequency to provide accurate temperature control within 1/2.degree. C. 
(E) The inventive system has a built-in onboard electronic system for 
controlling all internal functions. The electronic system includes a 
master computer driving an LCD display and having a removable keypad to 
input data and instructions. The master computer controls a plurality of 
digital controllers each of which has specific controlling functions 
within the entire system. For example, one digital controller may be 
provided to control all functions of the gas pathway while another digital 
controller may be provided to control all functions of the fluid pathway. 
Still another digital controller may be employed for thermal regulation 
while another digital controller may be provided to control administration 
of drugs or other substances and retrieval of data resulting from such 
administration. 
(F) In order to render the present invention completely self-sustaining and 
independent for long periods of time, a built-in power supply is provided 
which includes main batteries, back-up batteries as well as interface 
allowing connection to external power supplies. If the external power 
supplies fail, the internal back-up and emergency systems instantaneously 
take over providing uninterrupted power supply to the various systems and 
sub-systems of the inventive device. 
As such, it is a first object of the present invention to provide a system 
for automated culturing and testing of cells and tissues. 
It is a still further object of the present invention to provide such a 
device including up to four levels of containment to completely isolate 
the inner workings of the inventive device from the surrounding ambient 
environment. 
It is a still further object of the present invention to provide such a 
device having an independently controlled fluid pathway allowing supply of 
nutrients and other media to living cells and tissues. 
It is a still further object of the present invention to provide such a 
device with an independently operable gas pathway providing life 
sustaining gasses to living cells and tissues while exhausting spent 
gasses and waste products from the bioreactors while maintaining venting 
of such spent gasses and waste products through the outer levels of 
containment of the device to the ambient environment by means of a 
positive pressure gradient from inside to out. It is a still further 
object of the present invention to provide such a device with thermal 
regulation which is operable both in the influence of gravitational fields 
and in zero gravity. 
It is a still further object of the present invention to provide such a 
device having a built-in computer control allowing monitoring of all 
system aspects and functions. 
It is a still further object of the present invention to provide such a 
device which may be operated completely independently of outside 
involvement including the provision of an independently operable power 
supply and back-up power supply. 
These and other objects, aspects and features of the present invention will 
be better understood from the following detailed description of the 
preferred embodiment when read in conjunction with the appended drawing 
figures.

SPECIFIC DESCRIPTION OF THE PREFERRED EMBODIMENT 
Reference is first made to FIGS. 6 and 7 which show front and top views, 
respectively, of the present invention, as generally designated by the 
reference numeral 100. With reference to FIGS. 6 and 7, it is seen that 
the heart of the present invention consists of a plurality of rail 
assembly means generally designated by the reference numeral 1 and which 
will be described in greater detail hereinafter with reference to FIGS. 
1-5. 
While the present invention may be provided with four levels of 
containment, for ease of understanding, only three levels of containment 
are shown. In this regard, with reference to FIGS. 6, 7 and 17, it is seen 
that the inventive device 100 includes a third level of containment 
comprising an outer casing 47 preferably made of a strong, lightweight 
metal such as, for example, aluminum and which provides the outer 
containment. A second level of containment is provided by an inner 
containment vessel 19 having a sealed chamber in which is contained the 
first level of containment consisting of liquid or fluid pathways, gas 
pathways and thermal regulation as will be described in greater detail 
hereinafter. In addition, if desired, electrical circuitry may be provided 
within the inner containment vessel 19 but in a separate sub-chamber 
sealed from the other sub-systems contained therein to avoid corrosion or 
other contamination. As shown in FIG. 17, the inner containment vessel 19 
is sealed from the outside and has an access cover 19'. While the inner 
containment vessel 19 is sealed from incursion of any gas or substance 
from the outside, relief valves 61, to be described in greater detail 
hereinafter, allow venting of gasses from within the inner containment 
vessel 19 to the space surrounding the inner containment vessel 19 and 
within the outer containment 47. As also shown in FIG. 17, the outer 
containment 47 has a front panel 47' which may be placed over the front 
opening 63 thereof and, in conjunction with the resilient peripheral seal 
65, may hermetically seal the outer containment 47. Also contained within 
the inner containment vessel 19 are a digital computer controller 17 as 
well as a motor, solenoid and heater driver board 18. As noted above, if 
desired, these electronic components may be contained within a separate 
sub-chamber within the inner containment vessel 19. 
Reference numeral 31 refers to a plurality of test material reservoirs 
which, as the name suggests, are provided with test materials which may be 
selectively pumped into the bioreactor means consisting of the bioreactors 
8 in a manner to be described in greater detail hereinafter. 
With reference back to FIGS. 6 and 7, it is seen that within the inner 
containment vessel 19, a media reservoir/sump bag combination 2 is 
provided. The reservoir/sump bag 2 is surrounded by an elastic holster 83 
which squeezes the flexible reservoir/sump bag 2 so as to provide a 
positive pressure. Life sustaining gas preferably including some 
combination of oxygen, nitrogen and carbon dioxide is supplied to the gas 
pathway from one or more pressurized bottles 22 contained within the inner 
containment vessel 19. With particular reference to FIG. 7, it is seen 
that a pressurized bottle 22 has an outlet valve 67 with an actuating 
handle 69 so that movements of the handle 69 control flow through the 
outlet nozzle 71. The outlet nozzle 71 is fluidly connected to a 
proportioning-type pressure regulator 20 which conveys regulated gas 
through the filter cartridge 35, the filter 36, the filter 21, the 
humidifier cartridge 37 and the gas enable valve 28. These components will 
be described in greater detail with reference to FIG. 14. 
With further reference to FIG. 7, it is seen that the relief valves 61 may 
take on diverse configurations in keeping with diverse characteristics. 
Thus, for example, the valves 61 may be selected to open at differing 
levels of pressure threshhold. For example, one such valve 61 may be 
designed to open at .DELTA. psi above ambient pressure while another such 
valve 61 may be designed to open at a threshhold of 1/2 psi above ambient 
pressure. The valves 61 maintain a pressure in the vessel 19 slightly 
above ambient pressure to preclude incursion of contaminants therewithin. 
These are merely examples of the threshholds which may be chosen for the 
relief valves 61. In any case, the relief valves 61 vent gasses from the 
inner containment vessel 19 to the space within the outer containment 47. 
As shown in FIG. 6, the inventive device 100 may include a power and 
display module 48 including a control panel pulse switch 25, an external 
display 26, an on-off power switch 73, a power jack 138, and other desired 
aspects as shown. 
With particular reference to FIGS. 1-5, the specific details of a single 
rail assembly means such as those depicted in FIGS. 6 and 7 will now be 
described in great detail. 
As shown in FIGS. 1-5, the rail assembly means 1 includes a plate 16 on 
which all of the mechanical components thereof are assembled. Applicant 
has found that an aluminum reinforced printed circuit plate is suitable 
for use as the plate 16 due to its lightweight, strength and corrosion 
resistance. A support member 77 extends from the plate 16 and carries a 
series of selector valves 33 and 10 which will be described in greater 
detail hereinafter. Additionally, extending from the same face of the 
plate 16 from which supports 77 emanate, a circulating peristaltic pump 5 
is mounted. Additional high precision metering pumps 4, for a purpose to 
be described in greater detail hereinafter, are also mounted adjacent the 
peristaltic pump 5. 
A parallel lumen oxygenator 6 is mounted within the block 13 and, in 
surrounding relation therearound, four large bioreactors 8 are mounted 
within recesses in the block 13. These recesses 79 are best seen with 
reference to FIG. 15. 
The block 13 comprises a solid aluminum heat conductor in which the 
bioreactors reside during operation. As shown in FIG. 1, a Peltier 
heater/cooler 14 is directly mounted on the block 13. FIG. 15 particularly 
shows the opening 81 in the plate 16 which allows the heater/cooler 14 to 
extend through the plate 16 and into direct contact with the block 13. If 
desired, instead of employing four large bioreactors 8, a larger number of 
smaller capacity bioreactors may be employed, such as, for example, 
sixteen small capacity bioreactors (not shown). The valve 7 provided to 
control gas flow to the parallel lumen oxygenator 6 is best seen with 
reference to FIGS. 1, 2 and 5. FIGS. 1 and 2 show the port selector valves 
9 which control outflow from the bioreactors 8 as well as flow routing 
valves 10, while FIG. 4 schematically shows one of the selector valves 33, 
while FIGS. 1, 2 and 5 show open-cell foam bubble arrestors 32 which are 
provided, as will be described in greater detail hereinafter, to prevent 
frothing of the media and loss of dissolved proteinaceous components. 
The above schematic description of a rail assembly means 1 will become 
better understood with the following description of the various 
sub-systems of the present invention. 
In this regard, reference is now made to FIGS. 8-13 so that the description 
of the liquid flow paths of the present invention may be described in 
great detail. 
With reference, first, to FIG. 8, a general description of the fluid 
pathway will now be described. As explained above, a single rail may 
contain as many as sixteen bioreactors 8. As should be understood from the 
following description, each bioreactor has an individual fluid pathway 
which is parallel and non-communicating with the fluid pathways supplying 
and exhausting other bioreactors 8 at least beyond the point of media 
division manifolds 29 and until the sump collection manifolds 30 are 
reached. Thus, description of a single such fluid pathway is 
representative of all of the fluid pathways supplying and exhausting each 
bioreactor. 
With reference to FIG. 8, the flexible plastic bladder 2 is surrounded by 
an elastic sleeve 83 to pressurize the contents thereof. The bladder 2 is 
gas impermeable and the elastic holster 83 additionally prevents bubble 
formation. The bladder 2 has an outlet port 85 to which is coupled a flow 
passage 87 which is coupled, through suitable coupling means (not shown), 
with a manifold 29 having a single inlet port 89 and a plurality of outlet 
ports 91 of which four are shown in FIG. 8. As explained above, downstream 
of the manifold 29, each individual bioreactor 8 is individually supplied 
without interconnection with other bioreactors 8. As shown in FIG. 8, the 
outlet port 91 corresponding to the bioreactor 8 shown therein has a flow 
passage 93 coupled thereto which supplies media from the bladder 2 to a 
first inlet port 95 of the three-way solenoid activated selector valve 
described in the claims as a "second valve means", with the port 95 being 
that which is normally open when the solenoid (not shown) is deactivated. 
In addition, as shown in FIG. 8, a plurality of individual small bladders 
31 each provided with individual pressurizing sleeves 62 corresponding to 
the sleeve 83 are provided. Each bladder 31 may supply a drug, hormone or 
chemical which is to be used to perform tests in conjunction with living 
tissue contained within the bioreactor 8. Each bladder 31 supplies the 
stored substance via a fluid conduit 64 to a manifold 68 which, as should 
be understood by those skilled in the art, includes an internal valve 
allowing only one of the bladders 31 to supply fluid to the flow passage 
66 which leads to the normally closed inlet port 97 of the valve 3. Thus, 
the valve 3 may be left in the position shown in FIG. 8 to allow supply of 
media downstream thereof or, through activation of the solenoid thereof, 
may be switched to a position allowing supply of substance from one of the 
bladders 31 therepast. 
Downstream of the valve 3, a flow passage 70 supplies fluid to the inlet 
port 72 of a high precision diaphragm pump 4. The above described aspects 
of FIG. 8 comprise a first supply means. The outlet port 74 of the pump 4 
connects via a flow passage 76 to the inlet port 80 of a T-union 78 which 
contains a check valve 82 which is biased by the spring 84 in a direction 
normally closing the port 80 in the absence of applied fluid pressure from 
the flow passage 76. The T-union 78 also includes a second inlet port 86 
which is fluidly connected to the output port 88 of a peristaltic 
circulation pump 5. As should be understood, the circulation pump 5 
includes a plurality of rollers which may be rotated by the pump motor to 
progressively compress sections of flexible tubing captured therein to 
permit pumping to take place. The rollers are made sufficiently wide 
enough to accommodate a number of flexible tubes corresponding to the 
number of bioreactors 8 included in one rail assembly means 1. The pump 5 
comprises a portion of recirculation means permitting recirculation of 
fluid exiting said bioreactors 8 back to the inlet ports 96 of the 
bioreactors 8. 
The single outlet port 90 of the T-union 78 is fluidly connected to the 
inlet port 92 of the membrane oxygenator 6. The membrane oxygenator 6, as 
should be understood, has a tortuously spiraling gas exchange device which 
receives fluid from the port 92, dilutes the fluid, equilibrates it with 
gas conveyed as will be described in greater detail hereinafter with 
reference to FIG. 14, and, also brings the equilibrated fluid to an 
appropriate life sustaining temperature. 
Applicant has found that out gassing is not a concern in the membrane 
oxygenator 6 since the cellular elements are located external to the 
capillaries and the capillaries are impermeable to bubbles. If desired, 
however, in order to be absolutely sure that bubbles which are toxic to 
sustanation of life are not formed, an open-cell foam bubble arrestor 32 
may be provided downstream of the outlet port 94 of the oxygenator 6 to 
prevent frothing of the media and loss of dissolved proteinaceous 
components. 
With further reference to FIG. 8, properly adjusted media exiting the foam 
bubble arrestor 32 is supplied at the inlet port 96 of the bioreactor 8. 
As should be understood, the material flows through the intracapillary 
space and then diverts and equilibrates with the extracapillary space of 
the bioreactor 8. The media then exits the bioreactor 8 either via the end 
port 98 which is directly connected to the intracapillary volume or via a 
side port 99 which is connected to the extracapillary volume. The route of 
flow through the bioreactor 8 is determined by a three-way port select 
valve 9 having a port 103 which is normally open when the solenoid 
actuator thereof (not shown) is deactivated and a normally closed port 105 
which is opened through activation of the solenoid. The valve 9 has a 
common outlet port 107. 
Liquid exiting the common port 107 of the valve 9 is conveyed via the flow 
passage 109 to the single inlet port 111 of the first valve means 
comprising a three-way solenoid actuated routing valve 10. The normally 
open port 113 of the valve 10 supplies fluid from the flow passage 109 to 
the filter unit 11 which then supplies the fluid to the inlet port 115 of 
the pump 5 so that the fluid may be recirculated through the closed path 
shown. When the valve 10 is moved to the normally closed position 
interconnecting the inlet port 111 with the normally closed outlet port 
117, fluid in the flow passage 109 is diverted to the flow passage 119 and 
thence to the inlet port 121 of the three-way solenoid actuated collection 
valve 33 (third valve means). The valve 33 has a normally open port 123 
which supplies fluid via the flow passageway 125 to the manifold 30 which 
returns the fluid to the sump 2. When the valve 33 is activated to 
interconnect the ports 121 and 127, fluid, instead, is supplied to the 
fraction collection manifold 27 and thence through activation of the 
fraction collection manifold to a chosen one of a multiplicity of aseptic 
piston-cylinder collection tubes 34. These tubes may be provided with 
fixatives or preservatives to stabilize the analyte of interest in the 
drawn aliquot. 
In the preferred modes of operation of the fluid pathway, there are five 
different combinations of settings of the valves and pumps which are 
operable to cause various modes of operation of the inventive fluid 
pathway to occur. Table A shows fluid path logic indicating which 
components are activated or deactivated. The identifying words for each 
mode as set forth in Table A will be used in specifically describing the 
various modes hereinbelow. 
TABLE A 
______________________________________ 
Fluid Path Logic Table 
Mode Valve 3 9 10 33 Pump 4 5 
______________________________________ 
Recycle 0 0 0 0 0 1 
Add 0 X 1 X 1 0 
Media 
Add Test 1 X 1 X 1 0 
Material 
Collect X 0 1 1 1 0 
Effluent 
Collect X 1 1 1 1 0 
Cells 
Mode Valve 3 9 10 33 Pump 4 5 
______________________________________ 
X = Optional 
0 = Off 
1 = Energized 
With reference to FIG. 9, the inventive fluid pathway has been organized in 
the "recycle mode". In this mode, the valves 3, 9, 10 and 33 are in their 
de-energized positions, the pump 4 is de-energized and the pump 5 is 
energized. In this mode, a volume of media is retained within the closed 
circulation path shown and is repeatedly provided at the inlet port 96 of 
the bioreactor 8. A mobile fraction is drawn from the outlet 98 of the 
bioreactor via the valve 9 port 103, is supplied to the port 111 of the 
valve 10 and exits the valve 10 at the port 113 where it is supplied 
through the filter 11 to the inlet port 115 of the pump 5. After exiting 
the pump 5 at the port 88, the media flows through the T-union 78 and 
enters the membrane oxygenator 6 via the input port 92 thereof. The media 
is re-oxygenated and brought up to proper temperature and then is applied 
to the inlet 96 of the bioreactor 8 thus completing the loop. 
With reference to FIG. 10, the pumps and valves are organized in a 
configuration termed the "add media mode". In this mode, with reference to 
Table A, the valve 3 is deactivated, the valve 10 is activated, the pump 4 
is activated and the pump 5 is de-activated. The position of the valves 9 
and 33 is at the option of the user. This is because the bioreactor 8 may 
be evacuated via either port 103, 105 of the valve 9. Furthermore, the 
valve 33 may be moved to whichever position is desired, depending upon 
whether fluid is being returned to the sump 2 or is being collected at the 
collection tubes 34. 
In the "add media" mode, a volume of fresh media is introduced into the 
system and an equivalent volume of spent waste-laden media is removed to 
either the sump 2 or the fraction collector 34 depending upon the desires 
and needs of the user. Media is drawn from the reservoir 2 via the 
manifold 29. The media is conveyed via the selector valve 3 to the 
operating pump 4 which pumps the fluid through the T-union 78 and thence 
to the inlet port 92 of the oxygenator 6. The deactivated pump 5 prevents 
reverse flow through the recirculation line. The media flows through the 
oxygenator 6 and thence to the bioreactor 8. The media flow may exit from 
either the end port 98 or the side port 99 of the bioreactor 8 depending 
upon the desires of the user with the flow traversing the selector valve 9 
and being applied to the inlet port 111 of the valve 10 which, as 
energized, fluidly connects the inlet port 111 with the outlet port 117 
thereof supplying fluid to the inlet port 121 of the valve 33. The valve 
33 is either activated or de-activated, as desired, to supply the media 
either to the sump 2 via the manifold 30 or to the fraction collection 
tubes 34 via the manifold 27. Total fluid circuit volume is unchanged 
during this mode of operation. 
With reference to FIG. 11, the "add test material" mode will now be 
described. With reference to Table A, in this mode, the valves 3 and 10 
are activated, the pump 4 is activated and the pump 5 is de-activated. The 
valve 9 is either activated or de-activated depending upon the needs of 
the user. In this mode, a volume of fluid is withdrawn from one of the 
test material reservoirs 31 which has been selected through actuation of 
the rotary manifold 68. The test fluid flows via the energized selector 
valve 3 from the port 97 to the flow passage 70, thence to the pump 4 and 
thereafter through the T-union 78 to the membrane oxygenator 6, thence to 
the bubble arrestor 32 and to the inlet port 96 of the bioreactor 8. 
Following sufficient addition of test material, the manifold 68 may be 
moved to a position wherein it may be supplied with plain media to wash 
all residual material from the flow lines associated therewith in 
preparation for the next test. If desired, reagents may also be introduced 
to the system in this manner. Otherwise, this mode operates the same way 
as the "add media" mode described above. 
With reference, now, to FIG. 12, a description of the "collect effluent" 
mode will now be described. With reference to Table A, it is seen that the 
valve 9 is de-energized, the valves 10 and 33 are energized, the pump 4 is 
energized and the pump 5 is de-energized. The position of the valve 3 is 
at the option of the user, depending upon whether the user desires to 
supply fluid from the media reservoir 2 or from the test material 
reservoir 31. Fluid flows via the selector valve to the energized pump 4 
and into the fluid circuit via the T-union 78. Fluid exiting the 
bioreactor 8 passes through the de-energized selector valve 9 via the 
ports 103 and 107 and through the energized selector valves 10 and 33 via 
the ports 111 and 117 of the valve 10 and via the ports 121 and 127 of the 
valve 33. The manifold selector switch 27 is temporarily re-positioned to 
a tap connected to the sump manifold to allow flushing of retained fluid 
left in the circuit from the last collection into the sump. This volume is 
dependent upon system geometry but is typically less than 100 
microliters. Following this purging, the manifold selector 27 is 
repositioned and fluid is diverted to appropriate collection tubes 34. In 
this manner, an aliquot of fluid characteristic of the system charge is 
obtained. An alternative method for non-random access collection devices 
is possible in which the selector valve 33 is temporarily de-energized at 
initiation of collection. This diverts fluid initially located between the 
valves 10 and 33 to be sent to the sump. In this way, only fluid retained 
between the valve 33 and manifold 27 is mixed with the sample. The fluid 
passageway connecting the valve 33 and the manifold 27 is extremely short 
in length making such mixing extremely minor. This latter explained 
procedure may be appropriate only when minor contamination of sample with 
dead volume material is of no concern. 
With reference to FIG. 13, the "collect cells" mode will now be explained. 
With reference to Table A, in this mode, the valves 9, 10 and 33 are 
energized, the pump 4 is energized and the pump 5 is de-energized. The 
position of the valve 3 is at the option of the user. 
In this mode, the selector valve 9 may be temporarily energized to allow a 
desired volume of cells on carrier beads to exit the extracapillary space 
of the bioreactor 8 via the port 99. Following attainment of this sample 
volume, the valve 9 may be de-energized to prevent further exhaustion of 
available cell-carrier bead supplies. Temporary re-positioning of the 
manifold 27 or de-activation of the selector valve 33 is maintained to 
flush the dead volume of material between the valve 9 and the manifold 27. 
Thereafter, the selector valve 33 may be energized and the aliquot of 
cells collected from the bioreactor 8 may be conveyed into an appropriate 
tube 34 via the valve 10, the valve 33 and the manifold 27. The 
appropriate tube 34 may contain fixatives and/or preservatives previously 
added at the discretion of the user. 
With reference, now, to FIG. 14, in particular, an explanation of the gas 
pathway of the inventive system 100 will now be described in greater 
detail. As shown, the main portions of the gas pathway are contained 
within the inner chamber 19. As shown in FIG. 14, pressurized gas is 
contained within a bottle 22 which carries a valve 67 having an actuating 
handle 69 and an outlet nozzle 71 fluidly connected to a pressure 
regulator 20. The pressure regulator 20 reduces gas pressure to working 
levels whereupon the gas passes through an activated charcoal/molecular 
sieve cartridge 35 designed to remove potential organic contaminants. 
Thereafter, the gas mixture is passed through a 70 micron sintered bronze 
particulate filter 36 which protects the system from dust which might be 
generated within the cartridge 35. 
As also shown in FIG. 14, supplementary gas input is permitted via a quick 
disconnect coupling 39 which extends through the containment 19 as well as 
the outer wall 47 at an access opening 104. The coupling 39 is fluidly 
connected to an unvalved T-union 106 via a flow passageway 108 and a 
one-way check valve 102 precluding reverse flow out the coupling 39. In 
the preferred embodiment, the check valve 102 is designed to open at a 
pressure greater than 10 psi above ambient. 
Downstream of the T-union 106, a 0.2 micron HEPA filter 21 is provided to 
remove any residual particulates which might happen to pass through the 
particulate filter 36. At this point, the gas mixture is as clean as 
possible and is thereafter passed through a humidifier cartridge 37 in 
which water vapor equilibrates across a membrane and saturates a charge of 
gas passing therethrough. Downstream of the humidifier cartridge 37, a 
valve 28 is provided which may be remotely controlled by the master 
computer as pre-programmed. Downstream of the valve 28, a flow passage 110 
conveys the gas mixture to a series of serially disposed T-unions 38 along 
with an L-shaped union 112 at the termination of the flow passage 110. 
Each of the unions 38, 112 conveys the gas mixture to a high pressure valve 
7 which may have a solenoid actuator remotely controllable by a digital 
computer controller, as will be described in greater detail hereinafter. 
Downstream of each valve 7 is an individual membrane oxygenator 6. 
The oxygenation means comprises an oxygenator 6 (FIG. 18) and consists of a 
cylinder 161 of acrylic or polycarbonate tubing of defined diameter. A 
removable insert spool 163 with one end cap 165 the size of the inner 
diameter of the cylinder and the other end having a flange to allow only 
partial insertion into the cylinder constitute the fluid path proper of 
the oxygenator. The insert spool is composed of two interlocking plates 
167, 169 which form an X-shaped cross-section. Appropriate holes and 
undercuts allow threading of the exchange membranes and permit alignment 
of input and outlet ports for unambiguous hook-up. 
The input end cap is provided with transit holes 171, 173 through which the 
membranes pass and are sealed with potting compound. Hose barb connectors 
are attached to the input side of the oxygenator. The flanged end may be 
beveled, for example, 22.degree. and may be provided with threaded luer 
lock connectors for economy of space when bubble arrestors are not used. 
This configuration allows removal of the unit without the total 
disassembly of a rail assembly means 1. Each membrane oxygenator 6 is 
fully autoclavable. The inventive membrane oxygenator 6 is unique in 
providing discrete paths for parallel simultaneous treatment of the liquid 
content of the several individual paths on a rail assembly means 1. The 
gas is introduced in a counter current manner with respect to the flow of 
media in order to achieve maximum efficiency of gas uptake. Dissolved 
oxygen and/or acidity are sensed by indwelling detectors as well as flow 
cell pH electrodes. On the basis of these data, software controlled 
decisions are made to refresh the volume of gas in an oxygenator 6. 
Alternatively, timed introduction of fresh gas to the volume of the 
oxygenator is available for maintenance of the acidity of the media. While 
commercial membrane oxygenator units may also be utilized in conjunction 
with the present invention, the improvements over known oxygenators which 
are included in the inventive oxygenator 6 render it extremely 
advantageous in the present invention given the requirements of the 
present invention for sterility, complete oxygenation of media, resistance 
to bubble formation, etc. 
In each oxygenator, maximum transfer of gas to media occurs. Gas which is 
not transferred to media is released from each oxygenator 6 via an outlet 
check valve 114 attached thereto and into the general chamber defined by 
the containment vessel 19. This chamber fluidly connects with the chamber 
formed by the outer containment 47 via a series of check valves 61 
protected by filter units 23, such as, for example, a 0.22 micron filter 
unit which effectively prevents moisture, infectious agents and aerosols 
from exiting the inner containment 19. Two such cascaded check valves 61 
are best illustrated in FIG. 7 and, for example, may be designed to open 
at 1/3 psi above ambient and 1/2 psi above ambient, respectively. This 
configuration maintains the chambers defined by the containments 19 and 47 
at a positive pressure above ambient, thereby preventing incursion of 
contaminants. 
All of the components of the gas pathway as particularly illustrated in 
FIG. 14 are critically cleaned of all solvents, lubricants and coatings 
prior to assembly so that essentially pure gas may be provided to the 
living tissues contained within the bioreactors 8. 
With reference, now, to FIG. 15, the specific means for thermal regulation 
comprising thermal control means will now be described. As shown in FIG. 
15, and as described hereinabove, the casing 13 has a plurality of 
recesses 79 with each recess being sized to snugly receive a bioreactor 8 
therewithin. The casing 13 is made of a material such as, for example, 
aluminum and comprises a solid conductive heat sink. As shown, a single 
membrane oxygenator 6 is provided for each casing 13 and serves to supply 
life sustaining gasses to all of the bioreactors 8 contained within the 
casing 13. 
As also shown in FIG. 15, the plate 16 of the rail 1 has an opening 81 
therethrough which allows insertion of a Peltier-type heating/cooling unit 
14 so that the unit 14 is in direct physical engagement with a wall of the 
casing 13, as shown. As is known, Peltier heating/cooling units consume 
low amounts of power. In the present invention, each Peltier unit 14 is 
operated in a pulsing on/off manner with fine control being exerted by 
controlling the rate of switching on and off. This control is exercised by 
a digital controller incorporated in the present invention and as will be 
described in greater detail hereinafter. A sensor 15 is buried within the 
casing 13 and senses casing temperature, conveying this information to the 
digital controller so that, under software control, the digital controller 
can control activation and de-activation of the Peltier device 14. The 
temperature sensor 15 may be a thermistor or a dedicated integrated 
circuit. As should be understood, an A/D converter is interposed between 
the temperature sensor 15 and the digital controller. 
As should be understood from the above explanation, output from the sensing 
element is applied to the digital controller via the A/D converter. In 
accordance with software preprogramming, evaluation is made of thermal 
data and, responsive to such evaluation, the Peltier unit 14 is operated 
to maintain the temperature of the casing 13 within a narrow temperature 
range conducive to maintenance of living tissue viability. 
As is known, the direction of heat conduction in a Peltier unit is 
controlled by the polarity thereof. Thus, in one direction of polarity, 
the Peltier unit 14 will supply heat to the casing heat sink 13. By 
reversing the polarity of the Peltier unit 14, heat may be removed from 
the casing heat sink 13. 
As shown in FIG. 15, and as explained above, the Peltier unit 14 extends 
through an opening 81 in the plate 16 and is in direct engagement with the 
casing heat sink 13. The opposite face of the Peltier unit 14 engages a 
metallic sub-plate 40 designed to assist in maintaining radiation of 
energy from the Peltier unit 14 toward the casing heat sink 13. 
Additionally, all non-contact surfaces of the casing heat sink 13 are 
meshed with an insulator such as, for example, metallized MYLAR which 
rejects greater than 80% of heat which would attempt to transfer 
therethrough. Through the use of the sub-plate 40 and the insulator 41, 
thermal short circuiting is prevented. Transfer of heat through the 
various levels of containment is by direct metal to metal contact aided, 
where appropriate, through application of heat sink compound. As shown in 
FIG. 15, a finned heat exchanger 118 is provided and is located adjacent a 
ventilation fan 120 which is controlled by the digital controller as will 
be explained in greater detail hereinafter. The region designated by the 
reference numeral 42 in FIG. 15 may be provided with appropriate filters, 
if desired, to purify any air which is being moved by the ventilation fan 
120. 
The solid metal heat sink 13 is advantageous for many reasons. Firstly, 
this medium prevents rapid temperature changes which can be life 
threatening. Additionally, the solid material thereof permits effective 
heat transfer even in zero gravity conditions. Other advantages also 
exist. 
With reference, now, to FIG. 16, an explanation of the electronic circuitry 
of the present invention including the computer control means will now be 
explained. With reference to FIG. 16, the electronic system includes a 
power supply 43 which is designed, in a well known manner, to provide 5 
volt DC and 12 volt DC power. The power supply 43 may be connected to an 
external source of power and may be provided with appropriate internal 
circuitry to convert power received from an external source to 5 volt and 
12 volt DC power. Since the various levels of gas supply, media supply and 
temperature control must be maintained within narrow ranges, to sustain 
tissue life, a back-up power supply 44 is provided outside the outer 
containment 47 and is preferably designed to be capable of sustaining 
operation of the entire device 100 for up to 72 hours should power to the 
power supply 43 be interrupted. In the preferred embodiment of the present 
invention, the back-up power supply 44 includes a multiplicity of lithium 
bromine complex batteries arranged in a plurality of parallel stacks. 
Preferably, each battery and each stack is properly fused and diode 
protected. The batteries are also protected by a thermal fuse between each 
pair of batteries and the entire power supply 48 contains a control panel 
with a power switch. The power supply 48 also contains various indicators 
and a sensing means designed to sense interruption of power to the power 
supply 43 so that activation of the back-up power supply 44 is 
instantaneous. 
The front panel of power supply 48 is provided with pilot, status and 
warning indicators which give continuous indication of the status of the 
power supply. An audible alarm exemplified by the enunciator (not shown) 
is also employed to inform the user that the back-up power supply 44 has 
been activated. 
If desired, the digital circuitry of the inventive electrical circuit can 
be pre-programmed to telephonically alert someone at a remote location of 
an extended power interruption or any other critical fault condition 
sensing in the entire system 100. The keypad 45 may be interfaced to allow 
manipulation of the software to allow troubleshooting and diagnosis 
without interruption of main program execution. The display 26 allows 
visual output of information. 
The heart of the electrical circuitry comprises the master computer 17 
which may be controlled via the keypad 45 or programmed via RSR32 port 138 
and which sends signals through the lines 124 to control the system. The 
batteries 46 are directly linked to the master computer 17 internally to 
provide direct back-up power to maintain integrity of any and all soft 
memories. These soft memories may temporarily store data which is 
desirable to maintain even when power is interrupted. 
The master computer 17 is pre-programmed so that if power is lost to the 
power supply 43 and the back-up power supply 44 fails or otherwise 
discharges, the batteries 46 will provide an additional increment of time 
to protect the master computer 17 until an attendant can arrive to 
replenish the back-up power supply 44 and/or correct the failure of the 
power source which is supplying the power supply 43. 
In FIG. 16, a single digital controller 128 is illustrated. It should be 
understood that a plurality of parallel connected digital controllers are 
provided each directly connected to the master computer 17 via printed 
circuit mother board 24 (FIG. 6) and each having multiple control 
functions. Each digital controller 128 is interconnected with various 
detectors and sensors in the individual rails 1 or in other locations in 
the device 100 via an analog-to-digital converter 132 which converts the 
analog signals to digital signals which are readable by the digital 
controller 128. These detectors include small pH sensors in the liquid 
pathway, pressure sensors within the inner containment 19, the thermal 
sensor 15 within the heat sink 13, indicators of proper operation and 
position of the valves and pumps, and all other sensors. The detectors are 
schematically represented in FIG. 16 as are the various pumps and valves 
all of which are shown as located on a particular rail assembly 1. 
Also schematically represented in FIG. 16 is the circuitry board 18 which 
has contained thereon the driver mechanisms for the various pump motors, 
solenoid valve operators, driver mechanism for the Peltier unit 14, 
controllers for the ventilation fan 120, and all other system functions. 
An additional system driver board 18' is also depicted directly connected 
to the master computer 17 and emitting control signals designed to control 
the solenoid valve for control of the gas control valve 28 in the gas 
pathway as illustrated in FIG. 14 and to control the fraction collector 
device 27 in the fluid pathway as illustrated in FIGS. 8-13. 
As should be understood, the electrical circuitry is provided with 
appropriate test point access for diagnostic and repair purposes as well 
as integral LED indicators to assist in troubleshooting of electronic 
problems. The electrical connectors generally designated by the reference 
numerals 140 and 142 are specifically designed to be water-tight so that 
they are not affected by any changes in system humidity or any potential 
possible leaks. 
As should be understood from FIG. 16, the electrical circuitry is organized 
into a network comprised of the master computer 17 which provides for 
system associated services and which coordinates the activities of the 
individual slave computers identified as digital controllers 128 in FIG. 
16. The master computer 17 also senses power availability and regulates 
power utilization while providing user interface services by virtue of the 
keypad 45 and the display 26. 
A log of programmed event completion, user interface traffic, and general 
system conditions is kept by the master computer 17 in an internal memory 
thereof and may be accessed and displayed as desired. Additionally, 
although not shown, a printer may be associated with the master computer 
17 to permit printing out of data including test results, system 
condition, information as to power supply continuity, etc. The digital 
controllers 128 coordinate and control activities on each individual rail 
1 and provide equivalent data recording and event logging services which 
may be transmitted to the master computer 17 for storage and/or display. 
Although the description of the electronic circuitry as illustrated in FIG. 
16 is quite schematic, when this explanation is taken in conjunction with 
the specific details of the specific system components as described 
hereinabove, understanding of the electrical circuitry should be clear. 
For example, all of the valves have been described in terms of which 
position the valve head takes when the solenoid is deactivated and which 
position the valve head takes when the solenoid is activated. Clearly, one 
skilled in the art with the schematic description of the electrical 
circuitry as set forth above would understand how to manipulate the keypad 
45 to set the valves and pumps for any one of the modes of operation 
illustrated in FIGS. 9-13, for example. In another example, one skilled in 
the art would easily understand that the keypad 45 may be manipulated to 
control activation of the valve 28 in the gas pathway as well as to allow 
manipulation of the valve 67 and to control the set point of the pressure 
regulator 20. In the context of the entire system, all of these features 
take on increasing significance in combination. 
With the above description of the preferred embodiment having been made, 
the specific details of the structure and manner of operation of the 
present invention should be well understood. With such understanding in 
mind, numerous applications of the present invention are possible and 
feasible. Prior to the commencement of any application of the inventive 
automated cell culture and testing system, the inventive device must be 
rendered sterile. Several methods of sterilizing the inventive device may 
be carried out. A first method consists of circulating cold chemical 
sterilants such as, for example, a sterilant known by the Trade Name 
"ACTRIL", through the various branches of the flow path of each rail 
assembly means in accordance with the recommended procedures as provided 
by the manufacturer. Following this treatment, the inventive device is 
flushed first with sterile water until no detectable residue is found, 
followed by minimal essential media for 24 hours. After this treatment 
regimen has been completed, the system is ready to accept cellular 
material to be loaded into the bioreactors 8 thereof in a manner which 
should be understood by those skilled in the art. 
Alternatively, each rail assembly means may be irradiated with gamma rays 
to effect sterilization. This method is highly reliable and effective, 
however, the gamma rays accelerate aging of elastomeric and plastic 
components to a high degree. Thus, the use of gamma irradiation to effect 
sterilization significantly increases costs since many replacement parts 
must be frequently installed. 
Finally, if desired, the individual flow path components may be 
individually autoclaved and then re-assembled under aseptic conditions. It 
must, again, be stressed, that thorough decontamination of the entire 
system is essential to assure experimental success. 
Once the system has been thoroughly sterilized, it is ready for use. The 
suggested examples of applications of the inventive device as set forth 
hereinbelow are merely exemplary, and the potential applications of the 
inventive device are only limited by the imaginations of its users. 
The inventive device 100 is particularly suited to the testing of candidate 
therapeutics against standardized cells, which may be infectious, 
malignant or pathologic. In an application where cells carry an infectious 
agent, aliquots of the material to be tested are presented to a 
standardized battery of cell types at specific concentrations and for a 
specific designed duration of time. One bioreactor in each group on a 
single rail assembly means is not subjected to the treatment and serves as 
a vehicle/system control. Cellular material is removed from the system via 
the side port 99 of the bioreactor 8, the material is circulated through a 
flow cell/light scattering device to measure turbidity, and is thereafter 
returned to the extracapillary volume of the bioreactor 8. Growth 
characteristics of the ferments are obtainable from the light scattering 
results. In addition, cellular material may be sampled at specific points 
in time to monitor the progress of any experiment. Each bioreactor can 
thus serve as an internal control. Once removed from the fraction 
collecting assembly, cells can be subjected to standard histological and 
biochemical testing to determine effects. An entire experiment can be 
conducted without breaking sterile barriers due to the self-contained 
nature of the system 100. An application such as described above enables 
testing both for antibiotic activity or resistance/susceptibility of an 
infectious isolate to various standard antibiotics. 
In a further intended application for the inventive device 100, activity of 
natural product extracts or synthetic chemicals may be tested. A standard 
battery of transformed cell types can be challenged with the material 
under test, and growth/metabolic characteristics can be measured to 
determine effects. Standard testing procedures can be used on cells 
recovered from the system to grade effects. At the same time, normal cells 
can be included in the battery to determine toxicity to non-transformed 
tissues. The invention can be modified to allow morphological analysis by 
including an automated microscope/CCD camera system, bioreactors with 
integral optical surfaces and a robotic X-Y-Z translator application. In 
this way, a derivative of the invention can be used to allow direct 
microscopic observation of living cells during study. 
In a further application, any of the currently approved cell-based tissue 
toxicity, genotoxicity or carcinogenicity testing protocols may be 
implemented through the use of the inventive device 100 to allow rapid, 
multiple, and reproducible studies of the toxic, mutational and cancer 
causing potentials of various liquid, solid or gaseous materials. The 
various levels of containment and automated test material 
application/sampling ability makes the present invention especially useful 
for this particular application. Solid material can be placed in-line to 
test the effects of leachates on target tissues. Biotransformation of 
applied materials can be accomplished by connecting, in series with the 
test bioreactor, a second bioreactor cartridge containing hepatic tissue 
or S9 extracts of such cells. Aliquots of effluent may be removed to be 
subjected to analytical testing. In this manner, the chemical nature and 
exact dose applied to the cells can be determined with a high degree of 
certainty. Cells may also be observed directly as above. Cells can also be 
removed from the system periodically to determine activation of oncogenes, 
etc. Permanent mounts can be produced from cells removed from the system 
to provide a record of the experiment. The system can be programmed, as 
desired, so as to not permit deviation from established protocols. In this 
way, strict compliance with FDA GLP guidelines can be enforced. These 
aspects permit all experimentation employing the inventive device 100 to 
be reproducible. 
The present invention is especially well suited for automated cell-based 
experimentation in outer space. The inventive device 100 meets all 
established NASA electromagnetic radiation, mechanical and safety 
requirements. The mechanisms of the inventive device are attitude 
independent, can operate in zero gravity, and the system can operate on 
the power and voltages available from the Shuttle Orbiter in the mid-deck 
area thereof. In a configuration specifically adapted for use in the Space 
Shuttle, the unit is made of a size occupying less than 2 cubic feet. 
In a further aspect, the present invention may be easily modified to 
function as a stand-alone automated cell culture device capable of 
precisely following programmed protocols. Such use of the present 
invention will permit isolated biomedical researchers to conduct detailed 
experiments without the need for technical assistance. At present, the 
technique of growing living cells and tissues is quite labor intensive, 
requiring one or more technicians to be present throughout the entire 
process. Thus, the productivity of an individual experimenter may be 
increased significantly through the use of the present invention. 
In a further aspect, the inventive device may also be used for automated 
pre-screening of candidate compounds for FDA required premarket acute and 
chronic toxicity testing. Significant savings in time and finances may be 
gained by using cultured tissue tests to eliminate toxic and mutagenic 
compounds from consideration prior to testing in whole animal models. The 
inventive device can employ a variety of culture vessels with the 
selection depending upon the nature of the cells and the intended 
analytical treatment of cultures. In one design, hollow fiber techniques 
may be employed which allow growth of cells directly on slides/coverslips. 
The grown cells may be treated at the proper time in situ with inhibitors, 
drugs, fixatives and stains to allow facile gross morphological analysis, 
observation of chromosomal morphology and obvious deletions, detection of 
the presence of micronuclei, disclosure of sister chromatid exchange or 
other approved methods for analysis of genotoxicity. 
In the course of development of the present invention, the following types 
of tissues have been grown and tested: primary cultures of human bone 
marrow, human hybridoma cells, primary cultures of rat calvarial bone 
cells, primary cultures of rat cardiac myocytes, the Yaffe rat L-8 
myoblast line, HL-60 human premyelocytic leukemia peripheral blood 
lymphocyte line, and P388D mouse lymphode neoplasm (which has been used by 
the National Cancer Institute for cancer chemotherapy screening studies). 
Additional tissues which are clearly feasible for study in accordance with 
the teachings of the present invention include primary culture human 
umbilical vein endothelial cells, human chondrocytes, tendon and synovial 
cells, whole amphibian embryo, pre-implantation mouse embryo, and 
co-culture of rat myocytes and neuronal tissue. 
Of course, all of the applications set forth above are merely exemplary of 
the potential applications of the teachings of the present invention. It 
should now be understood that any experimentation and testing involving 
any type of living tissue may be carried out through the use of the 
inventive device 100 and in accordance with the teachings of the present 
invention. 
As such, an invention has been disclosed in terms of a preferred embodiment 
thereof and applications thereof, which fulfill each and every one of the 
objects of the invention as set forth hereinabove and provide a new and 
useful automated cell culture and testing system of great novelty and 
utility. 
Of course, various changes, modifications and alterations in the teachings 
of the present invention may be contemplated by those skilled in the art 
without departing from the intended spirit and scope thereof. 
As such, it is intended that the present invention only be limited by the 
terms of the appended claims.