Method of permanent removal from cell suspension in a porous container

A process and apparatus in which cryoprotectant in cryopreserved samples is removed with the use of turbulence and convective-dispersion around the cells within the primary cryopreservation container. Convective-dispersion is applied via the combined action of gravity and pulsatile transmembrane flow of buffer through the pores of containers fabricated from appropriate membrane material such as that disclosed in U.S. Pat. No. 5,026,342 and U.S. Pat. No. 5,261,870, as incorporated herein by reference above. By using mechanical forces to aid the process of cryoprotectant removal, the benefits of slow cryoprotectant removal are retained while the actual removal time is markedly decreased.

FIELD OF THE INVENTION 
The present invention relates to mechanically assisted methods, and 
apparatus therefor, to remove cryoprotectant from suspensions of cells in 
need of cryoprotection. 
BACKGROUND OF THE INVENTION 
Cryopreservation is a procedure for preparation of a suspension of cells, 
or a group of cells such as an embryo, for storage. The procedure normally 
incorporates adding cryoprotectants to the cells to be preserved, cooling 
of the suspended cells, long-term storage of the cell suspension at 
temperatures below about -80.degree. C., warming of the cells to normal 
cell temperatures, and removal of cryoprotectant from the cells. 
Cryopreservation of sperm or other cells from common mammals is a 
deceptively simple-appearing process which succeeds despite certain 
serious obstacles. This success depends on the use of one or more 
cryoprotectants in the context of certain procedural parameters. 
Overall cryopreservation procedures thus generally include: preparation of 
a suspension of cells for low-temperature storage by incorporation of 
cryoprotectants, and placement of individual units into vials or "straws"; 
cooling (sometimes called "freezing") at an appropriate rate; long-term 
storage of the suspension of cells at a temperature lower than -80.degree. 
C. and often between -180.degree. C. and -196.degree. C.; distribution at 
low temperature to intermediaries or users; warming (sometimes called 
"thawing") at an appropriate rate to the normal cellular temperature; and 
controlled removal of cryoprotectant plus any other medium or other 
adjustments needed to render the cells ready for in vivo use. The goal is 
not just to keep cells alive (viable), but to optimize retention of all 
cellular attributes such as normal life span, oxygen-carrying potential 
(especially in the case of erythrocytes) and fertilizing potential (in the 
case of spermatozoa or oocytes), for which the cells are being preserved 
in the first place. 
Notwithstanding cell type, species of origin or the various protocols used, 
prior art cryopreservation protocols traditionally result in about 30% 
mortality (or worse) of cells being preserved. Many cells traditionally 
did not survive the cooling and rewarming, and those which did suffered 
further damage during removal of the intracellular cryoprotectant. Damage 
can result from any or all of improper rates of temperature changes during 
cooling and rewarming, formation of ice crystals, reduction in temperature 
per se, toxicity due to high concentrations of solutes within and around 
the cells, the nature and concentration of the cryoprotectant(s) used, 
rates of addition and removal of cryoprotectants from within the cells, 
and other lesser known but empirically evident factors. 
A cryoprotectant is a molecule which allows a substantial percentage of 
cells to survive a freeze-thaw cycle and to retain normal cell function. 
Cryoprotectants which pass through the cell plasma membrane, and which 
thus act both intracellularly and extracellularly, are termed penetrating 
cryoprotectants. Non-penetrating cryoprotectants act only extracellularly. 
Glycerol is the most effective penetrating cryoprotectant for certain 
types of cells and for sperm from most species. Glycerol is of low 
toxicity, relative to the alternative penetrating cryoprotectants such as 
ethylene glycol, propylene glycol and dimethylsulfoxide. All penetrating 
cryoprotectants pass through a cellular membrane at a rate slower than 
water does, and each of these rates is itself temperature dependent. 
Non-penetrating cryoprotectants include proteins (such as milk or egg 
proteins used with mammalian sperm); sugars such as lactose, fructose, 
raffinose or trehalose; synthetic polymers such as methyl cellulose; and 
amide compounds. Most penetrating cryoprotectants, such as glycerol, serve 
as a solute (and cause osmotic flow of water) and a solvent (to dissolve 
salts and sugars) miscible with water. All non-penetrating cryoprotectants 
are solutes or colloids, and cannot themselves also serve as solvents. 
Both water and glycerol, as well as other solvents, pass through the 
membrane of cells and eventually equilibrate at the same concentration in 
all internal structures, so that the intracellular and extracellular 
concentrations are the same. 
The solute role of a penetrating cryoprotectant is believed to cause damage 
due to the induced osmotic flow of water. The solvent role is beneficial, 
however, because the penetrating cryoprotectants such as glycerol have a 
freezing point much lower than that of water. In the presence of glycerol, 
the portion of the solvent mixture remaining unfrozen at any given 
temperature is greater than if water were the only solvent. Hence, at any 
given temperature there is more "space" for the cells in channels of 
unfrozen solvent and a lower concentration of solutes (the same amount of 
solutes is contained in more liquid). This phenomenon occurs both inside 
and outside the cells. Further, the presence of glycerol probably reduces 
formation of micro-fractures in the ice and this, in turn, minimizes 
damage to cells. Non-penetrating cryoprotectants, such as sugars and 
lipoproteins, typically are present in relatively high concentrations. 
They typically act by modifying the plasma membrane, so that it is more 
resistant to temperature-induced damage, or simply by acting as a solute 
to lower the freezing point of the solute/solvent combination. 
Conventional procedures for preservation of many cells involve abrupt 
addition of a penetrating cryoprotectant, such as glycerol, to a cell 
suspension despite both long-standing and recent warnings that penetrating 
cryoprotectant should be added slowly. Similarly, the benefit of slow 
removal of penetrating cryoprotectant from cells is well known. Damage 
associated with the rapid addition or removal of a penetrating 
cryoprotectant is a direct consequence of extreme changes in cell volume, 
resulting from rapid movement of water, and formation of irreversible 
"tears" in the plasma membrane. Slow removal of cryoprotectants from 
within thawed cells generally has not been used, either because of 
ignorance or lack of a convenient approach to achieve it. 
Depending on the number of cells required for a functional "unit" after 
thawing, cells traditionally are packaged as individual units using glass 
ampules, plastic vials, plastic straws, or appropriately sized plastic 
bags. These packages all require removal of the cell suspension from the 
primary container before slow removal of cryoprotectant. Alternatively, 
technology disclosed by Hammerstedt et al. (U.S. Pat. No. 5,026,342 and 
U.S. Pat. No. 5,261,870, both incorporated herein by reference) allows 
slow removal of cryoprotectant while the cells remain within the primary 
container, via open pores in a special membrane formed to provide a 
primary container which allows exchange of fluid across the membrane. 
Although efficacious, because this approach is diffusion-limited the 
process can require up to two hours to reduce the concentration of 
cryoprotectant within the cells to a desired level. 
Apart from the widely practiced stepwise dilution used to address this 
problem, slow removal of cryoprotectant from cells in a suspension can be 
accomplished by placing the suspension, after thawing, into a conventional 
dialysis membrane and suspending the dialysis unit in a large volume of a 
salts solution. Alternatively, special plugged-pore containers, such as 
those disclosed in the U.S. patents incorporated by reference above, can 
be processed after thawing in a manner to open the pores of the 
membrane-container and to allow movement of molecules across the membrane. 
In both cases, movement of cryoprotectant or water through the membrane of 
the primary container is via diffusion and "down" the concentration 
gradient (i.e., away from the locus of highest concentration). Such 
diffusion-based processes effectively limit the rates at which composition 
of the medium immediately surrounding the cells is altered as water and 
cryoprotectant diffuse, in and out respectively, across the primary 
container membrane. Consequently, this limits rates of movement of water 
and cryoprotectant across the membranes of the cells to flux degrees which 
are not damaging to the cells. Reliance on simple diffusion through the 
membrane container requires a commercially unacceptably long 1-2 hour time 
period. 
The alternative assisted cryoprotectant removal method uses continuous flow 
centrifugation, such as is discussed in U.S. Pat. No. 4,221,322. This 
requires transfers from the original cryopreservation packaging, thus 
affording opportunity for contamination and requiring excessive 
processing. 
A need thus remains for a method for introducing and removing one or more 
cryoprotectants, to and from a cell or group of cells in need of 
cryopreservation, in which the cellular damage, potential contamination 
and/or long processing times of the prior art are avoided. 
SUMMARY OF THE INVENTION 
In order to meet this need, the present invention embodies a process and 
apparatus in which cryoprotectant in cryopreserved samples is removed with 
the use of turbulence and convective-dispersion around the cells within 
the primary cryopreservation container, applied via the combined action of 
gravity and pulsatile transmembrane flow of buffer through the pores of 
primary containers fabricated from appropriate membrane material such as 
that disclosed in U.S. Pat. Nos. 5,026,342 and 5,261,870, as incorporated 
herein by reference above. By using mechanical forces to aid the process 
of cryoprotectant removal, the benefits of slow cryoprotectant removal 
without transferring out of the original primary container are retained, 
while the actual removal time is markedly decreased and sterility 
maintained.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention embodies a process and apparatus in which 
cryoprotectant in cryopreserved samples is removed with the use of 
turbulence and convective-dispersion around the cells within the primary 
cryopreservation container, applied via the combined action of gravity and 
pulsatile transmembrane flow of buffer through the pores of containers 
fabricated from appropriate membrane material such as that disclosed in 
U.S. Pat. No. 5,026,342 and U.S. Pat. No. 5,261,870, as incorporated 
herein by reference above. By using mechanical forces to aid the process 
of cryoprotectant removal, the benefits of slow cryoprotectant removal are 
retained while the actual removal time is markedly decreased. 
Because of considerations of biohazard and sterility, optimally the means 
for providing the mechanical facilitation of cryoprotectant removal is a 
means not directly contacting the internal surfaces or contents of the 
primary container. This is particularly true in the case of human cells 
(e.g., spermatozoa, oocytes, embryos, fetal cells or blood cells), any or 
all of which should not be allowed to come in contact with the technician 
or other operator handling the containers. The following description 
presents two versions of a new device for rapidly reducing the 
concentration of cryoprotectant, within a separate container configured 
according to U.S. Pat. No. 5,026,342 and U.S. Pat. No. 5,261,870, by the 
combined use of gravity, turbulence and convective-dispersion. 
For certain types of cells (e.g., erythrocytes) destined for infusion into 
the vascular system or other body compartments, maintenance of sterility 
throughout the freeze-thaw and cryoprotectant removal process is 
desirable, but currently difficult. Cryoprotection can not be removed 
while the cells are within the original blood cell storage bag. For that 
reason, the FDA restricts the interval between thawing and deglycerolation 
of human erythrocytes and their infusion into humans to 24 hours. The 
primary problem is the need to transfer the thawed cells from a primary 
container (plastic bag) for freezing to a disposable system for 
cryoprotectant removal which includes interconnected containers of sterile 
buffer, a centrifuge bowl and cell-receiver. Similar problems hamper 
cryopreservation of other cell types. Hence the present invention provides 
in part for a single device in which a primary container stores the cell 
suspension and additional components enable rapid removal of 
cryoprotectant by combined actions of gravity, turbulence and 
convective-dispersion. The primary container also enables maintenance of 
sterility throughout the entire freeze-thaw and cryoprotectant removal 
process, and does not dilute the cell suspension. 
The present process is best understood in the context of the simple 
diffusion over which it represents an improvement. Traditionally, 
conventional dialysis devices containing cells and cryoprotectant, or 
primary containers fabricated as in patents incorporated by reference 
herein were diffused in buffer to remove the cryoprotectant through small 
pores or other membrane transport interstices provided in the walls of the 
vial or straw. When buffer flow was provided continuously outside the 
container of cells, the cryoprotectant concentration in the flowing buffer 
remained low and the cryoprotectant in the cell suspension predictably 
passed "down" the concentration gradient across the porous wall of the 
container, and into the external buffer. 
By contrast, the present process does not rely on simple diffusion across 
the container wall for removal of cryoprotectant. Further, simple 
diffusion of cryoprotectant across the cell plasma membrane is facilitated 
by convective-dispersion within the primary container. This is achieved 
via an appropriate blend of the processes of turbulent flow and diffusion 
through a porous membrane, created by pulsatile applications of pressure 
to transport fluid into and through the primary container. Inside the 
primary container, the turbulent flow and convective-dispersion of 
cryoprotectant-free buffer in cryoprotectant-rich buffer results in global 
spreading of inhomogeneity, as aided by gravity acting on the relatively 
dense cryoprotectant, modifies the microenvironment near a cell in a 
controlled manner. The extent of mixing is controlled, in part, by: the 
frequency and volume of pulses of cryoprotectant-free buffer entering the 
primary container; the zone of buffer entrance and/or penetration relative 
to the lower or upper portions of the contents within the primary 
container (due to the natural settling of cryoprotectant-rich buffer to 
the bottom of the container); orientation of the "entrance" membrane of 
the primary container (because gravity can affect redistribution and 
dispersion of cryoprotectant-rich buffer within the chamber); and optional 
movement of the primary container induced by mechanical means. Pulsatile 
flow can be applied with only inward pressure, allowing minor retrograde 
flow during the inter-pulse interval, or with a combination of inward and 
withdrawal pressures--positive and negative flow--directly inducing both 
direct and retrograde flow. 
Parameters were developed as follows. Assuming that removal of 
cryoprotectant by diffusion requires 6 container volumes (6.times.1 ml) of 
buffer over a thirty minute period, minimum flow rate to accomplish the 
same cryoprotectant removal would require 0.2 ml/min and a flow rate of 
1-3 ml/min would accomplish the task in under twelve minutes assuming 
actual flow of buffer during one-half (direct flow) of the elapsed time. 
Under these conditions, mass transfer is not limited by the transport rate 
associated with diffusion of cryoprotectant from the primary container and 
then the cells. Rather, mass transfer out of the primary container and 
mixing of cryoprotectant-rich with cryoprotectant-free buffer within the 
primary container occur via convective-dispersion, and cryoprotectant 
moves out of the cells by simple diffusion down a concentration gradient 
changed at an appropriate rate by convective-dispersion. The 
convective-dispersive transport of cryoprotectant will display 
approximately equal dependence on convection and diffusion at a flow rate 
of approximately 1.2 ml/min, assuming a membrane area of approximately 4 
cm.sup.2, with increasing dependence on diffusion at slower flow rates. To 
minimize accumulation of cells on the down-stream membrane face, and to 
facilitate occurrence of convective-dispersion, an oscillating 
(start/stop) flow is used; the pulsatile flow can range from 1 to &gt;60 
pulses/min, but oscillation frequencies of 2-40 pulses/min often is 
appropriate. Further, it is often advantageous to initiate cryoprotectant 
removal with low frequency (e.g., 1-2 pulses/min) and low volume (e.g., 
0.05-0.1 ml) pulses and gradually increasing the pulse frequency, pulse 
volume or both in a "ramp-like" manner. This reduces the concentration of 
cryoprotectant in the microenvironment of a cell at a rate which is 
non-damaging to the cell, but which facilitates diffusion of 
cryoprotectant out of the cells in a commercially viable time frame. 
Hence, the process provides an appropriate blend of turbulent flow, 
convection and diffusion to provide global spreading of inhomogeneity of 
the fluid environment near a cell in a controlled manner. With such 
approaches, cryoprotectant can be safely removed from cells in a 1-ml 
container in less than 15 minutes. 
The preferred embodiment of the present invention is a cryoprotectant 
removal device designed to accommodate a separate primary container 
incorporating two major faces fabricated from membranes as disclosed in 
the above-incorporated patents. At the time of cryoprotectant removal, a 
turbulent flow of small volumes of buffer is applied through the open 
pores of the primary container and via convective-dispersion within the 
primary container a gradual mixing of cryoprotectant-free and 
cryoprotectant rich buffer ensues, with the slow discharge thereafter of 
diluted cryoprotectant out of the primary container. To induce appropriate 
turbulence and convective-dispersion, and also to minimize accumulation of 
cells on the down-stream membrane face, an oscillating (start/stop) flow 
of cryoprotectant-free buffer is used. Hence, the device provides an 
appropriate blend of the processes of turbulent flow, convection and 
diffusion to provide global spreading of inhomogeneity of the fluid 
environment near a cell in a controlled manner. This reduces the 
concentration of cryoprotectant in the microenvironment of a cell at a 
rate which is non-damaging to the cell, but which facilitates diffusion of 
the cryoprotectant out of the cell. 
The simplest device according to the present invention is shown in FIG. 1. 
A primary container 1 containing walls 2 having pores therein and 
including cryoprotectant-containing cells 3 inside the walls 2 is infused 
with buffer by means of an exterior compression diaphragm 4. The exterior 
compression diaphragm 4 is powered by any known means such as cyclic 
vacuum to give pulsatile compression of the buffers into and out of the 
primary container 1. The inward pulsatile pumping of buffer and the 
resultant convective-dispersion which takes place inside the primary 
container 1 changes the cryoprotectant concentration within the cells 3 
and within the container 1 more rapidly than could simple diffusion, but 
at a rate still tolerated by the cells 3. Movement of water into and 
cryoprotectant out of the cells 3 is by diffusion across the cell plasma 
membrane, and the concentration gradients are steeper than with 
conventional methods. 
A second embodiment of the present cryoprotectant removal device is shown 
in FIGS. 2a and 2b. FIG. 2b shows the same structures as appear in FIG. 
2a, but in dissembled array. The same primary container 1 as shown in FIG. 
1 is shown in position within a split cube device 200. The primary 
container is a porous container whose previously plugged pores are now, at 
the time of cryoprotectant removal, open according to means disclosed in 
U.S. Pat. No. 5,026,342 and U.S. Pat. No. 5,261,870 and by other means 
known in the art. The split cube device 200 has two chambers 202 and 204 
describing inflow chamber 206 and outflow chamber 208, respectively. A 
pair of gaskets 230 seal the chambers around the primary container 1. The 
chambers 206 and 208 are connected to inlet port 232 and outlet port 234 
as illustrated. The inflow chamber 206 is connected via the inlet port 232 
to a pump 238 capable, by means of a controller known to those skilled in 
the art, of delivering a programmed sequence of pulses of desired buffer, 
with each pulse having a specific volume, duration, duty cycle and 
inter-pulse interval. Buffer is drawn from the reservoir 240 by the pump 
238, is passed into the inflow chamber 206, from thence through the 
primary container 1, and out through the outflow chamber 208 and the 
outlet port 234 for recycling. 
The structures of FIGS. 2a and 2b are generally plastic or polymer prepared 
by various means including sheet fabrication, injection molding, or any 
other method. Other materials such as metal or glass or other composites 
may also be used. In operation, and depending on the characteristics of 
the primary container and the cells therein, positioning of the split cube 
device will provide the desired orientation of the porous surfaces of the 
primary container 1 and the optimal direction of the buffer flow into and 
through the primary container 1. With some cell types it is advantageous 
to orient buffer flow into the lower region of the primary container 1, so 
that gravity can aid in mixing cryoprotectant-rich and cryoprotectant-poor 
buffer within the primary container 1. Additional movement of the split 
cube device 200 by any known means, but within defined parameters, further 
enhances convective-dispersion within the primary container 1. 
A more complex rapid cryoprotectant removal device is designed for a 
particular application such as removal of cryoprotectant from human sperm 
frozen and thawed in a CryoCell.RTM. container according to U.S. Pat. No. 
5,026,342 or U.S. Pat. No. 5,261,870. A system for this application 
addresses the provision of: (a) a one-time use of a sterile, integral unit 
containing necessary buffer, collecting waste, and allowing easy disposal 
as a biohazard; (b) complete automation via microprocessor control upon 
placement of the primary container 1 in the device; (c) fluid flow 
parallel to the membrane faces of the primary container 1 while the 
plugged pores open, followed by slow and controlled turbulent 
transmembrane flow thereafter; (d) controlled addition of desired 
additives; and (e) temperature control with optional warming, in addition 
to commercial features such as attractive features and minimized cost. 
In furtherance of such a specialized system, a third embodiment of the 
invention is shown in FIG. 3. An overall housing 30 incorporates a 
microprocessor controller 32, a thermoelectric cooler 34, piston driver 36 
and piston 38 as a mechanical means of creating pulsatile flow, a cam 
driver 40, a receptacle 42 formed as a recess in the housing 30 to accept 
a disposable insert 44. The receptacle 42 is temperature controlled and 
shaped to position the disposable insert 44 firmly against the piston 38 
and the spring-loaded cam driver 40. The disposable insert 44 contains 
buffer reservoir 46 and is attached as a unit to the treatment chamber 48 
having a removable cap 50 and a sleeve 52 adapted to receive a primary 
container 1 according to FIG. 1. The primary container 1 can be rotated 
within the sleeve 52 at 90.degree. about its own vertical axis, to 
reorient the membrane surfaces of the primary container 1 with respect to 
the direction of buffer flow. The spring loaded cam driver mates with the 
primary container 1 to facilitate this rotation, which can alternatively 
be performed manually in when the cam driver 40 is not present. A waste 
container 54 with a vent maintaining sterility is connected to the 
treatment chamber 48 by a waste tube 56, and all can be integral to the 
disposable insert 44. 
In the third embodiment of the invention, the disposable insert 44 and its 
associated structures can be sterile to begin with, and with both 
sterility and disposability the possibility of cross-contamination of 
samples is minimized or eliminated. Disposable single units such as this 
always minimize biohazard exposure to personnel, as well. Piston driver 36 
may be a stepping motor, solenoid-driven micro-pump, or other force 
exertion means known in the art. In operation, pulse rate and buffer 
volume can either be constant or more preferably begin at a pulse rate of 
from 1-3 pulses per minute to increase to a pulse rate in excess of 10 
pulses per minute over a total interval of 2 to 10 minutes. Flow can also 
optimally be increased from an initial flow rate of about 0.05-0.1 ml per 
pulse to 0.1-0.2 ml per pulse over the same 8 to 10 minute interval. 
The disposable insert 44 may be configured so that treatment chamber 48 is 
sealed with a peel-away closure (outer layer) known in the art. After 
removal of the seal, the cap 50 is removed and the primary container 1 is 
inserted into the sleeve 52 in a position to provide for initial buffer 
flow parallel to the membrane surfaces of the primary container 1. Buffer 
control is provided by the microprocessor 32, the piston driver 36 and the 
piston 38, with the microprocessor implementing preset pulse and volume 
rates. After a predetermined time, the primary container 1 can be rotated 
(either manually or by microprocessor guidance) within the treatment 
chamber 48 at 90.degree. relative to its own vertical axis, optionally in 
conjunction with rotation of the spring loaded cam driver 40, to create a 
normal (perpendicular) buffer flow with respect to the porous surface, 
rather than a parallel flow. After buffer treatment is complete, ready 
lights and ready alarms may be used to assure that the primary container 1 
is removed for further appropriate handling. The entire process requires 
less than 30 minutes. Because only 2-3 minutes is required to replace the 
disposable insert 44 in the housing 30, as few as 2-4 units described in 
the third embodiment would provide enough equipment even for a high volume 
clinic. 
A fourth embodiment of the invention addresses an alternative to the 
spring-loaded cam rotation discussed above to redirect buffer flow from 
parallel to normal (perpendicular with respect to the member surface). A 
fourth embodiment of the present apparatus is shown in FIGS. 4a, 4b and 
4c. 
For certain types of cells, such as erythrocytes, destined for infusion 
into the vascular system or other body compartments, maintenance of 
sterility throughout the freeze-thaw and cryoprotectant removal process is 
desirable and damage to the cells must be minimized. With prior art 
procedures this was difficult, because transfer of thawed cells to a 
counter-flow centrifugation washing system was necessary to remove 
cryoprotectant slowly from the thawed cells and to provide a suspension 
appropriate for infusion into a patient. The fourth embodiment of the 
present invention is adapted to address this medical challenge. A single 
unit combines a primary cell container, with membranes excluding most 
microorganisms (such as 0.2 micrometer pore size), and a rapid 
cryoprotectant cell removal device; the entire device is designed as a 
single-use, disposable item. Consequently, this device: (1) enables 
maintenance of sterility throughout the entire freeze-thaw and 
cryoprotectant removal processes; (2) provides removal of cryoprotectant 
by combined actions of diffusion and convection-dispersion at a rate which 
is conveniently short but also sufficiently slow so as not to damage the 
erythrocytes; and (3) allows direct infusion of processed erythrocytes 
from the container into the patient because it does not dilute the cell 
suspension or require post-thaw centrifugation of cells. 
The device of the fourth embodiment includes an outer housing 400 having 
therein supporting elements 402, two membranes 404 (as described in U.S. 
Pat. No. 5,026,342 or U.S. Pat. No. 5,261,870) providing major faces which 
characteristics selected for the particular application, and supporting 
screens 408 of adequate strength and porosity for the same particular 
application (such as thawing of erythrocytes). One inside major face of 
the outer housing 400 incorporates one or more types and sizes of baffles 
500 to distribute buffer entering the inflow chamber 502 of the device and 
to help to direct fluid through the membrane 404 forming the lower face of 
the inflow chamber 502 after the bypass tubes 504 are occluded by the 
clamp 506. The outflow chamber 508 may likewise be fitted with baffles 
(not shown). One end of the housing 400 has one or more tubes 510 leading 
to the inner chamber formed by structures 402, 404 and 408, and through 
which a suspension of cells 512 in buffer of appropriate composition and 
with appropriate cryoprotectants, as known to those skilled in the art, is 
placed. Another tube 514 later supplies additional buffer used for removal 
of cryoprotectant, as desired, with tube waste tube 516 providing egress 
when needed. The tubes are fabricated of suitable "sterile docking" 
materials known in the art. The unit is radiation sterilized (with tubes 
having been sealed) prior to use. The components may be made of virtually 
any material, preferably plastic or polymer. The two membranes 404 are 
fabricated from a porous membrane appropriate for the application, i.e., 
with composition, strength, pore diameter and percentage of pores being 
optimized for the particular application. The pores in the membranes are 
initially plugged as described in U.S. Pat. No. 5,026,342 and U.S. Pat. 
No. 5,261,870 with material which will maintain the pores in a plugged 
state during the interval required to fill the inner chamber with a 
suspension of cells (such as erythrocytes), to cool the contents to 
cryogenic temperature and to rewarm the cells to above 0.degree. C. and 
for a desired interval thereafter (such as 5 to 10 minutes), but which 
will dissolve from the pores after 3-10 minutes exposure to buffer 
circulating across the membrane face in chambers 502 and 508. 
Variations on the fourth embodiment include the possibility of the outer 
housing being a balloon or bladder structure itself, with any baffles 
being optional. 
A fifth embodiment of the present invention appears in FIGS. 10a and 10b. A 
reusable housing 1000 incorporates a microprocessor controller 1002, a 
thermoelectric cooler 1003 and insulation (not shown). A linear stepping 
motor 1004 drives a roller or bar 1055 over a bladder 1088 containing 
buffer 1010, which is in turn connected to a port 1024 in one-half of the 
treatment chamber 1013. The treatment chamber 1013 is a two-part right 
cylinder, with a short height, in which each half represents a cylindrical 
structure having a cubic aperture therein, wherein when the two halves are 
adjoined via a locking mechanism the dual apertures hold a primary 
container 1 as shown in FIG. 1. The port 1025 in the other half of the 
treatment chamber 1013 is connected to a waste bladder 1089 containing 
waste buffer 1014. The primary container 1 would representatively have an 
internal volume of 0.5 to &gt;5.0 ml, depending on the cell type and species, 
and preferably would be a thin (2.5 to 4.0 mm) rectangular, square, or 
cylindrical construct with the special membranes forming the major faces. 
In a further variation, the primary container 1 has a small port, sealed 
with UV glue after placing cells in the container before cooling, and a 
thin septum, punctured to remove cells after removal of cryoprotectant. 
Alternatively, a thin-walled tube, approximately 1 mm in diameter and as 
long as 10 or 15 mm, is used to fill the primary container 1, to provide a 
seal with a thermal bonding agent or adhesive plug, and to allow removal 
of cells after removal of cryoprotectant and cutting off the sealed 
portion where it protruded from the treatment chamber 1013. 
To assemble the device according to the fifth embodiment of the invention, 
the two halves of the treatment chamber 1013 are locked around a primary 
container 1, by finger counter rotation and means of interlocking 
mechanisms 1023 shown in detail. Sterile buffer can then be brought to 
contact both membrane faces of the primary container 1 from the 
surrounding bladders 1010 and 1089. In practice, after a few 
minutes'exposure to the buffer, the pores open, and automatic activation 
of the stepping motor 1004 brings about controlled buffer circulation as 
described for the other embodiments of the invention, with the same 
controllable variability of pulse and flow. 
The use of the devices disclosed herein improves the utility of containers 
including plugged pore membranes by coordinating buffer flow both to 
remove the pore plugs and to treat the contents of the container, and by 
providing pulsatile flow of buffer inducing turbulent flow and 
convective-dispersion within the primary container. 
The invention is further illustrated by means of the ensuing example. 
EXAMPLE 1 
The device according to FIG. 2 was tested as follows. The inflow chamber 
206 was pyramidal in shape, as was the outflow chamber 208. Buffer was 
moved through the device via a solenoid pump delivering 50 or 100 
microliters per stroke, at a stroke frequency of 3-120 strokes per minute 
(computer controlled). The device was operated with the primary container 
1 positioned so that its major faces--two opposing porous surfaces--were 
horizontal and the inflow chamber was below or above the primary container 
1, or with the major faces vertical. 
Initial tests monitored removal rates of glycerol from primary containers 
under various operating conditions (stroke volume, stroke rate, 
configuration and volume of the inflow and outflow chambers, starting 
glycerol concentration, presence or absence of bull or human sperm). 
Egress rates of glycerol from the primary chamber were controlled. 
FIG. 5 shows removal rate of glycerol from a 1 ml primary container 
containing bull sperm (100 million) in buffer containing 0.88 M glycerol, 
averaged across 4 runs. A slower removal rate and evidence for more 
initial mixing with the primary container with upward flow, rather than 
downward flow, were evident. Definitive tests compared cryopreserved human 
sperm deglycerolated: (a) by conventional 1:10 dilution with buffer, 
followed by centrifugation at 350 times gravity for 15 minutes; (b) an 
8-step process to achieve 1:10 dilution with buffer, followed by 
centrifugation at 350 times gravity for 15 minutes; versus (c) a rapid 
cryoprotectant removal device (RCRD) operated with the inflow chamber 
below the primary container. Evaluations of sperm quality, for cells 
deglycerolated by the 3 procedures, included percentage of motile sperm 
based on visual or computer-based analysis (using a Hamilton-Thorn 
Research, IVOS, system), percentage sperm bound to an egg membrane 
substrate (see U.S. Pat. No. 5,743,206), and penetration of zona-free 
hamster oocytes, using procedures known to those skilled in the art. Based 
on 8 replicate runs, 6.8 minutes were required to reduce glycerol 
concentration around the sperm to &lt;0.03 M, whereas the prior art 
procedures required greater than 18 minutes to achieve the same result. 
FIGS. 6, 7, 8 and 9 show that the rapid cryoprotectant device shown in 
FIG. 2 provides sperm of equal or better quality than the 1-step or 8-step 
dilution methods of the prior art. 
Although the invention has been described with particularity above, the 
invention is only to be limited insofar as is set forth in the 
accompanying claims.