WO1992003046A1

WO1992003046A1 - Improved cell cryopreservation method

Info

Publication number: WO1992003046A1
Application number: PCT/US1991/006010
Authority: WO
Inventors: Mary Anne Whyte; Paul J. Price
Original assignee: Somatix Therapy Corporation
Priority date: 1990-08-28
Filing date: 1991-08-22
Publication date: 1992-03-05

Abstract

The present invention provides a method for freezing and thawing cells that substantially enhances the viability of organ digests and primary cell cultures.

Description

IMPROVED CELL CRYOPRESERVATION METHOD

FIELD OF THE INVENTION The present invention relates to a method for freezing and thawing cells, particularly organ digests and primary cell cultures.

BACKGROUND OF THE INVENTION

A major obstacle encountered in organ transplantation is the need to transplant the organ shortly after removal from the body of the donor. This limited time period makes it difficult to type the tissue, ship over long distances, or detect the presence of viral contaminants.

One solution to this problem has been to attempt to freeze organ digests or to culture organ digests to obtain desired cells while providing a period of time for analysis of the organ. A problem with cultured digests is that the cell composition can change during culture and fibroblasts may predominate over differentiated parenchymal cells. Furthermore, parenchymal cells may dedifferentiate in culture and lose their specialized function.

Cryopreservation would allow testing while preserving the organ digest or primary culture before the cell composition undergoes vast changes. However, organ digest and primary cell cultures tend to have very poor viability following freezing and thawing. For example, fetal liver organ digests tend to have about 15% viable cells following a freeze/thaw process.

A freeze/thaw process that substantially increased the viability of organ digest and primary cells would allow time for quality control procedures and transportation of cells over long distances. In addition, such methods would facilitate banking cells for later transplantation when a suitable- recipient was available. The methods would also facilitate research, permitting studies requiring a portion of the cells isolated from a single organ to be performed over a period of time.

DESCRIPTION OF THE PRIOR ART D'Alessandro et al, Diabetes 38:7-9 (1989) and Wahlberg et al, Transplantation 43:5-8 (1987) demonstrated the successful preservation for 72 hours of canine pancreas for autotransplantation using U solution (also known as University of Wisconsin solution and Belzer's solution) . Henry et al, Transplantation 45:73-75 (1987) describe the successful preservation for transplantation of a cadaveric kidney using Belzer's solution.

Ja ieson et al, Transplantation 46:512-516 (1988) and Jamieson et al, Transplantation 46:517-522 (1988) describe the successful preservation for transplantation of rabbit liver and canine liver, respectively using UW solution.

Novicki et al, In Vitro 18:393-399 (1982) describe freezing isolated adult rat hepatocytes in freezing media formulated with glycerol or DMSO. The DMSO- containing freezing medium produced better viability after thawing. Viabilities of 23 and 28% after 24 hours in culture (depending on DMSO concentration) were achieved in comparison to 42% for non-frozen control cells.

Fuller et al, Cryoletters 1:139-146 (1980) describe recovery of frozen isolated rat hepatocytes. Dye exclusion and urea production were maintained better than the process of protein synthesis. Use of DMSO, glycerol and methanol as cryoprotectants and different freezing regimens were examined. Use of 1 M glucose in the thawing medium is described.

Moshage et al, J. Hepatol . 7:34-44 (1988) describe that the efficiency of cell plating of frozen and thawed adult human hepatocytes in comparison to freshly prepared hepatocytes was decreased. The use of an extracellular matrix obtained from normal liver of kidney donors significantly improved the deleterious effects of deep- freeze storage on attachment efficiency and survival.

Attachment of cryopreserved hepatocytes was reduced to about

57% in comparison to 75% survival for freshly prepared hepatocytes. Coundouris et al, Biochem . Soc. Transactions 14:692-693

(1986) describe optimal condition for freeze-thawing of rat and human hepatocytes. The reported viabilities for human hepatocytes at 24, 48 and 72 hours post-plating, as determined by trypan blue dye exclusion, were 59%, 48% and 48%, respectively.

SUMMARY OF THE INVENTION The present invention provides a method for cryopreservation of cells that substantially enhances the viability of organ digests and primary cell cultures. The method comprises suspending the cells in a freezing medium comprising an effective amount of DMSO and Hana freezing solution at 0 to 4°C to form a cell suspension; gradually lowering the temperature of the cell suspension to at least -1 °C to produce a frozen cell suspension; and storing the frozen cell suspension, preferably in liquid nitrogen.

Hana freezing solution comprises potassium lactobionate at a concentration in the range of from about 60 to about 140 mM, glutathione at a concentration in the range of from about 1 to about 4 mM, raffinose at a concentration in the range of from about 15 to about 40 mM, and insulin at a* concentration in the range of from about 2.5to about 10 μg/ l. The frozen cell suspension is thawed until the suspension contains both solid and liquid. The partially thawed suspension is combined with at least an equal volume of a thawing medium to finish thawing the suspension while diluting the concentration of DMSO. The thawed cell suspension is pelleted. The supernatant medium is removed, and the cell pellet is resuspended in thawing medium and plated in a culture vessel. Prefera^bly the cells are allowed to attach in the thawing medium for not more than about four hours and the thawing medium is replaced with growth medium.

The method has been used successfully to cryopreserve digests of human fetal hepatocytes which, previously, did not survive in culture after freezing.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides an improved method for freezing and thawing organ digests and primary cell cultures which greatly improves the viability of the cells. The method comprises suspending the cells in a freezing medium comprising a freezing solution and an effective amount of DMSO at 0 to 4°C to form a cell suspension; gradually lowering the temperature of the cell suspension to at least -12°C to produce a frozen cell suspension; and storing the frozen cell suspension, preferably in liquid nitrogen. The frozen cell suspension is thawed until the suspension contains both solid and liquid. The partially thawed suspension is combined with at least an equal volume of a thawing medium to finish thawing the suspension while diluting the concentration of DMSO. The thawed cell suspension is pelleted. The supernatant medium is removed, and the cell pellet is resuspended in thawing medium and plated in a culture vessel. Preferably the cells are allowed to attach in the thawing medium for not more than about four hours and the thawing medium is replaced with growth medium.

Defxn tions

As used herein, an organ digest refers to cells (single cells and cell aggregates) obtained from the intact tissue of an animal. As used herein, a primary cell culture refers to cells obtained from the intact tissue of an animal and grown on glass or plastic surfaces for the first time.

The cell freezing and thawing method can be used for organ digests and primary cell cultures. The method is also useful for early passage cells which have poor viability on freezing and thawing. The method can be used with cells of any organ from any animal species and is particularly useful with human fetal hepatocytes and human fetal islet cells. Although the method is also effective for use with established cell lines, less expensive and less time consuming methods provide suitable results with cell lines.

Freezing Medium and Thawing Medium Freezing Medium The freezing medium for the present method is Hana freezing solution with an effective amount of DMSO. Hana freezing solution is a physiological solution that comprises potassium lactobionate at a concentration in the range of from about 60 to about 140 mM; glutathione at a concentration in the range of from about 1 to about 4 mM; raffinose at a concentration in the range of from about 15 to about 40 mM; and insulin at a concentration in the range of from about 2.5 to about 10 μg/ml. A preferred formulation of Hana freezing solution is shown below in Table 1.

TABLE 1 Hana Freezing Solution

Component Concentration K Lactobionate 100 mM Glutathione 3 mM Raffinose 30 mM Insulin 5 μg/ml

Osm: 227 mMol/Kg

In another embodiment, Hana freezing solution additionally comprises Na KH₂P0₄ at a concentration in the range of from about 10 to about 35 mM; adenosine at a concentration in the range of from about 0.6 to about 10 mM; MgS0₄at a concentration in the range of from about 3 to about 6 mM; allopurinol at a concentration in the range of from about 0.5 to about 1.5 mM; and bactrim at a concentration in the range of from about 0.1 to about 1.0 ml/L. A preferred formulation for each of the additional components is listed below in Table 2.

TABLE 2 Supplements for Hana Freezing Solution

Component Concentration

Na KH₂P0₄ 25 mM Adenosine 5 mM MgS0₄ 5 mM

Allopurinol 1 mM

Bactrim 0.5 ml/L

In yet another embodiment, Belzer's solution can be used as the freezing solution. Belzer's solution is an organ transport solution that additionally contains hydroxyethyl starch. Belzer's solution is described in D'Alessandro et al., Diabetes 38:7-9 (1989), which article is incorporated herein by reference in its entirety. The formulation for Belzer's solution is shown below in Table 3. Belzer's solution is available commercially from DuPont. TABLE 3 Belzer's Solution

Component Concentration K Lactobionate 100 mM Na KH₂P0₄ 25 mM Adenosine 5 mM MgS0₄ 5 mM Glutathione 3 mM Raffinose 30 mM Allopurinol 1 mM Insulin 100 U/L Bactrim 0.5 ml/L

Hydroxyethyl starch 5 g% Na: 30 mM K: 120 mM Osm: 320-330 mOsm/L

Effective amounts of DMSO for freezing cells are well known and can vary from about 7.5 to 20% (v/v), and are preferably about 10%. The freezing medium optionally includes an antibiotic at an effective concentration.

Thawing Medium

The thawing medium of this invention is the growth medium for the type of cell that is being thawed, preferably supplemented with DNase and, for use with organ digests, preferably, a substance to increase the osmolality. The DNase is present at a concentration which is effective to prevent clumping due to DNA released from dead cells. A concentration of about 100 μg/ml DNase is effective.

For use with organ digests, the osmolality of the solution is preferably high to prevent swelling and subsequent rupture of cells during the thawing process and the first few hours thereafter. Use of a sugar, preferably glucose at a concentration of from about 1.0 to about 1.5 M, more preferably about 1 M glucose, to enhance the osmolality is preferred. Use of 1 M glucose for increased osmolality is well known. Other suitable substances for use in increasing osmolality in tissue culture solutions or cell processing solutions are well known and can be used in place of glucose.

When thawing a primary culture rather than an organ digest, the osmolality enhancer is preferably not present in the thawing medium. The osmolality enhancer is detrimental to cells which were plated prior to freezing.

The thawing medium is preferably also supplemented with serum for use with cells having a serum-free growth medium. A preferred serum is fetal calf serum at a concentration of at least about 10% (v/v) .

The choice of a growth medium for an organ digest or primary cell culture is not affected by the method of this invention. A preferred growth medium for use with hepatocytes and pancreatic islet cells is H₃H Basal medium. A most preferred thawing medium for those cells is listed in Table 4.

TABLE 4 A Preferred Thawing Medium

H₃H Basal medium¹

100 μg/ml DNase

10% FCS

1 M glucose

^JThe formulation for H₃H Basal medium is listed below in Table 5. TABLE 5 H₃H Basal Medium

F12K-Arg¹

3 X10"^ ornithine

0.05 mg/ml gentamicin sulfate trace elements²

2 mM glutamine

H₃ supplement³

10 ^xHam, Proc. Natl . Acad . Sci . 53:288-293 (1965), which article is incorporated herein by reference in its entirety. ²Listed below in Table 6. 15 ³Listed below in Table 7.

TABLE 6 Trace Elements

Concentration in

20 Component H₃ Supplement ( /ml) sodium metasilicate 70 molybdic acid 0.62 stannous chloride 0.047 nickel sulfate 0.26

25 cadmium sulfate 0.023 cobalt chloride 0.024 lithium chloride 0.024 ammonium metavanadate 0.60 manganese chloride 0.10

30 TABLE 7 H₃ Supplement

Final Concentration 6-fatty acid Bovine Serum Albumin 1 mg/ml

EGF (Epidermal Growth Factor) 0.05 μg/ml

Glucagon 0.001 μg/ml

Hydrocortisone 1.265 μg/ml

Insulin 10 μg/ml Somatotropin 0.0065 μg/ml

Transferrin 5 μg/ml

Se0₂ selenium oxide 0.035 μg/ml

CuS0₄ ^#5H₂0 cupric sulfate 0.0025 μg/ml

ZnS0₄ ^β7H₂0 zinc sulfate 0.00144 μg/ml

Freezing Process The organ digest is prepared by a standard method for preparation of a primary cell culture for the species and type of organ. The cell suspension can contain single cells and small cell aggregates. For use with a primary cell culture, the cell suspension is prepared by standard methods such as trypsinization, scraping and the like.

The cells are pelleted and counted. Then the cells are resuspended in a sufficient amount of freezing medium at 4°C to provide a concentration of from about 10⁵ to 2X10⁷ cells/ml. If there will be a delay before freezing the cells, preferably the cells are suspended in freezing medium without DMSO, since DMSO is toxic to cells at temperatures of 0°C or above. Conveniently, the cells are suspended in DMSO-free freezing medium and then diluted with an equal volume of freezing medium having twice the desired final concentration of DMSO just prior to freezing.

The temperature of the cell suspension in freezing medium is sequentially lowered to about -100°C prior to placing the vials in liquid nitrogen storage. The temperature is slowly lowered, preferably at about 1°C per minute, to the phase change where the liquid supernatant begins to freeze. The temperature at which the phase change occurs depends on the freezing medium used, particularly the DMSO concentration. For freezing media containing 10% DMSO, the phase change occurs from about -10°C to about -12°C. During the phase change, preferably the temperature is lowered rapidly, preferably at about 10°C per minute, more preferably at more than 10°C per minute, until the cell suspension is solid. After the phase change, the temperature is gradually lowered, preferably at from 2 to 5°C per minute. A most preferred sequential temperature lowering scheme is listed below. Departures from the scheme can be tolerated, particularly after the cells are frozen.

PREFERRED TEMPERATURE LOWERING SCHEME lower temperature at l°C/minute to -4°C lower temperature at 25°C/minute to -10°C lower temperature at 3°C/minute to -40°C lower temperature at 5°C/minute to -100°C

Once the temperature of the cells reaches about -100°C, the vials are transferred to liquid nitrogen storage. The cells should remain frozen for at least about a week prior to thawing.

Thawing Process

To thaw the cells, the frozen cell suspension is preferably thawed until there is both liquid and solid freezing medium in the vials. Most preferably the vials are thawed to about a half crystalline, half liquid state. The frozen cell mixture is diluted with an excess of thawing medium to dilute the concentration of DMSO in the supernatant medium to at least about one-half, preferably to about one-fifth, preferably to about one-tenth or less of the original DMSO concentration of the freezing medium. Preferably the thawing medium is at room temperature.

The diluted cells are pelleted, and the DMSO-con ining supernatant is removed. The cell pellet is resuspended in thawing medium. The cells are preferably counted prior to plating. The cells are plated in thawing medium for about 2 to 4 hours post-thawing to permit attachment of the cells. The cells preferably do not remain in the thawing medium more than about 4 hours. Thereafter, the medium is aspirated and replaced with growth medium.

This invention is further illustrated by the following specific but non-limiting examples. Temperatures are given in degrees Centigrade and concentrations as weight percentages unless otherwise specified. Procedures that are constructively reduced to practice are described in the present tense, and procedures that have been carried out in the laboratory are set forth in the past tense.

EXAMPLE 1

Effect of Serum Concentration in Freezing Medium Freezing media having varying concentrations of FCS were compared to determine the effect of the FCS concentration on the viability of human fetal hepatocytes. The hepatocytes were an organ digest prepared the day the study was performed. The cell suspension, freezing vials and freezing media were kept at 4°C until just before addition to a controlled rate freezer (Cryomed freezer with a thermocouple probe) . The cell suspension was pelleted and resuspended in half of the final volume of freezing medium without DMSO. The cells were frozen at a final concentration of 1X10⁶ in a final volume of 1 ml. The freezing media used were:

1) F12K with CHO supplement, 10% DMSO and 20% FCS

2) F12K with CHO supplement, 10% DMSO and 10% FCS

3) F12K with CHO supplement, 10% DMSO and 5% FCS 4) F12K with CHO supplement, 10% DMSO and 0% FCS (CHO supplement is a cell culture medium supplement which is commercially available from Irvine Scientific, Irvine CA) .

The four replicate cell suspensions for each of the freezing media were maintained on ice until the freezer reached 4°C. The remaining half of the freezing media with 20% DMSO

(twice the desired final concentration of DMSO) was added to each cell suspension, quickly transferred to a 2 ml freezing vial, capped and placed in the freezer. The probe was placed in the chamber. Once the sample and freezing chamber were within 2 to 4°C of each other the freezing program was started. The temperature was lowered as follows:

1) lower temperature at 3°C/minute until -4°C

2) lower temperature at l°C/minute until -10°C

3) lower temperature at 3°C/minute until -40°C 4) lower temperature at 5°C/minute until -1?0°C

When the temperature reached -120°C, the vials were immediately transferred to a liquid nitrogen storage container.

Four days later, one vial of each of the freezing media was thawed by agitating the vials in a 37°C water bath until a small ice crystal remained in the liquid medium. At that point, the contents of the vials were transferred to a 10 ml conical tube containing 10 ml of a room temperature thawing medium, H₃H basal medium with 10% FCS (H₃H basal medium is a preferred, serum-free growth medium for hepatocyte cultures) , gently mixed by inversion and pelleted. The cells were resuspended in thawing medium for plating on rat tail collagen-coated 35 mm plates. A 0.1 ml aliquot of each cell suspension was used in a trypan blue dye exclusion test for a cell viability determination. Viabilities were:

20% FCS: 18.8% 10% FCS: 10.5% 5% FCS: 40.5% 0% FCS: 11.2% At four hours post-plating, the supernatant was removed and the plates were rinsed with phosphate buffered saline (PBS) to remove non-attached cells. The cells were fed with H₃H basal medium with 10% FCS. The following day, only the 5% FCS plate had significant numbers of hepatocytes. Two days post-plating, the plates were examined. Each plate was sparsely populated with hepatocytes. The cells in 5% FCS appeared healthiest. All of the plates were discarded.

The study was repeated using a second vial for each of the serum concentrations. The thawing procedure was the same as described above except that the thawing medium used was α-MEM with 1 M glucose and 10% FCS. In this study, cells in freezing medium with 10% FCS looked best. Cells from 20% and 0% FCS freezing medium looked worst. Plates were discarded at 24 hours post-plating.

The study demonstrated that there is a large range of viability associated with cells frozen under identical conditions.

EXAMPLE 2 Effect of Thawing Medium The study of Example 1 was repeated with the following changes. The thermocouple probe of the freezer was placed in a "blank" vial which contained freezing medium with cells. The freezing medium used was H₃H basal medium with 5% FCS, CHO supplement and 10% DMSO. Four vials of cells were used to evaluate each of the following two thawing media. 1) MEM with 1 M glucose and 10% FCS 2) H₃H basal medium and 10% FCS

For each of the thawing media, the cells contained numerous vacuoles at 24 hours.

EXAMPLE 3

Evaluation of Freezing Media The study of Example 2 was repeated with the changes described below to evaluate the effects of freezing media of the cells. The freezing procedure was modified so that the rate of freezing from -4°C to -10°C was 25°C/minute. The thawing media used was 125 ml H₃H basal media; 13.8 ml FCS; 25 g glucose and 2.5 ml H3 supplement. The freezing media evaluated were:

1) H₃H media with CHO supplement, 10% FCS and 10% DMSO

2) H₃H media with CHO supplement, 5% FCS and 5 10% DMSO

3) Wicomb's media with 10% DMSO

4) Belzer's transport solution with 10% DMSO

5) L-15 media with 10% FCS and 10% DMSO Wicomb's media is described in Wicomb et al, Transp. Proc ,

10 21:1360-1368 (1989). L-15 media was purchased from GIBCO (Grand Island, NY) .

When the cells were thawed, diluted in thawing medium and pelleted, the pellets were resuspended in 1.6 ml of thawing media prior to plating in an attachment factor-

15 coated (Matrigel, Collaborative Research; diluted 1:200 in PBS; 300 μl/well) 24 well plate. Of the 1.6 ml, 0.5 ml was plated in one well to which 0.5 ml of thawing medium was added; 1.0 ml was plated in a second well; and 0.1 ml was used for a viability determination. The viability for each 0 of the freezing media is shown in Table 8. Duplicate samples were counted for number 4.

At two hours post-plating, cells frozen in media 4 appeared healthiest. The other wells had few attached hepatocytes. Another 1.0 ml of thawing media was added at that time to dilute any DMSO remaining in the media. At three hours post-plating, the media was removed, the wells were rinsed to remove debris, and the media was replaced with H₃H basal media with CHO supplement. At about 24 hours post-plating, only cells frozen in media 4 had hepatocytes attached to the wells. Those cells survived one week in culture.

This study demonstrated that Belzer's transport solution with 10% DMSO was the most effective freezing solution tested.

EXAMPLE 4 Evaluation of Thawing Media Two vials of cells frozen in media 4 in Example 3 were used to determine whether FCS or 1 M glucose, an osmolality enhancer, in the thawing medium was more effective for preserving the viability of the cells. The vials were thawed as described in Example 3, divided into three aliquots per vial and plated in six wells of a 24 well plate. The thawing medium used for each of the wells was H₃H basal media with H3 supplement, as in Example 3, and also included 10% FCS and 1 M glucose. At three hours post- plating, the medium from one well for each vial was removed and replaced with three different thawing media. Each tested thawing media included H₃H basal media with H3 supplement, as previously, and in addition included the following:

1) 1 M glucose

2) 10% FCS 3) 1 M glucose and 10% FCS

At 24 hours post-plating, the cells in both media containing glucose (with and without FCS) were detached and dead. The cells in media with FCS and without glucose looked healthy and survived in culture for two weeks. The study demonstrated that thawing media with glucose was needed for organ digest for an initial period post- thawing to provide an environment with appropriate osmolality for freshly thawed cells. However, high osmolality is not conducive to cell attachment and survival. The high osmolality appeared toxic to the cells when it remained on the cultures for extended periods following thawing.

EXAMPLE 5

Evaluation of Freeze/Thaw Method The study of Example 4 was repeated using Belzer's solution with 10% DMSO as the freezing medium and H₃H basal media with H3 supplement, FCS and glucose as the thawing medium for plating the cells. At two hours post-plating, the 1 ml of thawing was supplemented with an additional 1 ml to dilute residual DMSO. At three hours post-plating, the thawing medium was removed and replaced with H₃H basal media with CHO supplement. By three hours post-plating, the cells looked healthy and appeared to be attached, but were rounded.

The study was repeated. Viability on plating was 74%, as determined by trypan blue dye exclusion. At 17 days post-plating, the cultures were healthy and appeared to be proliferating.

EXAMPLE 6

Evaluation of Freeze/Thaw Method The study of Example 5 was repeated using three versions of the freezing medium:

1) Belzer's solution and 10% DMSO

2) Supplemented Hana freezing medium (formulated as listed in Tables 1 and 2) and 10% DMSO 3) Hana freezing solution (formulated as listed in

Table 1) and 10% DMSO

Duplicate samples of each freezing medium were prepared. The cells used had been passaged prior to freezing. Therefore, the osmolality enhancer was omitted from the thawing medium. The thawing medium was as described in Example 5, except that the medium included 20% FCS and 100 μg of DNase. The cells were plated onto 35 mm rat tail collagen-coated plates. The cell count and viability for each tested freezing medium are shown below.

Total Cells fx 10⁴) % Viability la) 81 100 lb) 79 99

2a) 113 94 2b) 97 95

3a) 57 89

3b) 57 85

At 1.5 hours post-plating, the cells were attached and looked healthy. No differences between the three formulations of freezing medium were apparent. Few viable eels were observed clumped together or to dead cells using thawing media with DNase. This was an improvement in comparison to the number of clumped, viable cells using thawing media without DNase.

The studies described in the examples demonstrate that the method of the present invention improves the viability of organ digest and primary cell cultures of human fetal hepatocytes. In particular, organ digests of human fetal hepatocytes are well known for failing to survive after freezing. By the methods of the present invention, survival rates of 70% or more were achieved.

Claims

WHAT IS CLAIMED IS:

1. A method for freezing cells comprising: a. suspending said cells in a freezing medium comprising a freezing solution and an effective amount of DMSO at 0 to 4°C to form a cell suspension, said freezing solution comprising potassium lactobionate at a concentration in the range of from about 60 to about 140 mM, glutathione at a concentration in the range of from about 1 to about 4 mM, raffinose at a concentration in the range of from about 15 to about 40 mM, and insulin at a concentration in the range of from about 2.5 to about 10 μg/ml; b. gradually lowering the temperature of said cell suspension to at least -12°C to produce a frozen cell suspension; and c. storing said frozen cell suspension.

2. The method of Claim 1 wherein the temperature of said cell suspension is sequentially lowered to at least -100°C.

3. The method of Claim 1 wherein said cell suspension is stored in liquid nitrogen.

4. The method of Claim 1 wherein step (a) comprises: a. suspending said cells in a freezing solution comprising potassium lactobionate at a concentration in the range of from about 60 to about 140 mM, glutathione at a concentration in the range of from about 1 to about 4 mM, raffinose at a concentration in the range of from about 15 to about 40 mM, and insulin at a concentration in the range of from about 2.5 to about 10 μg/ml at 0 to 4°C to form suspended cells; and b. diluting said suspended cells in an equal volume of said freezing solution additionally containing 20% DMSO at 0 to 4°C to form a cell suspension.

5. The method of Claim 1 wherein step (b) comprises: a. slowly lowering the temperature until the cell suspension begins to freeze; b. rapidly lowering the temperature during the phase change until the cell suspension is frozen; and c. gradually lowering the temperature until the suspension reaches about -100°C.

6. The method of Claim 5 wherein the temperature is lowered by at least 10°C per minute from about -4°C to about -10°C.

7. The method of Claim 1 wherein said freezing solution comprises 100 mM potassium lactobionate, 3 mM glutathione, 30 mM raffinose, and 5 μg/ml insulin.

8. A method for preparing a suspension of cells for culture, said cells being frozen in a freezing medium comprising a freezing solution and an effective amount of DMSO, said freezing solution comprising potassium lactobionate at a concentration in the range of from about 60 to about 140 mM, glutathione at a concentration in the range of from about 1 to about 4 mM, raffinose at a concentration in the range of from about 15 to about 40 mM, and insulin at a concentration in the range of from about 2.5 to about 10 μg/ml; said method comprising: a. thawing said suspension until said suspension contains both solid and liquid to produce a partially thawed suspension; b. combining said partially thawed suspension with at least an equal volume of a thawing medium to produce a thawed cell suspension; c. pelleting said thawed cell suspension to form a cell pellet and supernatant medium; d. removing said supernatant medium from said cell pellet; e. resuspending said cell pellet in said thawing medium to form a cell suspension in thawing medium; and f. plating said cell suspension in said thawing medium in a culture vessel.

9. The method of Claim 8 wherein said method additionally comprises: a. maintaining said cell suspension in said culture vessel until said cells attach to said vessel to form attached cells; b. removing said thawing medium from said attached cells; and c. replacing said thawing medium with growth medium.

10. The method of Claim 9 wherein said cell suspension is maintained in said culture vessel for fewer than four hours.

11. The method of Claim 8 wherein said thawing medium is a serum-free growth medium for said cells which has been supplemented with calf serum.

12. The method of Claim 11 wherein said thawing medium additionally comprises an osmolality enhancer.

13. The method of Claim 12 wherein said osmolality enhancer is glucose at a concentration of from 1.0 to 1.5 M.

14. The method of Claim 11 wherein said thawing medium additionally comprises DNase at a concentration which is effective to prevent clumping due to DNA released from dead cells.

15. The method of Claim 8 wherein the volume of thawing medium is at least about ten times the volume of said suspension.