CRYOBIOLOGY, 10, 502-504 (1973)

BRIEF COMMUNICATION

Cryoprotection of Mammalian Cells in Tissue Culture with Pluronic Polyols ~

M. J. ASHWOOD-SMITH, W. A. G. VOSS, AND C. WARBY

The Department of Biological Sciences, University of Victoria, Victoria, British Columbia, Canada and, The Surgical Medical Research Institute, University of Alberta, Edmonton, Alberta, Canada

Pluronics (ethylene oxide polypropylene glycols) are used routinely in heart-lung machines in medical practice. Concentra- t-ions used are normally in the range of 0.4% (w/v) . The effectiveness of this rather un- usual group of nonionic surfactant polymers in preventing erythrocyte hemolysis during freeze-thawing procedures has been noted (2). We wish to report that one member of the pluronic series, the polymer F-68, which has the lowest molecular weight in the series ( av MW ,8350 daltons (4) ), has cryo- protective properties for mammalian cells in tissue culture.

METHODS

The pluronics used in this study were kindly provided by the BASF Wyandotte Corporation, Wyandotte, MI 48192, Di- methyl sulfoxide was obtained from the Baker Chemical Company and the organic buffer tricine [N-tris [hydroxymethyl] methyl glyeine] was obtained from the Sigma Chemical Company, St. Louis, MO. The tissue culture cell line and the tech- niques of freezing, thawing, and assessing viability have been described previously (1). The mean rate of cooling was approxi-

Received July 10, 1973.

1Supported by Canadian Medical Research Council Grant.

mately 175 ° C/min (the value for - 1 5 ° C to - 6 0 ° C was 345 ± 2 0 ° C/rain). The equivalent mean thawing rate was 150 ° C/rain (from - 6 0 ° C to - 1 5 ° C, 3 0 0 ± 15 ° C/min).

RESULTS

The increasingly general use in tissue culture of organic zwitterion buffers rather than sodium bicarbonate CO2 buffers prompted us to investigate their influence, if any, on eryoprotective agents. This in- fluence of a tricine buffer system compared with the standard sodium bicarbonate buf- fer system present in the tissue cul- ture medium was investigated using di- methyl sulfoxide as the cryoprotective agent. The results of these experiments are illustrated in Fig. 1 and it is apparent that the substitution of tricine for bicarbonate as buffer for the tissue culture medium in no way detracts from the effectiveness of dimethyl sulfoxide as a cryoprotective agent used at concentrations up to 10% (v/v) . The effectiveness of the pluronic F-68 com- pared with dimethyl sulfoxide at a concen- tration of 10% is illustrated in Fig. 2. Be- cause of the interesting property that solu- tions of pluronics are more viscous at high temperatures, the experiment was divided into two stages. In the first stage compari- sons of protection with and without DMSO

502

Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

CRYOPROTECTION WITH PLURONIC POLYOLS 503

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T R I C I N E

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NQHCO 3 TRICINE NoHCO 3

Fro. 1. Effect of .tricine and bicarbonate buffer systems on the protective action of different concentrations of dimethyl sulfoxide against freeze-thaw damage in Chinese hamster cells in tissue culture. Ordinate: percentage of protection ( cell survival). Abscissa: concentration (v/v) of dimethyl sulfoxide. Results expressed as percentage survival compared to unfrozen cells. Separate controls as shown were used for each set of experiments. Bars indicate standard deviation.

and with and without the pluronic were made when the cells were incubated in the presence of either the pluronic or the di- methyl sulfoxide for approximately 15 min at 22 ° C. In the second series of experi- ments the conditions were identical except that the incubation tempera ture was 4 ° C when the viscosity of the pluronie solution is less than at 22 ° C. The protective effect of 10% dimethyl sulfoxide is statistically the same whether incubation was carried out at 22 or 4 ° C. F-68 gave approximately 65% protection to mammal ian cells against f reeze- thaw damage irrespective of whether the incubation conditions prior to freezing were 4 ° C or 22 ° C. F-68 is clearly not as good as the dimethyl sulfoxide as a cryo- protective agent. These results have been repeated several times; no toxic effect of the pluronics at the concentrations used were observed. Occasionally, however, cells would not at tach as well to the surface of the plastic unless the pluronic was diluted below a concentration of approximately 0.5%. Provided this simple precaut ion was taken, the degree of protection afforded tissue culture cells from f reeze- thaw dam- age by pluronics is always a little less than

t h a t which can be achieved with dimethyl sulfoxide. The degree of protection, how- ever, is usually of the same order afforded

by a 10% solution of polyvinylpyrrol idone (PVP K-30).

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I I I 0 CONTROL DMSO

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FIG. 2. Protective action of pluronic (F-68) against freeze-thaw damage in Chinese hamster cells; comparison with dimethyl sulfoxide and effect of incubation at 22 ° C or 4 ° G prior to freeze-thaw regimen. Ordinate: percentage of pro- tection (cell survival). Abscissa: cells incubated at the stated temperature for 15 min in the pres- ence or absence of the protective agents prior to freezing and thawing. Results expressed as percent- age of survival compared to unfrozen cells. Bars indicate standard deviation.

504 ASHWOOD-SMITH, VOSS, AND WARBY

DISCUSSION

There has been one previous report con- cerning the cryoprotective action of plu- ronics toward erythrocytes; the phironic concentration ranged from 5 to 20% (2). The remarkable lack of toxicity of the pluronics and the use of these compounds for priming heart-lung machines (3) cou- pled with their cryoprotective properties suggest that the use of the pluronics for organ preservation at low temperatures might be fruitfully investigated.

A wide range of pluronie molecules are available from manufacturers and hence this group of polymers presents an interest- ing series for theoretical studies in cryo- biology.

SUMMARY

Chinese hamster cells in tissue culture are protected equally well by dirnethyl suff- oxide against freeze-thaw damage when an

organic buffer, tricine, is substituted for the normal bicarbonate buffer system. The pluronic polyol, F-68 has pronounced cryo- protective action for tissue culture cells.

REFERENCES

1. Ashwood-Smith, M. J., Warby, C., Connor, K. W., and Becker, G. Low temperature preser- vation of mammalian cells in tissue culture with polyvinylpyrrollidone (PVP), dextrans, and hydroxyethyl starch ( HES ). Cryobiology 9, 441--449 (1972).

2. Glauser, S. C., and Talbot, T. R. Some studies on freezing and thawing human erythrocytes. Amer. J. Med. Sci. 75-81 (1956).

3. Hymes, A. C., Safavian, M. H., Arbulu, A., and Baite, P. A comparison of pluronic F68, low molecular weight dextran, mannitol, and saline as primary agents in the heart-lung apparatus. 1. Thorac. Cardiovasc. Surg. 56, 16-27 (1968).

4. Pluronie Handbook. Lib. Congr. Card No. 70-150738. BASF Wyandotte Corp. Michi- gan (1971).