Studies on Preservation by Freezing of Human Diploid Cell Strains

Abstract

Summary Methods for optimal preservation by freezing of various tissue culture passages of human diploid cell strains have been investigated. Considerable loss of viability occurred when cells were slow frozen and stored at —70°C in glycerin medium. Storage loss at this temperature was reduced when DMS or a combination of glycerin and PVP was used as additive. Cell numbers per ampoule, in the concentrations tested, did not influence survival during freezing and storage at —70°C. In contrast to storage at —70°C, storage of frozen samples under liquid nitrogen refrigeration has not resulted in viability loss during a period of 1 1/2 years. Differences in viability due to the use of various additives were also smaller upon storage at these lower temperatures. Results of equilibration tests in additives prior to freezing indicate no toxicity of DMS for these cells, but prolonged exposure to glycerin was detrimental to survival. A constant cooling rate of l°C/min was not found to be critical in freeze-preservation of these cell strains, although under the conditions described, more rapid and variable cooling rates did not provide for greater cell recovery. Thawing of frozen samples in 30 or 60 seconds following storage under liquid nitrogen refrigeration gave comparable cell survival; somewhat higher viability was indicated with the faster warming rate after long term storage at —70°C. Plating efficiencies of these strains were low, but when compared in qualitative tests with pre-freeze controls, unstained cells of thawed suspensions had lost none of their ability to adhere to a plate and multiply. When returned to serial culture, recovered cells have retained full passage potential In vitro and all properties which characterize cultures of the non-frozen strains. The competent technical assistance of Mrs. Susan Kulina is gratefully acknowledged.