Abstract
The protection provided by a number of chemical agents against damage due to freezing and thawing was investigated by testing the reproductive integrity of cells from a tissue culture cell line after freezing. Plating efficiency of individual cells was determined with Puck's cloning technique. 3 Prevention of Freezing Damage by Calf Serum and Polyvinylpyrrolidone (PVP) Agent Concentration Surviving Cells ∗ % % Serum 5 † 16.8 12.5 26.6 25 24.0 50 35.3 PVP 0 † 18.6 0.125 27.6 0.25 21.8 0.5 30.8 1 23.1 2 27.9 4 38.8 ∗ Each value is the mean of two experiments. † Control. Cells frozen in normal tissue culture medium, consisting of 0.5% lactalbumin hydrolysate and 5% calf serum in Hanks' balanced salt solution, showed an average survival of 16.3%. The variation between different experiments had 95% tolerance limits of 6.8 and 34.5%. The protective capacity of every agent tested was investigated by adding it in different concentrations to the cell suspension. The best prevention of freezing damage was obtained with ethylene glycol, diethylene glycol, propylene glycol, glycerol, dimethyl sulfoxide, pyridine N-oxide, and hexamethylene tetramine. Less protective compounds were acetamide, dimethylacetamide, formamide, dimethylformamide, monoacetine, d-mannose, d-ribose, glucose, d-mannitol, sorbitol, and inositol. Little protection was observed with dimethyl sulfone, polyvinylpyrrolidone, and serum. Lower survival than in control experiments was found if methanol, ethanol, phenol, resorcinol, or sodium chloride was added to the growth medium. The applicability of this tissue culture method to testing the efficacy of protecting agents against freezing damage, and the results obtained in the present investigation, are discussed.
Published Version
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