Preservation at Subzero Temperatures of Mouse Fibroblasts (Strain L) and Human Epithelial Cells (Strain HeLa).∗

Summary Mouse fibroblasts of strain L (Earle) and human epithelial cells of strain HeLa (Gey) have been preserved successfully at −60° to −70°C for 6 and 7 months, respectively. Glycerol in the suspending liquid greatly increased the percentage of surviving cells. Solutions of glycerol (5% for strain L and 20% or 30% for strain HeLa) in a solution for maintenance of animal cells (MS), in balanced salt solution, or in serum (horse serum for strain L cells, adult human serum for strain HeLa cells), were of approximately equal value for the periods of preservation studied. The cells survived storage poorly at −60° to −70°C when suspended in undiluted serum or in 5% dextrose in water. Strain L cells were preserved in slightly greater numbers after freezing over a period of 1–1.5 hours than after freezing in 3-5 minutes; strain HeLa cells survived both freezing procedures equally well. Better preservation of both cell types resulted from storage at −60° to −70°C than at −20°C, and from thawing in 1–2 min. rather than in 30–60 minutes. The value of low temperature preservation for handling and maintenance of strain L and strain HeLa cells and for preservation of strain stability is discussed.