Increasingly, the cryopreservation of living cells is being attempted by researchers whose primary interest and experience is with the medical applications of those cells or tissues and whose prior experience with cryobiology may be negligible. It is therefore generally necessary to imitate some regimen used by others, perhaps with some other cell type and attempt to optimize the recovery empirically. This article makes no attempt to provide specific protocols for the many individual cell types. Rather it is a primer that may help to give such investigators an insight into the basic principles of cell freezing, cryoprotectants, and, particularly, their addition and removal. Finally, the article summarizes the five different approaches to applied cryopreservation: ultrarapid freezing and thawing, controlled-rate freezing, freezing with nonpenetrating polymers, vitrification, and equilibrium freezing.
A modification of previously reported methods for freezing red blood cells with a high concentration of glycerol is described. The limits of osmotic tolerance of human red blood cells range from one‐half to four times isotonic. The introduction and removal of glycerol creates transient osmotic gradients that can exceed these limits. It is shown that much of the cell lysis following thawing and during washing can be related to hypertonic injury incurred during the glycerolizing procedure. Refinements of this portion of the process produce significant gains in recovery. Problems associated with washing out a high concentration of glycerol by centrifugal means can be alleviated by a preliminary dilution with hypertonic sodium chloride which serves also to reduce cell volume and increase cell specific gravity improving sedimentation characteristics and affording more latitude for the hypotonic stresses associated with glycerol removal. Careful attenion to the osmotic limits during both glycerolizing and washing results in a process in which hemolysis from freezing, thawing and washing can be reduced to less than three per cent.
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