Effects of preezing on marrow stem cell suspensions: Interactions of cooling and warming rates in the presence of pvp, sucrose, or glycerol

Abstract

Based on results with microorganisms, one theory of freezing damage in cells states that the survival of a frozen-thawed cell is critically dependent on the rate at which it is cooled to subzero temperatures. The hypothesis suggests that the ultimate survival of a frozen and thawed cell is a function of the interaction of two factors that are oppositely dependent on cooling rate: 1) exposure to solution effects such as increased electrolyte concentration, pH changes, and dehydration and 2) the formation of intracellular ice during cooling and the extent of its recrystallization during warming. Using the colony-forming ability of the stem cells of mouse marrow as an assay of viability, we have sought to test the applicability of this theory to freezing damage in a nucleated mammalian cell. Stem cells suspended in each of three concentrations of glycerol, two of polyvinylpyrrolidone (mol wt, 40,000), or one of sucrose were frozen at rates varying from 0.3 to 500°C per min to −196°C, held for about 1 hr, and then thawed rapidly at about 900°C per min. In a second type of experiment, cells cooled at various rates were warmed either rapidly or slowly. Under all conditions, the survival of the colony-forming ability of the stem cells varied as a function of cooling rate, showing a distinct maximum at one rate. The optimum rate, however, varied with the specific additive and its concentration. For example, maximum survival was obtained for cells suspended in 1.25 m, 0.8 m, and 0.4 m glycerol and cooled at 1.5°C per min, 15°C per min, and 100°C per min, respectively. If cells in 1.25 m glycerol were cooled at 1.5°C per min, survival was about 65%, regardless of whether the suspensions were thawed at 2°C per min or 900°C per min, but if these cells were cooled at 200°C per min survival was 25% when the cells were thawed at 900°C per min and only 5% when they were thawed at 2°C per min. Good survival was also obtained in two additives that we believe to be nonpermeating, namely PVP and sucrose. Here, too, survivals showed distinct maxima as a function of cooling rate. Moreover, since the maximum survival was about 55% for cells suspended in 0.35 m sucrose but only 20% for cells in 0.4 m glycerol, it appears as if a nonpermeating additive is more effective on a molar basis in preventing freezing damage than is glycerol which has been assumed to permeate nucleated cells. All of these results are consistent with the “two-factor” theory of freezing damage developed from findings with microorganisms.