A two-factor hypothesis of freezing injury. Evidence from Chinese hamster tissue-culture cells.

Abstract When Chinese hamster tissue-culture cells are frozen in a variety of suspending media, the percentage of cells surviving is maximal at optimum cooling rates, rates that are 2–4 orders of magnitude lower than those used to freeze cells for subsequent processing by the electron microscopy techniques of freeze-cleaving and freeze-substitution. The existence of such optima suggests that at least two factors dependent on cooling rate interact to determine the ultimate survival of a frozen-thawed cell. Other data are consistent with the view that the causes of injury in rapidly and slowly frozen cells are different. First, cells frozen rapidly in 0.4 M solutions of sucrose, glycerol, and dimethyl sulfoxide, or in 0.004 M polyvinylpyrrolidone, are inactivated to a much greater extent by slow warming than are cells frozen slowly in those solutions; that is, cells frozen at rates greater than the optimum are considerably more sensitive to slow warming. Second, the inactivation rate of cells frozen rapidly in glycerol is greater at −40 °C than that of cells frozen slowly. Third, the temperatures at which cells are killed as they are slowly frozen are very different from those observed during the slow warming of rapidly frozen cells. The precise nature of the two factors remains uncertain, but indirect evidence suggests that cells cooled slower than optimum are killed by alterations in the properties of the extracellular and intracellular solution induced by ice formation (e.g., high solute concentrations), and that cells cooled faster than optimum are killed by the formation of intracellular ice and its subsequent recrystallization during warming. Such intracellular recrystallization may be a potentially serious source of artifacts in frozen material processed for electron microscopy at temperatures above −60 °C, and perhaps even above −100 °C.