Survival of hamster tissue culture cells after freezing and thawing

Summary The usual procedure for preserving the viability of nucleated mammalian cells by freezing involves suspending them in a presumably permeating additive such as glycerol and cooling them at about 1°C per min. This procedure was found to be highly deleterious to Chinese hamster cells. The optimal cooling velocity when 0.1-ml quantities of cell suspension were rapidly thawed was closer to 100°C per min than to 1°C per min. Furthermore, about 25% of rapidly cooled cells survived freezing to −196°C without any additive. However, even in the presence of an additive, the 0.1-ml samples of rapidly cooled cells survived poorly when warming was slower than 1100°C per min. “Rapid” warming with quantities larger than 0.1 ml is often considerably slower than 1100°C per min, a fact that might make the sequence of rapid cooling and “rapid” warming risky to use as a routine procedure for cell preservation. The highest survivals of slowly cooled cells were obtained not by using glycerol, but by using two additives (PVP and sucrose) that are apparently unable to penetrate the cells. Furthermore, one of these (PVP) has too high a molecular weight for one to be able to ascribe its ability to protect to its molar concentration.