Serum-free culturing of mammalian cells - adaptation to and cryopreservation in fully defined media

Abstract

Long term storage of living cells is a central issue in cell biology and medicine. In addition to the cryoprotectant dimethyl sulphoxide (DMSO), foetal bovine serum (FBS) is often added to the freezing medium for the cryoconservation of serum dependent cell lines. FBS, with its high protein content, protects cells against shear forces and gives the medium a desirable osmotic environment with a physiological viscosity. However, the harvesting of FBS is painful for the foetus and should be avoided for ethical reasons. In this work we describe the adaptation of several commonly used cell lines to serum and protein-free media; however, such cell lines should not be frozen in a conservation medium containing serum. We tested the synthetic surfactant “Pluronic F68™”, known to protect mammalian cells grown in serum-free bioreactors (Papoutsakis, 1991), as an active cryoprotectant. In samples containing 0.1 to 1% Pluronic F68, we found a significant increase in viable cells after thawing. Values up to 115% of starting cell number indicate that the cells proliferate within the first 24 hours after thawing, a property which was not observed in cryoconservation media without Pluronic F68.