Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.