1 Most previous attempts to cryopreserve hepatocytes have used suspensions stored at either - 70°C or in liquid nitrogen, and the major problem is that these do not, on subsequent thawing, attach well in culture. This limits their use in studies of drug metabolism and xenobiotic- induced toxicity. In this manuscript we demonstrate successful cryopreservation of rat hepatocytes as mono layers attached to a collagen film. 2 Monolayers can be frozen and thawed without significant loss of cells, and although damage to the internal and plasma membranes is evident immediately post-thaw, a remarkable repair process takes place over 24-48 h post-thaw. Immediately post-thaw only 10% of the cells exclude Trypan Blue, but by 48 h 80 - 90% of the thawed cells are viable, indicating that repair of the plasma membranes has taken place. 3 The cells post-thaw retain aspects of liver-specific function including cytochrome P450 content and albumin synthesis. However, cytosolic proteins are lost through the damaged membranes and, probably because of this, urea synthesis from ammonia is retained at only 25% of pre- freeze values. 4 A cryopreservation method based on adherent hepa tocytes on a collagen substrate overcomes the problems encountered with culture of cryopreserved hepatocyte suspensions, and may provide a practical means of establishing a 'bank' of hepatocytes from several donors and species.
Effect of 2,5-anhydro-d-mannitol on membrane potential in rat hepatocyte couplets and hepatocyte monolayer cultures