CRYOPRESERVATION OF RAT HEPATOCYTE MONOLAYER-CULTURES

1 Most previous attempts to cryopreserve hepatocytes have used suspens ions stored at either - 70 degrees C or in liquid nitrogen, and the ma jor problem is that these do not, on subsequent thawing, attach well i n culture. This limits their use in studies of drug metabolism and xen obiotic-induced toxicity. In this manuscript we demonstrate successful cryopreservation of rat hepatocytes as monolayers attached to a colla gen film. 2 Monolayers can be frozen and thawed without significant lo ss of cells, and although damage to the internal and plasma membranes is evident immediately post-thaw, a remarkable repair process takes pl ace over 24-48 h post-thaw. Immediately post-thaw only 10% of the cell s exclude Trypan Blue, but by 48 h 80 - 90% of the thawed cells are vi able, indicating that repair of the plasma membranes has taken place. 3 The cells post-thaw retain aspects of liver-specific function includ ing 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 prefre eze values. 4 A cryopreservation method based on adherent hepatocytes on a collagen substrate overcomes the problems encountered with cultur e of cryopreserved hepatocyte suspensions, and may provide a practical means of establishing a 'bank' of hepatocytes from several donors and species.