Development of an optimal method for the cryopreservation of hepatocytes and their subsequent monolayer culture

Adult rat hepatocytes, of good viability, were routinely isolated using a versatile biopsy perfusion technique. In order to develop an optimal cryopreservation regimen we investigated a variety of factors including the cooling rate, the constituents of the medium, the cryoprotective agents and the thawing conditions. The preferred freezing medium consisted of Leibovitz L15 culture medium supplemented with 10% (v/v) foetal calf serum, 10% (v/v) tryptose phosphate broth, 20% (v/v) dimethylsulphoxide, 100 mug/ml streptomycin and 100 U/ml penicillin. A freezing rate faster than the generally used 1-2 degrees C/min appeared to be optimal for subsequent hepatocyte attachment and survival in culture. Handling conditions after thawing were important for maximal recovery of cells in culture. To assess the functional integrity of hepatocytes in monolayer culture after cryogenic storage a number of parameters were monitored. Biochemical assays included lactate dehydrogenase leakage, protein synthesis, cellular ATP and ADP content, glutathione (reduced form), cytochrome P-450 content, ethoxycoumarin-O-deethylase activity, glucuronide and sulphate conjugation capacity. Cytochrome P-450 content was maintained at levels comparable with those in freshly prepared cultures for up to 24 hr in culture; ethoxycoumarin-O-deethylase activity was also similar to that of freshly prepared cultures. Changes in various biochemical parameters indicated that cryopreservation resulted in subtle damage to hepatocytes and that even cells that excluded dye did not survive as well as non-frozen cells. When human hepatocytes were subjected to the method developed for rat hepatocytes, they survived the trauma of cryopreservation but fewer cells subsequently attached and spread out to form a monolayer culture. These results indicate that human hepatocytes are more sensitive to freezing under the conditions developed for rat hepatocytes. This may reflect a greater sensitivity of the cells to cryopreservation and/or that the cryogenic conditions were not optimal for human hepatocytes.