VIABILITY AND FUNCTION IN PRIMARY CULTURE OF ADULT HEPATOCYTES FROM VARIOUS ANIMAL SPECIES AND HUMAN-BEINGS AFTER CRYOPRESERVATION

Cryopreserved hepatocytes from various animal species and human beings were tested for their ability to survive and function in primary cult ure. The freeze/thaw protocol primarily designed for rat hepatocytes w as used with slight modifications for the cells of all other species; it consisted of suspending parenchymal cells in the Leibovitz L15 medi um containing 10% fetal calf serum and 10% to 16% dimethyl sulfoxide. After transient storage at 4-degrees-C cell suspensions were transferr ed to - 20-degrees-C and then to - 70-degrees-C before being plunged i n liquid nitrogen. Hepatocytes were stored for a few weeks to 4 yr. Pr olonged storage did not augment loss of cell viability and function. C ell viability after thawing was estimated by the trypan blue exclusion test, and attachment efficiency to plastic was estimated by measuring intracellular lactate dehydrogenase content. Similar values were obta ined for most species tested; after cryopreservation cell viability an d attachment were decreased by 10% to 25% and by 40% to 50%, respectiv ely. A lower attachment rate was found with dog hepatocytes. Total cyt ochrome P-450 and protein synthesis were compared in fresh and cryopre served cells from four species after 4, 24, 48 or 72 hr of culture. Si milar values were found in both cells after 24 or 48 hr of culture. In addition, drug-metabolizing activities were measured in human hepatoc ytes from five donors. In most cases phenacetin deethylation activity was decreased whereas procainamide N-acetylation and paracetamol sulfo conjugation and glucuronidation were increased in cryopreserved cells. These results show that a simple and reproducible freeze/thaw protoco l can be used to cryopreserve hepatocytes from various species includi ng human beings and that after thawing a large fraction of the cells i s still viable and capable of expressing various functions in vitro.