Toxic. in Vitro Vol. 5, No. 5/6, pp. 479~t82, 1991 0887-2333/91 $3.00+ 0.00 Printed in Great Britain.All fightsreserved Copyright© 1991 PergamonPress pie USE OF CRYOPRESERVED ANIMAL AND HUMAN HEPATOCYTES FOR CYTOTOXICITY STUDIES C. CHESNI~*t~, C. GUYOMARD*~', L. GRISLAIN§, C. CLERC*, A. FAUTREL* and A. GUILLOUZO* *INSERM U 49, Unit6 de Reeherches H~patologiques and I"BIOPREDIC, H6pital Pontchalllou, 35033 Rennes Cedex and §Bio-pharmacie SERVIER, 5 Rue de Bel Air, 45000 Orleans, France Abstract--To evaluate the potential use of cryopreserved hepatocytes for toxicological studies, rat, dog and human hepatocytes were frozen in Leibovitz medium containing 20% foetal calf serum and various concentrations of dimethylsulphoxide and stored in liquid nitrogen. 50% or more of the hepatocytes that attached and survived immediately after isolation still possessed these properties after freezing/thawing. Thawed hepatocytes from the three species showed only a slight reduction in their ability to metabolize phenacetin or to conjugate paracetamol with glucuronic acid. Sensitivity to the toxic effects of erythromycin was not affected by the MTT and neutral red assays. Similar observations were made with rat hepatocytes for five other toxic agents--chloramphenicol, chlorpromazine, acrylamide, chloroqulne sulphate and p-chloromercuribenzoic acid. These results suggest that, after cryopreservation, isolated hepatocytes represent a suitable model for drug metabolism and toxicity screening studies. Introduction Isolated hepatocytes, either in suspension or in pri- mary culture, are now extensively used in pharmaco- toxicological research. Numerous studies have demonstrated their usefulness for the evaluation of metabolism and toxic responses to xenobiotics (Guil- louzo, 1986 and 1988). Because of frequent variations in the routes and the rates of drug metabolism between laboratory animals and humans, data ob- tained with animal liver cells cannot be extrapolated without risk to the human situation. Ideally, metab- olism of toxic chemicals by human and animal hep- atocytes should be compared and the different toxic mechanisms elucidated to give a more rational extra- polation of toxic risk to man. However, comparisons between human and animal hepatocytes are usually difficult and unrealistic be- cause of the erratic availability of human hepatocytes and their individual variation in metabolic functions. As large numbers of cells can be obtained from a single perfusion, storage of viable hepatocytes in excess could at least partly solve these problems. This would allow studies to be performed in parallel on cells from different donors. Cryopreservation cur- rently represents the only way of storing viable cells for a prolonged period (Guillouzo et al., 1987). Most cryopreservation protocols have been devised for rat hepatocytes (Chesn6 and Guillouzo, 1988) and the few reports on parenchymal cells from other species show that these cells are more sensitive to freez- ing/thawing conditions than their rat counterparts (Powis et al., 1987). By using a well defined protocol first designed for rat hepatocytes and later applied to hepatocytes from other species, including man :~To whom correspondence should be addressed at BIOPREDIC, H6pital de Pontchaillou, 35033 Rennes Cedex, France. Abbreviations: DMSO = dimethylsulphoxide; FCS = foetal calf serum; LDH = lactate dehydrogenase. (Chesn6 and Guillouzo, 1988; C. Chesn6 et al., un- published data) we found that more than 50% of hepatocytes able to attach to plastic and survive in culture when plated just after isolation still possessed these properties after freezing in liquid nitrogen and thawing. Whether cryopreservation affects the sensi- tivity of adult hepatocytes to toxic compounds has yet to be evaluated. In this study we have assessed the toxicity of chemicals in rat, dog and human hepato- cytes before and after freezing. Materials and Methods Cell isolation. Hepatocytes from various species were isolated by the two-step collagenase perfusion method (Guguen-Guillouzo and Guillouzo, 1986; Seglen, 1975). Rat hepatocytes were prepared from the whole liver of male Sprague--Dawley rats weigh- ing 180-200 g. Beagle dog hepatocytes were obtained by dissociation of a 10-g wedge liver biopsy. Human hepatocytes were also isolated from wedge biopsies; the two (female) donors were 52 and 58 yr old and histological examination did not indicate morpho- logical alterations of liver samples. Cell viability was estimated by the trypan blue exclusion test; in all cases it exceeded 80%. Cryopreservation and thawing. Isolated bepatocytes were suspended at a density of 4 x 106 cells per ml in Leibovitz (L-15) medium containing 20% foetal calf serum (FCS). An equal volume of L-15 medium containing 20% FCS and dimethylsulphoxide (DMSO), the cryoprotective agent, was then slowly introduced. DMSO was added at a final concen- tration of 16, 14 and 12% for rat, dog and human hepatocytes, respectively. The cells were distributed in freezing vials in 1.6 ml of medium per vial, trans- ferred at -20°C for 12 min then to a -80°C freezer for 1 hr and plunged in liquid nitrogen. For recovery storage, vials were warmed in a water-bath at 37°C until ice disappeared, and cell suspensions were gently pipetted. Each cell sample 479