XENOBIOTIC-METABOLIZING ENZYME-ACTIVITIES IN ISOLATED AND CRYOPRESERVED HUMAN LIVER PARENCHYMAL-CELLS

Liver parenchymal cells (hepatocytes) of human organ donors were isola ted using a two-step collagenase perfusion technique. The average viab ility of the freshly isolated liver parenchymal cells, as judged by tr ypan blue exclusion, was 82% (SD = 7%; n = 6). The inter-individual di fferences in the determined enzyme activities were less than a factor of 7.5, despite the different sexes and ages of the donors. Freshly is olated parenchymal cells (PC) were cryopreserved using a computer-cont rolled freezing protocol. After thawing, cryopreserved cells had a mea n viability of 57% (SD = 18%; n = 6). The activities of xenobiotic met abolizing enzymes in freshly isolated and cryopreserved cells were com pared using PC from two donors. The enzyme activities of phenol sulfot ransferase, 1-naphthol UDP-glucuronosyltransferase and microsomal epox ide hydrolase were well maintained after thawing (87-117% of activitie s in freshly isolated cells), whereas the activities of glutathione S- transferase, monitored with the broad spectrum substrate 1-chloro-2,4- dinitrobenzene, and the major broad spectrum cytosolic epoxide hydrola se were moderately but markedly reduced after cryopreservation (34-64% and 45-89% of levels in fresh cells, respectively). The decrease of b oth activities was dependent on the viability after thawing. When cryo preserved cells were purified by a Percoll centrifugation after thawin g, the viability was increased from 67 to 92% for cells from one of th e donors and from 88 to 98% for PC for the other donor. Subsequently t he activity of glutathione S-transferase in Percoll-purified PC from t he two donors was increased to 71 and 96% of levels in freshly isolate d cells. It is concluded that the use of cryopreserved liver parenchym al cells of humans and other species represents a valuable tool in pre dicting which animal species best represents humans in hepatic metabol ism and therefore should be the preferred species for investigations o f metabolism and metabolism-dependent toxicities.