Cryopreservation of adult rat hepatocytes

Abstract

Human hepatocytes in suspension or in primary culture provide an useful model for xenobiotic metabolism study and toxicity assays. A major problem with studies of human liver is the limited supply of fresh normal human liver and its irregular availability. As a preliminary step to using human hepatocytes, various factors of cryopreservation procedure were investigated using adult rat hepatocytes. The viability assessed by trypan blue exclusion was optimal using dimethylsulfoxide at concentration of 10\%(v/v), an ultrarapid freezing procedure, and a quick thawing protocol at 37$^\circ$C. Using this method the recovery of hepatocytes was 68\% and the cell viability was 58\%. The postthaw viability was not varied until 35 days. Hepatocytes recovered from the ultrarapid freezing and thawing had a relatively high activity in ethoxyresorufin 0-deethylase compared to that of the fresh hepatocytes during 4-hr suspension culture. 7-Ethoxycoumarin 0-deethylase and pentoxyresorufin 0-dealkylase activities were similar to those of the freshly prepared suspension culture. 7-Ethoxycoumarin was used as a substrate to measure integrated xenobiotic metabolizing activity by hepatocytes on suspension culture. Total 7-hydroxycoumarin formation, a mixed function oxidase activity, was maintained in cryopreserved hepatocytes at 95\% and conjugate formation at 35\% of activity in fresh hepatocytes. It is concluded that cryopreservation offers a way of storing rat hepatocytes, at least, for short-term toxicity studies performed on suspension culture system.