Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation

Abstract

Little work in the literature focuses on the cryopreservation of primary hepatocytes as monolayer cultures, yet this technique offers many distinct advantages over other cryopreservation systems, including high recovery, high post-thaw nutrient penetration, and low numbers of trapped dead cells. This article investigates the cryopreservation of primary rat hepatocytes at −78 °C attached as monolayers to collagen coated culture dishes, and describes efforts to increase post-thaw viability and function through manipulation of the freeze/thaw protocol. Different concentrations of foetal calf serum (FCS) with 10% (v/v) dimethyl sulphoxide (ME 2SO) were tested as cryopreservation media, and high cryoprotectant serum levels were found to be important in maintaining membrane integrity and function in the cryopreserved rat hepatocyte monolayer cultures. Cultures cryopreserved with 90% (v/v) FCS plus 10% (v/v) ME 2SO maintain 79.7 ± 6.5% of the monolayer area as viable cells with normal morphology (by image analysis), 112.7 ± 14.2% protein concentration, 55.4 ± 4.2% carboxyfluorescein diacetate de-acetylation, 27.2 ± 7.5% kaempherol glucuronidation (a measure of UDP-glucuronosyl transferase activity), and 39.3 ± 7.3% testosterone hydroxylation (a measure of cytochrome P-450 activity) compared with non-cryopreserved controls. This method of cryopreservation may provide a simple, convenient means of long-term storage of hepatocytes for in vitro metabolism studies.