Cryopreservation of Immobilized Rat Hepatocytes for the Development of a Bioartificial Liver System

Liver transplantation, the only effective treatment for end-stage disease, is limited by donor availability. Cell transplantation offers the possibility to restore liver mass and function. Cryopreserved primary hepatocytes that can be thawed as needed might address this problem. Hepatocytes were harvested from a male Sprague Dawley rat, weighing approximately 250 g, using a 2-step in situ collagenase perfusion technique modified from the method described by Seglen. Hepatocytes were immobilized using a 100-mmol/L calcium with 1.5% alginate solution. Primary, immobilized hepatocytes transferred to various cryopreservation solutions containing 15% dimethyl sulfoxide were immediately placed into an isopropanol progressive freezing container at −80°C. We analyzed 4 cryopreservation solutions: Hormonally defined medium, histidine-tryptophan-ketoglutarate (HTK), fetal bovine serum (FBS), and HTK-modified cryopreservation solution (JH). After thawing, we measured viability, plating efficiency, urea synthesis, and albumin secretion to assess the effects of cryopreservation. Primary hepatocytes in HTK solution showed the better results in hepatocytes viability and urea synthesis after thawing. Cryopreserved immobilized hepatocytes in FBS maintained their viability and urea synthesis function. However, JH seemed to be the most effective medium for albumin secretion by both cyropreserved primary and immobilized hepatocytes.Our study suggested that HTK and JH cryopreservation solutions without FBS can be used to develop a bioartificial liver system.