Effect of Spheroid Aggregation on Susceptibility of Primary Pig Hepatocytes to Cryopreservation

Abstract

Bioartificial liver support systems, which use freshly isolated primary hepatocytes (PH), present severe logistical difficulties. Stored frozen PH that are thawed as required could answer this problem. The aim of this study was to develop a cryopreservation protocol for large-scale preparation of porcine PH. We cryopreserved single and spheroid hepatocytes. Harvested hepatocytes were cryopreserved in various concentrations of dimethyl sulfoxide (DMSO) using hormonally defined medium (HDM) and various presentation solutions, such as University of Wisconsin (UW) and fetal bovine serum. After thawing the hepatocytes, we measured the viability, plating efficiency, ammonia removal, urea synthesis, and albumin secretion. UW solution was most effective for cryopreservation, as evidenced by the viability and liver-specific functions of the thawed hepatocytes. The optimal DMSO concentration for porcine hepatocyte cryopreservation was 15%. After cryopreservation, spheroid hepatocytes maintained greater viability and functional activity compared with single hepatocytes. Moreover, spheroid hepatocytes showed native cell structures and maintained high levels of liver-specific functions. In conclusion, cryopreserved spheroid hepatocytes were superior to cryopreserved single hepatocytes in terms of viability and liver-specific functions.