Cryopreservation of Porcine Hepatocyte Cultures

Abstract

Cryopreservation of freshly isolated hepatocytes is regarded the standard procedure for long term storage of liver cells. However, a dramatic loss in cell number, viability, and differentiated cell function is usually inevitable, and, upon thawing, hepatocytes do not attach well in culture. Freezing of hepatocyte cultures has already been shown to reduce these hazardous effects, probably due to the beneficial impact of a repair phase that allows the cells to restore their membranes during the first days of culture prior to and after freezing. We have compared the performance of porcine hepatocytes from slaughterhouse organs that were frozen at day 3 of immobilizing gel culture and stored at −80°C for increasing time intervals up to 30 days (three groups;n= 3 each). Control groups consisted of batch identical hepatocyte cultures, either frozen at a fixed cooling rate (−1°C/min;n= 3), subjected to rapid freezing (−10°C/min;n= 3) or left without any freezing (n= 4). Upon thawing, light and electron microscopic studies revealed a cell survival rate of 70.3 ± 10.5% (mean ± SD;n= 9) in hepatocyte cultures after adjusted rate-controlled freezing. In these groups, the length of storage time did not influence results. Functional parameters, like albumin secretion values and ethoxycoumarin deethylase activity (cytochrome P450-IA1) were comparable to non–frozen cultures after a recovery time of 48 h post-thaw. Fixed cooling rates at −1°C and −10°C, respectively, showed a greater reduced viability (35.4 ± 7.4%; 9.87 ± 2.8%) and a severely impaired post-thaw functional performance of cultures. Cryopreservation of hepatocyte cultures could provide a practical means of establishing a bank of hepatocyte cultures. This could be extremely useful for the development of a hybrid artificial liver device, and it might support repeated investigations on batch-controlled hepatocyte cultures, especially with respect to human cells.