A Simple Isolation and Cryopreservation Method for Adult Human Hepatocytes

Abstract

Objective The objective of this study was to establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for the treatment of acute and chronic liver diseases by hepatocyte transplantation and bioartificial liver support systems, and for the use of hepatocytes as an in vitro model of the liver. Methods Adult hepatocytes of 20 separate donors were isolated with a two-step extracoporeal collagenase perfusion technique. Seven preincubation time points (2h, 6h, 12h, 24h, 36h, 48h and 72h) were selected, then the hepatocytes were transferred to HepatoZYME-SFM medium containing 10% FBS and 10% DMSO, and were immediately put into an isopropanol progressive freezing container at -80 °C overnight and immersed in liquid nitrogen the next day. During the postthawing culture period, viability, plating efficiency, albumin secretion and urea synthesis were analyzed. Results The viability and plating efficiency of hepatocytes after partial hepatectomy using two-step extracorporeal collagenase perfusion technique were 75.0±4.6% and 72.0±6.0% respectively. Preincubation at 4°C for 12 hours or 24 hours proved to be the optimal time at which the albumin secretion was higher than at other time points (p<0.05). Compared to the immediate cryopreservation groups (IC), we also found significant improvement in viability (61.4±4.8%/62.0±5.6% vs. 53.4±4.2%, p<0.05), plating efficiency (63.2±5.8%/62.6±3.6% vs. 55.2±4.6%, p<0.05), albumin secretion and urea synthesis (p<0.05) at these time points. Conclusions The two-step extracorporeal collagenase perfusion technique after partial hepatectomy provides a novel, simple, and reliable method for hepatocyte isolation. The results of the present study suggest that recovery of human hepatocytes after isolation preincubation at 4 °C for 12 hours to 24 hours prior to cryopreservation can obtain hepatocytes ideal for use in pharmacotoxicology bioartificial liver and cell therapy research purposes.