OBJECTIVETo establish a stable method of isolation, culture and cryopreservation of adult primary hepatocytes to provide potential hepatocyte resources for therapeutic usage in hepatocyte transplantation and bioartificial liver support systems for the treatment of acute and chronic liver diseases,and for experimental usage as an in vitro model of the liver.

METHODSAdult hepatocytes from 20 human donors undergoing partial hepatectomy were isolated using a two-step extracoporeal collagenase perfusion technique.Seven preincubation time points (2h,6h,12h,24h,36h,48h and 72h) were selected for optimization.After pre-incubation at 4 degrees C for 12-24h in HepatoZYME-SFM (the optimal condition),hepatocytes were microencapsulated using alginate-poly-L-lysine-alginate microcapsules,transferred to a complete medium containing 10% dimethyl sulphoxide and immediately placed into an isopropanol progressive freezing container for overnight freezing at -80 degrees C followed by immersion in liquid nitrogen the next day.During the post-thawing culture period,the cells were tested for albumin secretion,urea synthesis,cell cycling,transcription and protein synthesis (measuring mRNA and protein levels),and the morphological structure and pathology,for comparison with the features from before microencapsulated cryopreservation (PMC).

RESULTSThe viability and plating efficiency of the hepatocytes isolated using the two-step extracorporeal collagenase perfusion technique were 75.0+/-4.6% and 72.0+/-6.0%,respectively.The pre-incubation times of 12h and 24h (viability:61.4+/-4.8% and 62.0+/-5.6%; plating efficiency:3.2+/-5.8% and 62.6+/-3.6%,respectively) showed significantly higher albumin secretion than all other time points tested (F =40.3,all P less than 0.05).Compared with the immediate cryopreservation (immediately frozen control) hepatocytes,the PMC hepatocytes showed significantly better transcription and protein synthesis and higher albumin secretion and urea levels.The PMC group did not show a significantly different level of albumin production from the directly cultured hepatocytes (culture day 2:ll9.2ng/ml vs.131.36ng/ml,P =0.051; day 3:110ng/ml vs.120.4ng/ml,P=0.063; day 4:98.2ng/ml vs.109.8ng/ml,P more than 0.05).However,over culturing days 2,3 and 4,comparison of the PMC hepatocytes to the immediate cryopreservation hepatoeytes showed the former to have significantly higher secretion of albumin (119.2ng/ml vs.101.2ng/ml,110.0ng/ml vs.87.6ng/ml and 98.2ng/ml vs.73.8ng/ml; all P less than 0.05) and urea level (7.83 mug/ml vs.6.79 mug/ml,6.83 mug/ml vs.5.89 mug/ml and 5.85 mug/ml vs.4.83 mug/ml; all P less than 0.05).The post-thawed PMC hepatoeytes showed preservation of the morphological structure,while the immediate cryopreservation hepatocytes did not.

CONCLUSIONThe two-step extracorporeal collagenase perfusion technique after partial hepatectomy is a novel,simple,and reliable method for hepatocyte isolation.Pre-incubation at 4 degrees C for 12-24h before the microencapsulation cryopreservation allows for efficient recovery of functional and morphological integrity after thawing and provides viable hepatoeytes that may be useful for clinical applications in pharmacotoxicology,bioartificial liver therapy and cell therapy in humans.