Abstract: Objective To isolate and cryopreserve human hepatocytes from adult whole liver with heavy steatosis to provide human hepatocytes source for bio-artificial liver. Methods Primary human hepatocytes were isolated using a modified two-step collagenase retrograde perfusion via hepatic vein from adult whole livers with heavy steatosis. The isolated hepatocytes were under programmed and standard cryopreservation, respectively. The viability, adhesion rate, lactate dehydrogenase (LDH) releasing level and albumin synthesis ability were compared between the thawed hepatocytes with the 2 different cryopreservation methods.Results The average yield and viability of hepatocytes from livers perfused with N-acetylcysteine (NAC) was significantly higher than that without NAC [(7.4±0.5)×10⁶ cells/g vs. (5.6±0.8)×10⁶ cells/g and (81.4±3.4)%vs. (67.3±5.0)%, both P<0.05], respectively. Compared with hepatocytes under standard cryopreservation, hepatocytes under programmed cryopreservation showed significantly higher in the viability, adhesion rate, LDH release level and albumin synthesis ability (all P<0.05), respectively.Conclusion A modified two-step retrograde perfusion using collagenase with the NAC via hepatic vein could improve the yield and viability of isolated hepatocytes from adult whole livers with heavy steatosis. Besides, the programmed cryopreservation program could improve the quality of the isolated hepatocytes to meet the grossing need of hepatocyte for the bio-artificial liver.

Key words: Human hepatocytes, Isolation, Cryopreservation, Bioartificial liver