302.1: 30 years of human hepatocyte isolation and cryogenic preservation

Introduction: Despite 30 years since the first clinical infusion and more than 150 patients treated so far, Hepatocyte transplantation is still considered an experimental procedure, characterized by fluctuating outcomes in congenital or fulminant diseases. Our laboratory has always been active in the translation of hepatocyte transplant technology from bench to clinic. Our 30 years of experience on more than 2,000 human livers allowed us to standardize reagents and procedures in accordance with Good Manufacturing Practice (GMP). We performed hepatocyte isolation and cryopreservation on organs rejected from transplantation, explanted organs, fetal and neonatal liver tissues. We developed and validated rapid and sensitive functional assays to ensure freshly isolated hepatocytes, as well as cryopreserved products, function adequately. Methods: Different surgical procedures and enzymatic solutions have been tested during the past decades, since coming to a new collagenase-protease mixture specifically designed for the isolation of human hepatocytes. Both DMSO-supplemented and DMSO-free cryopreservation solutions were tested. Data from viability/apoptosis, recovery to plating adhesion, and hepatic function including basal and induced Cytochrome P450 (CYP) activities, phase II conjugation, and ammonia metabolism were collected on fetal, neonatal, pediatric, and adult hepatocytes.z Results: Human hepatocytes were isolated from 1315 post-natal and 706 fetal livers. Viability significantly improved from the first clinical applications in the early 90s (64±1%, mean±SEM) compared to the clinical transplants performed during the last decade (88±2%). The viability of fetal hepatocytes was significantly higher (91 + 9%), both immediately after isolation and after cryogenic procedure. High viability was also obtained from neonatal livers (83 + 16%), while adult liver have generated wide range of quality cells with an average value of 70 + 24%. Cryopreservation procedure was successfully performed on young cells, but poorly effective on adult and steatotic hepatocytes. CYP activity increased during gestational and postnatal age, but remained 1-10% of adult values. Phase II-conjugation and ammonia metabolism were null/low in fetal hepatocytes, but increased in vitro and in vivo. Conclusions: A large spectrum of primary cells has been analyzed to determine a normal range of activities to expect from isolated cells. Data collected on multiple hepatic functions worked as quality control and form the basis for patients’ need match criteria. Measurements on prenatal and postnatal liver cells allowed us to determine the normal range of metabolic activities and synthetic capacity for donor liver cells, guiding stem cell-derived hepatocyte-like validation. Our long-term database can serve as quality control and form the basis for an auxiliary strategy to validate clinical products but also evaluate stem cell sources proposed as hepatocyte replacement.