CRYOPRESERVED HUMAN HEPATOCYTES FROM CELL BANK: IN VITRO FUNCTION AND CLINICAL APPLICATION

Abstract

P658 Aims: the development of a National Bank of Human Hepatocytes, isolated from livers unwanted for transplantation at harvesting, allows to obtain live hepatic tissue usable for in vitro and clinical evaluation of cell therapy and transplantation and bioartificial organs. The aim of this study is to analyze in vitro function of isolated and cryopreserved human hepatocytes from cell Bank and define their potential clinical application in a bioartificial liver (BAL) device. Methods: 103 not transplantable livers were utilized for human hepatocytes isolation and cryopreservation from July 2001 to December 2003 in the Nord Italia Transplant area (NITp and AIRT). Hepatocytes were isolated by collagenase-enzymatic digestion. Isolated hepatic cells were analyzed in terms of yield, viability (tripan blue and XTT test), diazepam metabolism (cyt P450 activity) and production of human albumin (immunonephelometry and mRNA by RT-PCR) after isolation (fresh hepatocytes) and after cryopreservation (10% DMSO) in liquid nitrogen. Results: macrosteatosis >60%, ischemic damage due to prolonged donor hypotension or cardiac arrest and non-viral cirrhosis were the causes of refusal of the liver for transplantation respectively in 60%, 11% and 8% of cases. Cell yield averaged 7 million of hepatocytes per gram of liver tissue digested; mean viability was 80%±13%. The best results in terms of cell yield and viability were obtained from steatotic organs, while cirrhotic livers yielded less cell/gr of liver (3x106 vs 7x105 p<0.043 and 81% vs 66% p=0.27, respectively). Viability of cryopreserved hepatocytes after thawing averaged 50%. However thawed hepatocytes had a normal diazepam metabolism (fig 1) and human albumin synthesis was comparable to that of fresh cells (fig 2).FigureFigureCryopreserved human hepatocytes (CHH) were utilized as the biological component of a BAL device for temporaneous support of two patients affected by FHF awaiting for urgent transplant. A total of 3 BAL applications were performed. Ten to 13 billion viable CHH have been loaded in each BAL. No adverse events were recorded and vital functions remained table during the procedures. Biochemical liver functions showed bilirubin and ammonia reduction at the end of each treatment with CHH based BAL. One patient was successfully bridged to emergency OLTx performed after a single BAL; in the second case there was a spontaneous recovery of liver function after two BAL treatment and surgical arterialization of the portal vein. Both patients are still alive and well at home. Conclusions: the recovery of donor human livers unwanted for transplantation at harvesting, allowed isolation and cryopreservation of viable and functionally active human hepatocytes which have been banked and successfully used for clinical application of a BAL device.