Functional human hepatocytes: Isolation from small liver biopsy samples and primary cultivation with liver-specific functions (original) (raw)
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PLoS ONE, 2011
Successful and consistent isolation of primary human hepatocytes remains a challenge for both cell-based therapeutics/ transplantation and laboratory research. Several centres around the world have extensive experience in the isolation of human hepatocytes from non-diseased livers obtained from donor liver surplus to surgical requirement or at hepatic resection for tumours. These livers are an important but limited source of cells for therapy or research. The capacity to isolate cells from diseased liver tissue removed at transplantation would substantially increase availability of cells for research. However no studies comparing the outcome of human hepatocytes isolation from diseased and non-diseased livers presently exist. Here we report our experience isolating human hepatocytes from organ donors, non-diseased resected liver and cirrhotic tissue. We report the cell yields and functional qualities of cells isolated from the different types of liver and demonstrate that a single rigorous protocol allows the routine harvest of good quality primary hepatocytes from the most commonly accessible human liver tissue samples.
Isolation and culturing of hepatocytes from human livers
Journal of Tissue Culture Methods, 1992
Using a modified collagenase digestion procedure, we were successful in the isolation of viable hepatocytes from human liver surgical wastes, unused transplantable livers, and diseased livers from transplant recipients. The modified procedures for isolation involved collagenase perfusion of a sample of limited size (<50 g) with only one cut surface, perfusion via only one blood vessel, the use of an appropriate amount of collagenase, as well as manual "massaging" of the liver sample during critical stages of the collagenase perfusion. Using this modified procedures, we had close to 100% success rate in the isolation of viable hepatocytes from the human liver samples.
Large-Scale Isolation of Human Hepatocytes for Therapeutic Application
Cell Transplantation, 2005
During the last decade, hepatocyte transplantation has been suggested as a safe and potentially effective clinical option for the treatment of acute or decompensating chronic liver failure as well as for hereditary liver disease. Currently, one of the major limiting factors for clinical application is the insufficient access to suitable liver cell preparations. In cooperation with the German and Catalane organ procurement organizations, a routine procedure for the isolation of hepatocytes from donor organs rejected for transplantation (n = 117) has been established. The process is performed according to the current EC Guidelines for Good Manufacturing Practice (cGMP) and all corresponding national laws and regulations concerning donor organ and tissue procurement. In about 50% of the cases (n = 58) the three-step perfusion procedure has been completed with an average total cell yield of 5.9 × 10 9 cells per organ, the cell preparations displaying a mean viability of 64%. The mean specific yield was 3.6 × 10 6 total and 2.6 × 10 6 viable cells per gram liver tissue, respectively. Specific cell yields from three infantile donor livers were considerably higher. No correlation between isolation efficiency and cold ischemia time or donor age was found within the adult organ donors. In contrast, organs with a severe steatosis generally did not result in successful cell isolation. Results of sterility and endotoxin determination are also presented. In summary, a standardized and cGMP conform method of hepatocyte isolation from nontransplantable liver organs was established, which reproducibly yields large amounts of hepatocytes suitable for therapeutic application.
State of the art on human hepatocytes: isolation, preservation and clinical use
Current Opinion in Organ Transplantation, 2006
Purpose of review The offer of liver transplantation to many patients affected by liver failure is limited by organ shortage. The clinical application of human-based liver cell therapies such as bioartificial liver and hepatocyte transplantation might support liver transplantation. In this review we focus on the most recent development in the isolation, preservation and clinical use of human hepatocytes. Recent findings We have performed a Medline search (www.pubmed.gov) focusing on the isolation, cryopreservation and clinical use of human hepatocytes. We have identified 90 papers published from 2002 to 2005; of these, 35 articles have been used for the purpose of this review. Summary At present technical developments and innovations in cell biology allow the isolation of a definite amount of live and functioning human hepatocytes from livers donated by heart-beating and very recently also non-heart-beating donors deemed unusable for organ transplantation. Despite good results in the process of isolation, with viability greater than 70%, the currently available technique of cryopreservation and thawing allows the recovery of only a fraction of the isolated cells. Some case reports and small series preliminarily showed that isolated human hepatocytes could be used for clinical hepatocyte transplantation and bioartificial liver treatment.