Liver cell transplantation: basic investigations for safe application in infants and small children.

Liver cell transplantation (LCT) is a very promising method for the use in pediatric patients. It is significantly less invasive than whole organ transplantation, but has the potential to cure or at least to substantially improve severe disorders like inborn errors of metabolism or acute liver failure. Prior to a widespread use of the technique in children, some important issues regarding safety and efficacy must be addressed. We developed a mathematical model to estimate total hepatocyte counts in relation to bodyweight to make possible more appropriate dose calculations. Different liver cell suspensions were studied at different flow rates and different catheter sizes to determine mechanical damage of cells by shear forces. At moderate flow rates, no significant loss of viability was observed even at a catheter diameter of 4.2F. Addition of heparin to the cell suspension is favored, which is in contrast to previous animal experiments. Mitochondrial function of the hepatocytes was determined with the WST-1 assay and was not substantially altered by cryopreservation. We conclude that especially with the use of small catheters, human LCT should be safe and efficient even in small infants and neonates.

Enzyme induction in cryopreserved human hepatocyte cultures.

Freshly isolated human hepatocytes are considered as the gold standard for in vitro testing of drug candidates. Meanwhile also cryopreserved human hepatocyte suspensions are available. However, a drawback of these cells is the incalculability of attachment to the culture dish. Therefore, we established a technique freezing hepatocytes cultured on a collagen gel. After thawing damaged cells were removed to a certain extent by gentle washing with culture medium prior to adding an upper gel layer. The morphology of the resulting hepatocyte cultures could not be distinguished from that of non-frozen cells. However, basal activities of cytochrome P450 isoforms decreased in cryopreserved compared to non-frozen hepatocytes, as evidenced by analysis of testosterone hydroxylation (OHT) in positions 6beta, 16alpha, 2beta and 6alpha. Nevertheless, enzyme induction factors caused by 24 h incubation with 50 microM rifampicin were similar in cryopreserved and non-frozen hepatocytes. In cryopreserved hepatocytes rifampicin caused an increase in mean values of 6beta-OHT formation from 57.2 to 157.7 pmol/well/min (2.8-fold), compared to an increase from 115.8 to 269.1 pmol/well/min (2.3-fold) in non-frozen cells. Similarly, 16alpha- and 2beta-OHT showed induction factors of 2.4- and 2.3-fold in cryopreserved compared to 1.6- and 2.4-fold in non-frozen hepatocytes, respectively. In conclusion, human hepatocytes cryopreserved on collagen gels show a clear induction of CYP3A4 by rifampicin, although the basal activities are reduced compared to non-frozen cells.

Large-scale isolation of human hepatocytes for therapeutic application.

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 x 10(9) cells per organ, the cell preparations displaying a mean viability of 64%. The mean specific yield was 3.6 x 10(6) total and 2.6 x 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.