O0060 EFFECT OF SERA FROM CHILDREN WITH ACUTE LIVER FAILURE DUE TO NON A-E HEPATITIS ON CRYOPRESERVED HUMAN HEPATOCYTES

Abstract

Introduction: Acute liver failure (ALF) serum has been shown to have inhibitory effects on DNA and protein synthesis in cultured human HepG2 cells. Isolation and culture of hepatocytes has provided a source of functioning hepatocyte mass either for cell transplantation or for use in bioartificial liver assist devices but there are concerns about the effect of toxic substances accumulating in the ALF patients on the function of the hepatocytes. Our aim was to assess the effect of ALF sera from non A-E hepatitis patients on cryopreserved human hepatocytes. Methods: Cryopreserved human hepatocytes were thawed using a slow thaw technique. Collagen-coated 96 well culture plates were seeded with hepatocytes (30,000 cells /well) and incubated overnight at 37°C, 5% CO2. Sera obtained from 10 children with ALF due to non A-E hepatitis were pooled. The controls consisted of pooled sera from 10 age-matched normal children. The culture media was replaced with fresh media containing pooled normal or ALF sera at concentrations ranging from 0% to 80%. Plates were re-incubated for 24 h and cell activity and function were analysed using MTT, [14 C]-leucine incorporation and [3H]- thymidine incorporation assays. Results: There was no difference between the effects of ALF and normal serum on overall cell activity as determined by the MTT assay (20% ALF serum: mean 0.092 ± SD 0.01 OD units/well; 20% normal serum: 0.096 ± 0.004 OD units/well), and on the protein synthesis as assessed by [14C]-leucine incorporation assay (20% ALF: 94.9 ± 13.5 cpm/well; 20% normal sera: 97.1 ± 12.6 cpm/well). However, when the hepatocytes were incubated with both ALF and normal sera inhibitory effects were observed on DNA synthesis as assessed by the [3 H]-thymidine incorporation assay. The inhibitory effect of both normal and ALF sera increased with serum concentration, but greater effect was observed in the presence of ALF sera (20% ALF sera: 17.05 ± 3.9 cpm/well; normal sera: 229.87 ± 96.5 cpm/well; p<0.003). Conclusion: Sera from children with ALF due to non A-E hepatitis did not have toxic effects on overall cell activity and protein synthesis of cryopreserved human hepatocytes. As the main goal of hepatocyte transplantation or use of bioartificial livers in ALF is the immediate metabolic support, therefore the observed inhibitory effect of ALF sera on DNA synthesis on human hepatocytes is unlikely to be important.