Background: A clonal cell line that combines both stable hepatic function and proliferation capacity is desirable for in vitro applications that depend on hepatic function, such as pharmacological or toxicological assays and bioartificial liver systems. Here we describe the generation and characterization of a clonal human cell line for in vitro hepatocyte applications.

Anno 2004 freshly isolated or cryopreserved porcine or human hepatocytes have been most frequently used as bio-component in clinically applied bioartificial livers (BALs). Phase 1 studies of all bio-component modalities showed safety, feasibility, and improvement of biochemical, neurological, and hemodynamic parameters. However, both the pilot-controlled clinical trial with C3A cells and the randomized larger clinical trial with cryopreserved porcine hepatocytes did not show significant improvement of survival by intention-to-treat analysis. Because of the xenotransplantation-related disadvantages of porcine cells and the shortage of primary human hepatocytes, other sources of bio-components have to be explored. The future lies in the development of one or more human hepatocyte cell lines, which will have minimal immunogenicity, no risk of xeno-zoonosis, and the requested functionality and availability. Primary sources for the development of such human cell lines are liver-tumor-derived cell lines, immortalized fetal or adult hepatocytes, and stem cells of hepatic, hematopoietic, or embryonic origin. At present the most promising results for BAL application have been obtained by immortalization of human fetal liver cells by reconstitution of telomerase activity. However, in all cell types tested so far, the in vitro differentiation cannot be stimulated to such an extent that their functionality reaches that of primary human hepatocytes. More insight in differentiation-promoting factors and the influence of matrix and co-culture conditions is needed.

Acute liver failure (ALF) is a disease with a mortality of 60–90% depending on the cause. Only high-urgency liver transplantation is able to increase survival compared to standard intensive care therapy. Liver transplantation is hampered by the increasing shortage of organ donors, resulting in a high incidence of patients with ALF dying on the transplantation waiting list. Amongst a variety of liver assist therapies, bioartificial liver (BAL) therapy is marked as the most promising solution to bridge ALF patients to liver transplantation or to liver regeneration, since several BAL systems showed significant improvement of survival time in experimental animals with irreversible ALF. One of these systems has been developed at the Academic Medical Center in Amsterdam, The Netherlands – the AMC-BAL. This overview describes the development of the AMC-BAL based on porcine hepatocytes which was started 10 years ago. Positive results of in vitrofunctionality and in vivo safety and efficacy led to a successful phase I study in 12 ALF patients in Italy. However, xenotransplantation legislation in many European countries prohibits the use of porcine hepatocytes in clinically applied BAL systems. The future of the BAL, therefore, resides in the development of a human-derived hepatocyte cell line as biocomponent of BAL systems.

Mature human hepatocytes are not suitable for large-scale in vitro applications that rely on hepatocyte function, due to their limited availability and insufficient proliferation capacity in vitro. In contrast, human fetal liver cells (HFLC) can be easily expanded in vitro. In this study we evaluated the hepatic function of HFLCs under proliferative conditions, to determine whether HFLCs can replace mature hepatocytes for in vitro applications. HFLCs were isolated from fetal livers of 16 weeks gestation. Hepatic functions of HFLCs were determined in primary culture and after expansion in vitro. Clonal derivatives were selected and tested for hepatic functionality. Results were compared to primary mature human hepatocytes in vitro. No differences were observed between primary HFLCs and mature human hepatocytes in albumin production and mRNA levels of various liver-specific genes. Ureagenesis was 4.4-fold lower and ammonia elimination was absent in HFLCs. Expanding HFLCs decreased hepatic functions and increased cell stretching. In contrast, clonal derivatives had stable functionality and morphology and responded to differentiation stimuli. Although their hepatic functions were higher than in passaged HFLCs, functionality was at least 20 times lower compared to mature human hepatocytes. HFLCs cannot replace mature human hepatocytes in in vitro applications requiring extensive in vitro expansion, because this is associated with decreased hepatic functionality. Selecting functional subpopulations can, at least partly, prevent this. In addition, defining conditions that support hepatic differentiation is necessary to obtain HFLC cultures suitable for in vitro hepatic applications.

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In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4-20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.

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