Assessment of in vitro applicability of reversibly immortalized NKNT-3 cells and clonal derivatives

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