Morphology, Proliferation and Apoptosis of Mouse Liver Epithelial Cells Cultured as Spheroids

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Abstract
The MLEC10 is an epithelial cell line derived from an untreated, normal C3H/HeN mouse liver. We previously demonstrated that tumorigenic variants from this cell line produced moderately differentiated hepatocellular carcinomas in nude mice. However, it has remained unclear whether the parental MLEC10 cells represent immortalized hepatocytes or so‐called oval cells, both of which may serve as precursors for hepatocellular neoplasms. In this study, we performed 3‐dimensional, spheroid culture of the MLEC10 cells in order to facilitate histological assessment of their lineage. Spheroidal aggregates were formalin‐fixed and embedded in paraffin for routine light‐microscopic observation of hematoxylin and eosin‐stained sections. Histopathologically, the MLEC10 cells were indistinguishable from immature hepatocytes and distinct from oval cells. At the electron‐microscopic level, their hepatocytic nature was evidenced by bile canaliculus structures and glycogen storage. Intriguingly, the spheroids contained fragmentary material reminiscent of Councilman bodies, implying apoptosis of the hepatocytes. Although the cells significantly proliferated during the first three days of culture, apoptotic death then resulted in a 75 % decrease in viable cell number. Thereafter, both apoptosis and cell division appeared silent, the numbers being unchanged. Expression of the p53 tumor suppressor gene became gradually elevated, correlating positively with growth arrest, but negatively with apoptosis, suggesting that the cell death occurred independently of p53. Our results indicate that at least some liver epithelial cell lines derived from untreated murine livers exhibit a hepatocytic morphology in spheroid culture. Also, the present culture system provides a useful tool for investigating biological phenomena, e.g. apoptosis, specifically involving liver cells, under 3‐dimensional conditions.