Organ Culture of Fetal Rat Liver by the Circumfusion System In Vitro Differentiation of Hepatocytes

Hepatocytes cultured in vitro offer one of the best experimental models for examining the complex pathophysiology of various liver diseases. Until recently, however, efforts to maintain differentiated hepatocytes in vitro met little success. In the present study, small pieces of liver tissue from fetal rats were cultured and examined by phase contrast microscopy, light microscopy, electron microscopy, enzyme histochemistry and radioautography. Liver specimens were obtained from fetal rats 15 to 21days after gestation. The culture was maintained by the Circumfusion System designed by G. G. Rose. The culture medium was a mixture of TCM-199 and calf serum (4:1). The following results were obtained. 1) Epithelial cells with features of both hepatocyte morphology and hepatocyte enzymic content were maintained in vitro. 2) Epithelial cells occupying the central parts of the cultured tissue showed the positive staining of GOT and G-6-Pase. On the other hand, epithelial cells at the periphery of the cultured tissue arising probably as an outgrowth of the planted tissues showed the positive staining of only GOT. Epithelial cells of the latter group were thought to be partially differentiated hepatocytes because of the continuity between the two types of epithelial cells. 3) Enzymic differetiation of G-6-Pase was accelerated when culture specimens were collected from older fetal rats, and a more marked enzymic differentiation was found in cultures continued for longer durations. 4) One of the mechanisms resulting in cell differentiation in vitro seemed to be inhibiting cell proliferation as demonstrated by the results from the radioautographical study.