In Vitro Isolation, Culture And Intraspleen Transplantation Of Hepatic Oval Cells In Rats

Hepatic oval cells possess potent proliferating capacity and multipotential differentiating capacity, and reveal promising prospects in the treatment of hepatic failure. In this study, the rat model for hepatic oval cell proliferation was reestablished using the proceduce proposed by our laboratory, hepatic oval cells were isolated and cultured in vitro, and the intraspleen transplantation of hepatic oval cells was made in homogenous rats. The aim of this study is to observe the evolution and differentiation of hepatic oval cells in vivo and thus to provide experimental data for treating hepatic failure with hepatic stem cells. Male Wistar rats weighing 150g received daily oral gavage of AAF for 4 days before and up to 7 days after two-thirds hepatectomy. Two-thirds hepatectomy was performed after first 4 daily gavages and no dosing was performed on the day of surgery. Animals were dosed at 20mg/kg body weight. Obvious oval cell proliferation was observed during 10-16 days after surgery. Two-step perfusion was used to separate hepatic parenchymal cells and nonparenchymal cells and then, nonparenchymal cell suspension was loaded into Percoll gradients for centrifugation. The cell layer between 50% and 70% Percoll gradients was hepatic oval cells. Approximately 1.69×105 cells/ml were obtained from each rat and the viability of the cells was 95%. Image analysis showed that the average diameter of hepatic oval cells was (13±3.6)μm, theaverage area of the cells was (23±7.9) square micron, and the form factor of the cells was (1.9±1.1). The freshly isolated hepatic oval cells vary in size posses ovoid nuclei and scanty cytoplasma. After they were cultured 12 hours, hepatic oval cells proliferated actively and became larger, revealing the characteristics of epithelial cells. The freshly isolated and cultured hepatic oval cells were stained positive for cytokeratin 19, OV6, AFP and negative for leucocyte common antigen (LCA). Electron microscopy revealed that the cells had larger nucleus/cytoplasma ratio, a few short microvilli on the cell surface and few organell in the cytoplasma, revealing the characteristics of immature and undifferentiated cells. After transplantation, hepatic oval cells differentiated into liver tissue structure including hepatocyte cords and bile ducts, but transplanted hepatocytes could not evolve into bile duct structure.