Most previous methods of isolating KCs included two-step collagenase-pronase perfusion followed by gradient centrifugation. Although these methods provide certain numbers of KCs with reasonable purity, they require sophisticated skills, equipment and tedious cell isolation procedures. Furthermore, although the expansion of KCs has been demonstrated by zymosan stimulation, recombinant GM-CSF stimulation or two-thirds partial hepatectomy experimental models, few studies have attempted to demonstrate the self-renewal and subculture ability of normal KCs in vitro. Therefore, the aims of this study are to establish a simple and efficient method to isolate KCs as well as further investigate the mitotic potential of normal KCs in vitro. Most previous methods for KCs isolation utilized two-step collagenase perfusion in situ which were skillful and needed large amount of collagenase. The liver tissue often treated with pronase to eliminate parenchymal hepatocytes, however, pronase may destroys the lipopolysaccharide receptor CD14 on KCs. Finally, obtained cells by percoll density gradient differential centrifugation, though the procedures are sophisticated and tedious. In our protocol, we utilized one-step PBS perfusion in situ, the main purpose of which was to free the hepatic sinusoids from circulation blood cells. Overdigestion resulted in a low yield of viable cells, while underdigestion would made it difficult to separate the cells. The liver is 
comprised of parenchymal hepatocytes and non-parenchymal cells, and density as well as cell size are significantly different between the cell types. Due to this fact, we obtained the non-parenchymal cell fraction via differential centrifugation and cultured it. To purify KCs, we used the method of selective adherence to plastic, which is one of KC’s basic biological characteristics. Previous studies have demonstrated that hepatic non-parenchymal cells, such as sinusoidal endothelial cells, KCs and hepatic stellate cells, share certain biological characteristics, such as adhering to glass. However, the attachment time were significantly different. After cultivation for 2 h, we rinsed the culture plates with PBS to eliminate possible contamination with other hepatic non-parenchymal cell types. Adherent KCs were selectively harvested with high purity, which is similar to Valatas’ study using traditional methods. The origin of KCs remains controversial. Certain studies indicated that KCs are derived from blood monocytes and precursor cells in bone marrow and that, as a type of mature differentiated cells, are unable to divide. However, the expansion of KCs had been demonstrated by zymosan stimulation, recombinant GM-CSF and two-thirds partial hepatectomy experimental models. Consistent with the latter results, we also found that isolated KCs showed vigorous self-renewal and subculturing potential.