Isolation,Culture and Identification of Hepatocytes of Primary Rat

Objective To investigate a simple and feasible method for primary rat hepatocytes isolation and culture,and to identify its purity and biological activity. Methods Primary SD rat hepatocytes were isolated by using a modified two-step in-situ non-circulating perfusion method followed by multiple filtration and low-speed centrifugation.The hepatocytes were cultured in Dulbecco′s modified Eagle′s medium with high level of glucose.The viabilities of cells evaluated by Trypan blue exclusion before cells were seeded.Cellular morphological changes were observed using an inverted microscope.Periodic acid Schiff(PAS) staining was performed to evaluate the status of hepatic glycogen storage,combining with the immunocytochemical staining targeting cytokeratin 18(CK-18) to identify the purity of hepatocytes. Results A total of(1.38-1.74)×108 hepatocytes per rat were obtained.The viabilities of hepatocytes were more than 90% when seeded.The hepatocytes with enlarged and flattened round shape were attached to the surface of collagen-coated dishes 4 hours after seeding and then connected with surrounding cells forming islands or cords.Glycogen preserved in hepatocytes was dyed purple using periodic-acid-Schiff staining.Through the PAS and anti-CK-18 immunocytochemical staining of hepatocytes,the purity of hepatocytes was more than 95%. Conclusions Primary rat hepatocytes can be successfully isolated and cultured using a modified two-step in-situ non-circulating perfusion method,which is highly feasible.The quantity,viability and purity of cultured hepatocytes were high.This method is available to perform in labs with basic cell culture conditions.