Isolated Mice Hepatocyte For Primary Culture And Morphologic Studies Of Hepatocytes In Nutrient-Different Medium

1. Isolation and cultivation of primary mice hepatocytes:Objective: The purpose of this study was to search a simplified, inexpensive perfusion method in isolating mice hepatocytes.Methods: Liver cells of adult male Kun-Ming mice with in situ portal vein collagenase perfusion were used. The primary hepatocytes were put into RPMI 1640 medium supplemented with 10% fetal calf serum (PCS) and cultured in monolayer. The viability of the isolated hepatocytes was determined by trypan blue exclusion. The morphological change of the primary hepatocytes was investigated with inverted phase contrast microscopy.Results: 6.58X106 hepatocytes were obtained from per mice An average viability was 64.1%.The viable hepatocytes were plated in culture bottle with 10% PCS RPMI 1640 with penicillin, streptomycin medium. We examined the cells began to adhere 12 hr after the cells inoculated. The pseudopod were determined at 48 hr of culture. These cells adhered displayed typical epithelial cells morphological characteristics: the hepatocytes had a rich cytoplasm and were sometimes binucleate. We also observed the cell-cell contactthrough the pseudopod. These cells can be kept alive up to 1 week in 10% PCS RPMI 1640 medium, The cell contraction or size-minimized was determined after 5 days of culture and these cells began to fall from the bottle wall after 1 week. Consequently, the hepatocytes died or were replated by nonparenchymal cells.2. Morphologic studies of hepatocytes in different nutritive elements medium.Objective: This study was designed to determine if different nutritive elements, such as transferrin, insulin, nicotinamide, P -mercaptoethanol ( -Me) and hepatocytes growth factor (HGF) affected the morphology of the primary hepatocytes in short term culture.Methods: Hepatocytes obtained from mice with in situ portal vein collagenase perfusion were inoculated on two types of medium, which one was 10% PCS RPMI 1640 medium (common medium), the other was RPMI 1640 medium supplemented with 5 u g/ml tranferrin, 5 u g/ml insulin, 10nm nicotinamide, 5mM -Me, 40 u g/ml HGF ( special medium ). The morphological change of the primary hepatocytes in different medium was observed under an inverted phase contrast microscopy.Results: Hepatocytes adhesion was examined at 12 hr of culture. The number of adherent cells in special medium was more than that in common medium, but the morphologic difference between two groups was not significant at that time.The difference became significant after 24 hr of culture. Hepatocytes in common medium were triangle or spindle in shape and small in size. The number of non-viable cells was larger than in special medium. These cells displayed metabolic disturbance, such as rough cytoplasmic granules, many vacuolation. Nearly all cells in the common medium showed cytoretraction and pyknotic nuclei, and were detached from the plate after 5-7 days of culture. While most hepatocytes in special medium showed well condition. These cells were polygon and displayed morphologic characteristics of liver parenchymal cells at 24 hr of culture, such as a large round nucleus with a few nucleoli and many cytoplasmic granules, sometimes binucleate, hepatocyte was in direct contact with adjacent cells. Hepatocytes still grew well after 10-14 days of culture. Prolonged the culture term, hepatocytes both of experimental groups and control groups became pyknotic and were detached from the wall or fibroblasts became prominent in the culture plate. It was important to note that none of the condition caused an increase in the number of cell in the whole experimental process.Conclusion: The primary hepatocytes in medium of additional special nutritive may benefit in comparison with common medium.