Primary Culture And Characterization Of Human Peritoneal Mesothelial Cells

Objective To establish a modified enzymatic disaggregation method to isolate human peritoneal mesothelial cells in order to establish a invasion model of peritoneal metastasis in vitro .Method Human greater omenta was enzymatic disaggregated with 0.1%trypsin—0.02% ethylene diaminetetraacetic acid (EDTA), and cultured after isolation from red blood cells. Morphologic changes were deteted through downward microscope during cell culture. Morphologic types were observed by HE staining and the purity was calculated. The ultrastructure was observed by scanning electron microscopy(SEM). Isolated cells were characterized by immunohistochemical analysis. Cell growth under culture mediums contained different ingredients were determined by the dimethylthiazolzyl diphenyl tetrazolium bromide (MTT )assay.Result Cultured cells were multipolar and presented a cobblestone-like appearance when they reached confluence, with a purity 96%. SEM verified the abundant microvilli on the surface of the cells. Immunohistochemical studies showed positive staining for cytokeratin and vimentin, but negative staining for white blood cell CD45 antigen and factor Ⅷ associated antigen. All the characters of the isolated cells were coincided with mesothelial cells. Hydrocortisone(0.4%) and insulin(10μg/ml) had a exact effect to stimulate the growth of mesothelial cells.Conclusion Trypsin—EDTA enzymatic disaggregation method is a simple, effective and repetitive protocal for isolation of human peritoneal mesothelial cells. RPMI—1640, calf serum(20%), hydrocortisone(0.4%) and insulin(10μg/ml) are effective and cheap medium ingredients to culture human peritoneal mesothelial cells.