Experience in Primary Culture of Human Peritoneal Mesothelial Cell

To compare the growth condition between different sources and different culture environments, mesothelial cells were isolated from omentum and peritoneal dialysate effluent (PDE), seeded at different densities (5×105, 1×105, 5×104, 1×104, 5×103, 1×103 and 5×102 cells/cm2, respectively), supported with different fetal calf serum (FCS) concentrations (3%, 6%, 10% and 15%) and grown in dishes with and without gelatin pre-coating. Growth condition was evaluated by simple morphological observation. Cells phenotype was examined by immunofluorescent staining. The results showed that omentum-derived mesothelial cells generally showed a uniform growth pattern with good quality. Alternatively, there was a wide patient-to-patient variation in PDE-derived culture. Heterogeneous colonies composed of a mixture of large, small or abortive mesothelial colonies as well as fibroblastoid colonies were frequently observed. A minimum seeding density of 5 × 103 cells/cm2 is required for the omentum-derived mesothelial cells to grow to confluent monolayer (1-5×104 cells/cm2 for initial culture from fresh PDE). Appropriate seeding density is always associated with successful culture in omentumbased culture, but not in PDE-based culture. Mesothelial cells could grow to confluency regardless of FCS concentration and gelatin pre-coating. However, growth rate was slower in lower FCS concentrations and on dishes without gelatin coating. Most cells in culture expressed cytokeratin and vimentin, but not VWF. Alpha-smooth muscle actin frequently appeared in cytokeratin+ mesothelial cells, especially in higher FCS concentrations and in PDE-derived culture. Our data demonstrate that PDE, in contrast to omentum, provides a source of mesothelial cells with poor and unstable quality for primary culture. Healthy cell quality and sufficient seeding density seem to be the most important factors for successful culture of mesothelial cells. The frequent occurrence of epithelial-to-mesenchymal transition in cultured mesothelial cells indicates the feasibility of mesothelial cells to undergo phenotype change upon environment changes, especially following chronic exposure to uremic environment and dialysate in peritoneal dialysis patients.