Abstract:

BACKGROUND: Peripheral peritoneum mesothelial cells are served as an important component of peritoneum which secrete many kinds of cytokines, and play an important role in anti-inflammatory, immunoloregulation, and peritoneal fibrosis. How to obtain high-quality and homogeneous peritoneal mesothelial cells has become the key to solving problems.
OBJECTIVE: To develop an improved method for digestion culture of human peritoneal mesothelial cells (HPMCs).
METHODS: HPMCs were digested by 0.1% collagenaseⅠ, after eliminating erythrocytes, the cells were cultured by 10% fetal bovine serum of 1640 medium. Inverted microscope was used to observe the morphological structures of cells; CCK-8 was used to test the promotion of medium on the growth of mesothelial cell. The ultrastructure of cells was observed by transmission electron microscope (TEM), and isolated cells were identified by laser confocal immunofluorescence microscope.
RESULTS AND CONCLUSION: The isolated and cultured mesothelial cells were polygonal and confluenced gradually and grew well like the slab stone. The purity of cultured mesothelial cells was more than 90%; the cells grew well, quickly, and could be passaged to 4 and 5 generations. Microvilli of the cells were evident on the surface of cells under TEM. Immunofluorence analysis showed the cells were positively express cytokeratin and vimentin, but negative for leucocyte CD45 and factor Ⅷ fos-related antigen. All identified marks are accorded with the characteristics of mesothelial cells. The results indicated that collagenaseⅠdigestion method is simple, efficient, and reproducible separation method of HPMCs.