Expansion of human mesothelial progenitor cells in a longterm three-dimensional organotypic culture of Processus vaginalis peritonei.

A 3D culture system was used to investigate the behaviour of mesothelial cells present in the wall of human processus vaginalis peritonei. Small tissue frag- ments placed on collagen sponges were cultured for 7, 14 and 21 days in medium supplemented with 10% FBS, and analysed for the expression and distribution of cytokeratins (CKAE1-AE3, CK19), p63, Ki-67, vimentin, CD34, and HBME-1. Before culture, flat mesothelial cells displayed immunoreactivity for cytokeratins, vimentin and HBME-1, while p63 and CD34 were negative. Mesenchymal cells within the stroma were vimentin-positive and endothelial cells of small vessels displayed positive staining for CD34. Cytokeratins, p63 and HBME-1 were negative in all stromal cells. In cultured fragments, flat mesothelial cells positive for vimentin, cytokeratins and HBME-1 proliferated, lining the fragment surface and migrating into the sponge. Capillaries showed morphological alterations; however, their immunoreactivity was com- parable with the stroma prior to culture. Cells that had migrated into the sponge and displayed characteristics of mesothelial progenitors, predominantly spindle- shaped and stellate, showed heterogeneous expression of markers especially in late phases of cultivation. These cells were constantly positive for vimentin, a small fraction was cytokeratin-positive and a few dis- played HBME-1 immunoreactivity. CD34 was found in cells forming small cavities into the matrix, resembling newly formed blood vessels. Cells that had migrated into the sponge could be isolated and expanded in co- culture with feeder NIH.3T3 fibroblasts. This system is suitable for studying growth and behaviour of mesothelial cells within their natural environment,