Cortical fibroblast culture from human biopsies

Abstract

Background: Tubulointerstitial fibrosis is an integral part of progressive renal disease. Human cortical fibroblasts are believed to be key effector cells in fibrogenesis. Thus, a reliable culture of these cells is necessary for studies of their pathophysiology. Methods: Cortical fibroblast culture from routine kidney biopsies were analyzed and the cells were characterized. Indirect immunofluorescence staining was done after the first passage for cytokeratin, vimentin, alpha -smooth muscle actin, CD 44, CD 54, CD 68, collagen types I, III, and HLA-DR. We then assessed the utility of the putative fibroblast markers CD 90, prolyl-4-hydroxylase (P4H) and F1b in simultaneous stainings of tubular epithelial cells. Results: During the study period, 49 biopsy cores were cultured and cortical fibroblasts could be successfully established in 21 cases (42.9%). There was no relation between the success rate of culture and the degree of interstitial fibrosis, but an association was seen with the time of completion of the first passage. There was a negative correlation between the extent of scarring and the percentage of cytokeratin positive cells (r=-0.66, p <0.001). All primary fibroblasts were negative for factor VIII, HLA-DR, CD 68, and cytokeratin. They expressed alpha -smooth muscle actin and collagen types I and III to variable degrees. There was a robust correlation between die percentage of alpha -smooth muscle actin positive cells and interstitial scarring but no such association with collagen type I or type III positive cells. The three putative fibroblast markers did not prove useful in differentiating between tubular epithelial cells; aid fibroblasts. However, since only fibroblasts stained positive for CD 90 and negative for cytokeratin, these two markers may suffice to distinguish fibroblasts from other renal cellular elements. Conclusions: Cortical renal fibroblasts can be easily cultured from kidney biopsy cores, though the success rate of pure cultures is below 50%. Staining for CD 90 and cytokeratin may suffice for initial characterization of these cells.