The construction of renal glomerular tissue has provided an important tool not only for the understanding of renal physiology and pathology in blood ultrafiltration and cell dysfunction, but also in the application of tissue engineering to glomeruli regeneration and nephritic therapy. In this study, a novel method to reconstruct glomerular tissue combining cultured cells on a collagen vitrigel scaffold is described. The method consists of two newly developed techniques, one to isolate glomerular epithelial and mesangial cells rapidly from kidney, which facilitates the prolongation of cell population doublings and allows a long-term cell culture without losing cellular features, and another to prepare a stable and thin transparent collagen gel membrane termed collagen vitrigel that can facilitate three-dimensional cultures for reconstructing an epithelial-mesenchymal model. By combining the two methods, we cocultured glomerular epithelial and mesangial cells on both surfaces of the collagen vitrigel by the manipulation of two-dimensional cultures, resulting in the successful reconstruction of a three-dimensional glomerular organoid. The coculture results showed that the collagen vitrigel maintains cell growth and cell viability for more than I month, and surprisingly, the epithelial layer demonstrated polarity formation, which usually appears in in vivo normal epithelial cells existing at the glomerular basement membrane, but seldom appears in epithelial cells cultured in vitro. Moreover, the coculture results showed that fibronectin, an extracellular matrix component, and integrin beta 1, a receptor of fibronectin, were detected in high amounts on both cells, suggesting our collagen vitrigel can provide a suitable environment for cell-cell interactions that stabilize the cell structure and may contribute to the polarity formation of epithelial cells.