"CO-CULTURE" OF BLADDER SMOOTH MUSCLE AND UROTHELIAL CELLS ON SMALL INTESTINAL SUBMUCOSA : EVALUATION OF THE BEST CULTURE METHOD FOR IN VITRO TISSUE ENGINEERING TECHNIQUES

Background: Small intestinal submucosa (SIS) is a xenogenic, acellular, collagen rich membrane with inherent growth factors previously shown to promote in vivo bladder regeneration and support the individual 3 dimensional growth of bladder smooth muscle cells (SMC) and urothelial cells (UC). It is currently unknown whether in vitro seeding of SIS with cells will enhance the regeneration process and what the best method of seeding is for use with this technique. This study was conducted to evaluate the combined in vitro growth of SMC and UC on SIS and to determine the best culture method for in vitro tissue engineering use. Methods: Primary cultures of bladder SMC and UC were established from both humans and dogs using standard explant techniques. The following cell culture experiments were then carried out with both dog and human cells individually. Cultured cells were seeded on SIS at a density of 1x[10.sup.5] cells per [cm.sup.2] SIS, incubated, and harvested at 3, 7, 14 and 28 days. Four separate groups were studied: 1) UC seeded alone on the mucosal surface of SIS, 2) SMC seeded alone on the mucosal surface, 3) "co-culture": SMC and UC seeded simultaneously on the mucosal surface, and 4) "sandwich" culture: SMC seeded on serosal surface followed by seeding of UC on the mucosal surface 24 hours later. The SIS-cell constructs were formalin fixed at the time of harvesting and processed for routine histology including Masson's trichrome staining. The specific cell growth characteristics were studied with special attention to cell morphology, cell proliferation, cell adherence, and 3 dimensional patterns of growth. To help in the identification of cells in the groups where cells were seeded together, immunohistochemical analysis was performed with antibodies to smooth muscle [Alpha]-actin and cytokeratins AE1/AE3. Results: Both human and dog cells grew similarly on SIS. When seeded alone, SMC and UC grew in several layers with minimal matrix penetration. When cells were seeded on the SIS in "co-culture", there appeared to be a synergistic effect with respect to enhanced growth and penetration of the SIS membrane. Both SMC and UC layered more readily and had greater adherence to the SIS. Cells formed a thick stratified layer that was organized such that SMC were basally located with matrix penetrance while the UC grew in multiple layers with early polarity on top of the proliferating SMC. With the "sandwich technique" there was also enhancement of growth and