Material selection is one of the most important aspects to construct a novel template for tissue regeneration. In this research alginate, bacterial cellulose (BC) and gelatin were selected based on available material and published data to prepare membranes and then the morphology of Swiss mouse embryo fibroblast NIH/3T3 cells on the surface of these membranes was examined to select the best material for the development of a biodegradable skin tissue regeneration template. The cells on alginate membrane crosslinked with Ca2+ (AGM_Ca) showed a spherical morphology and growth retardation, probably due to high calcium content on and in the surface of membrane. This has been confirmed in control experiments in which calcium was added to the culture medium (DMEM medium). The NIH/3T3 cells grown on the BC membrane (BC_M) and membrane of glutaraldehyde (GTA) crosslinked gelatin (GTA_GM) had a polygonal morphology. The proliferation rate of cells on the GTA_GM was faster than that on the BC_M. Therefore the GTA_GM is better than the AGM_Ca and BC_M to apply as a skin tissue regeneration template. Cytotoxic effects of GTA were studied in GTA_GMs prepared using gelatins obtained from cow bones, pork skins, and fish skins. It was found that molar ratio of GTA to gelatin for preparation of membrane should not be higher than 0.033. Cytotoxic effects of CIA were observed on the GTA_GMs prepared with molar ratio higher than 0.033, except pork skin gelatin membrane with a molar ratio, 0.033, which showed the cytotoxic effects on fibroblast cells. The physical morphology of the membranes of cow bone gelatin and fish skin gelatin is stronger and more flexible than that of pork skin gelatin in wet forms. According to these results, it can be suggested that pork skin gelatin might have had less crosslinked points. NH2-sites of lysine molecules than cow bone gelatin and fish skin gelatin. These two gelatins are selected for further development of a template for skin tissue regeneration. (C) 2009 Elsevier Ltd. All rights reserved.