In previous work, we engineered functional cell sheets using bone marrow-derived mesenchymal stem cells (BM-MSCs) to promote islet graft survival. In the present study, we hypothesized that a cell sheet using dermal fibroblasts could be an alternative to MSCs, and then we aimed to evaluate the effects of this cell sheet on the functional viability of human islets. Fibroblast sheets were fabricated using temperature-responsive culture dishes. Human islets were seeded onto fibroblast sheets. The efficacy of the fibroblast sheets was evaluated by dividing islets into three groups: the islets-alone group, the coculture with fibroblasts group, and the islet culture on fibroblast sheet group. The ultrastructure of the islets cultured on each fibroblast sheet was examined by electron microscopy. The fibroblast sheet expression of fibronectin (as a component of the extracellular matrix) was quantified by Western blotting. After 3 days of culture, islet viabilities were 70.2 +/- 9.8%, 87.4 +/- 5.8%, and 88.6 +/- 4.5%, and survival rates were 60.3 +/- 6.8%, 65.3 +/- 3.0%, and 75.8 +/- 5.6%, respectively. Insulin secretions in response to high-glucose stimulation were 5.1 +/- 1.6, 9.4 +/- 3.8, and 23.5 +/- 12.4 mu IU/islet, and interleukin-6 (IL-6) secretions were 3.0 +/- 0.7, 5.1 +/- 1.2, and 7.3 +/- 1.0 ng/day, respectively. Islets were found to incorporate into the fibroblast sheets while maintaining a three-dimensional structure and well-preserved extracellular matrix. The fibroblast sheets exhibited a higher expression of fibronectin compared to fibroblasts alone. In conclusion, human dermal fibroblast sheets fabricated by tissue-engineering techniques could provide an optimal substrate for human islets, as a source of cytokines and extracellular matrix.