Genetically-engineered islets can serve as a potential means to modulate the regional immunity occurring in the transplant environment, although gene transfer into islets is not easy due to the extracellular matrix of islets. In this study, we aggregated single islet cells dissociated by trypsinization for successful gene transfection into islet cells. To enhance the functionality of aggregated islets, we optimized the conditions for the aggregation of dissociated islet cells using several supplements such as collagen, all-trans-retinoic acid (atRA) and the culture medium of pancreatic exocrine cells (ECM). The dissociated islet cells formed tight cell clusters containing the normal insulin secretion against glucose concentration and the aggregation yield was significantly improved by treatment with atRA and ECM. However, collagen did not improve cell aggregation. Therefore, this re-aggregation technology would be useful for the development of genetically-engineered islets.