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Recent advancements in tissue engineering shows that generating multicellular spheroids by combining different cell types can enhance their regenerative capacity. Human amniotic epithelial cells (hAECs) gained great interest in regenerative medicine due to their availability, safety, regenerative, immunomodulatory and anti-inflammatory properties. The aim of this study was to determine whether combination of hAECs with islet cells in the same spheroid would further improve islet cell survival and function in vitro under normoxic and hypoxic conditions.Functional Islet spheroids were generated on 3D agarose-patterned microwells. To form homospheroids dispersed rat islet cells (ICs) and hAECs (128,000 cells/mold and 500cell/spheroid) were seeded alone. Heterospheroids were formed by mixing ICs and hAECs at ratio of 1:1. Engineered islet homo- and heterospheroids were cultured under normoxic and hypoxic conditions for 16 h. For all conditions, cell viability, GSIS, total islet cellular insulin content were detected. Hypoxia-induced changes in gene expression were assessed by real time PCR analysis. Next we considered possible molecular mechanisms behind the beneficial effect of hAECs on islet cell function. For this purpose, the mRNA expression levels of Hif-1α, Casp3, Casp8, Casp9 and Bcl2 were determined.Confocal laser scanning microscopy showed uniform distribution of islet cells and hAECs throughout of hybrid spheroids, without evidence of cell loss. Quantifications of the insulin positive area ratios to nuclei, showed that heterospheroids expressed more insulin compared to homospheroids. Moreover, islet heterospheroids expressed significantly more E-cadherin, key cell-to-cell adhesion molecule then homospheroids. The stimulation index of the heterospheroids was significantly higher than those of homospheroids. Exposure to hypoxia rapidly caused fragmentation of homospheroids and augmented cell membrane permeability. In contrary considerably less dead signals were observed within heterospheroids. As anticipated, glucose responsiveness of homospheroids was seriously impaired; the average glucose stimulation index of the heterospheroids was significantly higher than that of the homospheroids (1.7±0.4 vs 0.9±0.5, p < 0.05). This was correlated with downregulation of apoptotic genes Casp3, Casp8 and Casp9 and 2 fold upregulation of antiapoptotic gene Bcl2.These data indicate that incorporation of hAECs into the islet heterospheroids improves the secretory function and viability of islet cells both in conventional culture and in hypoxic conditions.