Three-dimensional (3D) scaffold culture of pancreatic -cell has been proven to be able to better mimic physiological conditions in the body. However, one critical issue with culturing pancreatic -cells is that -cells consume large amounts of oxygen, and hence insufficient oxygen supply in the culture leads to loss of -cell mass and functions. This becomes more significant when cells are cultured in a 3D scaffold. In this study, in order to understand the effect of oxygen tension inside a cell-laden collagen culture on -cell proliferation, a culture model with encapsulation of an oxygen-generator was established. The oxygen-generator was made by embedding hydrogen peroxide into nontoxic polydimethylsiloxane to avoid the toxicity of a chemical reaction in the -cell culture. To examine the effectiveness of the oxygenation enabled 3D culture, the spatial-temporal distribution of oxygen tension inside a scaffold was evaluated by a mathematical modeling approach. Our simulation results indicated that an oxygenation-aided 3D culture would augment the oxygen supply required for the -cells. Furthermore, we identified that cell seeding density and the capacity of the oxygenator are two critical parameters in the optimization of the culture. Notably, cell-laden scaffold cultures with an in situ oxygen supply significantly improved the -cells' biological function. These -cells possess high insulin secretion capacity. The results obtained in this work would provide valuable information for optimizing and encouraging functional -cell cultures. (c) 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:221-228, 2017