Spherical three-dimensional (3D) cellular aggregates are valuable for various applications such as regenerative medicine or cell-based assays due to their stable and high functionality. However, previous methods to form aggregates have shown drawbacks, being labor-intensive, showing low productivity per unit area or volume and difficulty to form homogeneous aggregates. We proposed a novel strategy based on oxygen-permeable polydimethylsiloxane (PDMS) honeycomb microwell sheets, which can theoretically supply about 80 times as much oxygen as conventional polystyrene culture dishes, to produce recoverable aggregates in controllable sizes using mouse insulinoma cells (MIN6-m9). In 48 hours of culture, the PDMS sheets produced aggregates whose diameters were strictly controlled (?32, 60, 90, 150 and 280 mm) even at an inoculum density eight times higher (8.0x105 cells/cm(2)) than that of normal confluent monolayers (1.0x105 cells/cm(2)). Measurement of the oxygen tension near the cell layer and glucose/lactate analysis clearly showed that cells exhibit aerobic respiration on the PDMS-based culture system. Glucose-responsive insulin secretion of the recovered aggregates showed that the aggregates around 90 mm in diameter secreted the largest amounts of insulin. This confirmed the advantages of 3D cellular organization and the existence of a suitable aggregate size, above which excess organization leads to a decreased metabolic response. These results demonstrated that this microwell-based PDMS culture system provides a promising method to form size-regulated and better functioning 3D cellular aggregates of various kinds of cells with a high yield per surface area. (c) 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:178-187, 2014