A microstructure-based hydrogel was employed to study the relationship between spatial specificity and cellular behavior, including cell fate, proliferation, morphology, and insulin secretion in pancreatic beta-cells. To effectively form homogeneous cell clusters in vitro, we made cell-containing hydrogel membrane constructs with an adapted grid structure based on a hexagonal micropattern. Homogeneous cell clusters (average diameter: 83.6 +/- 14.2 mu m) of pancreatic insulinoma (MIN6) cells were spontaneously generated in the floating hydrogel membrane constructs, including a hexagonal grid structure (size of cavity: 100 mu m, interval between cavities: 30 mu m). Interestingly, 3D clustering of MIN6 cells mimicking the structure of pancreatic islets was coalesced into a merged aggregate attaching to each hexagonal cavity of the hydrogel grid structure. The fate and insulin secretion of homogeneous cell clusters in the hydrogel grid structure were also assessed. The results of these designable hydrogel-cell membrane constructs suggest that facultative in vitro beta-cell proliferation and maintenance can be applied to biofunctional assessments.