Patterned hydrogel films are attractive for the generation of 3D multicellular spheroids, a new in vitro cell model. Traditional methods for their synthesis, such as photolithography and micromolding, usually require a template and are difficult to fit into commercial cell culture plates. Here a simple method to synthesize patterned poly(2-hydroxyethyl methacrylate) (PHEMA) films were developed. First PHEMA films were synthesized in situ in the wells of cell culture plates by bulk redox polymerization of the monomer. Films with a corrugated surface were usually obtained. Fortunately a narrow range of initiator concentration was identified in which smooth films could be obtained. Then water was added to swell the films and wrinkling patterns were produced spontaneously. The shape and size of the patterns can be controlled by adjusting cross-link density and film thickness. Under optimized conditions, films with regular hexagonal patterns were obtained successfully. The patterned PHEMA films were further used to generate multicellular spheroids. The resulting spheroids exhibit high viability and narrow size distribution.