Three-dimensional multicellular spheroids are better in vitro cell models than two-dimensional cell monolayers; however, their applications are limited by their difficult production. PHEMA hydrogel films with honeycomb-like wrinkled patterns have been developed for high-throughput generation of multicellular spheroids with a uniform shape and size; however, the films are prepared by polymerization of the HEMA monomer, and should be washed extensively before use. Here to synthesize washing-free PHEMA hydrogel films, linear anthracene-functionalized PHEMA polymers were first synthesized by the free-radical copolymerization of HEMA and an anthracene-containing vinyl monomer. Smooth films were then prepared from the linear polymers in the wells of cell culture plates by solution casting. They were cross-linked via photo-dimerization of anthracene groups by exposing to 365 nm UV light, and patterned spontaneously by adding water to swell them. The swelling degree of the films and hence the shape of the wrinkled patterns could be facilely adjusted by adjusting the anthracene content in the linear polymers. If necessary, the cross-link density of the film and thus the shape of the wrinkled patterns could be callbacked by irradiating with 254 nm UV light, thanks to the reversibility of the photo-dimerization of anthracene. The as-prepared films exhibit a high biocompatibility and support cell growth well even without washing and sterilization. The high biocompatibility is attributed to the facts that no low molecular weight monomer, crosslinker and initiator were involved in the film preparation, and the photo-dimerization of anthracene groups produces no by-products. The films are sterilized simultaneously when cross-linked with UV irradiation, thus avoiding an additional sterilization step. PHEMA films with long-range ordered hexagonal wrinkled patterns were selected to generate multicellular spheroids of tumor cell lines. The resulting spheroids exhibit a narrow size distribution and high cell viability. Preliminary tests demonstrated their potential in the screening of anti-cancer drugs.