Fabrication, characterization, and application of micropatterned one-component poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA) brushes for monolayer cell and spheroid culture and temperature-triggered release are reported. Micropatterns of various shapes and sizes were designed to possess a unique functionality imparted by thermoresponsive thin PDEGMA patches, which are cell adhesive at 37 °C, embedded in a much thicker cell-resistant PDEGMA matrix that does not exhibit measurable thermoresponsive properties. Depending on the cell seeding density, PaTu 8988t human pancreatic tumor cells or spheroids were cultured area-selectively, confined by the 40 ± 4 nm thick passivating PDEGMA matrix, and could be released on demand by a mild thermally triggered brush swelling in the 5 ± 1 nm thin regions. As shown by surface plasmon resonance (SPR) measurements, in contrast to the thinner brushes, the thicker brushes exhibited virtually no fibronectin adhesive properties at 37 °C, whereas at 25 °C, both areas showed similar protein resistant behavior. The quasi-2D thickness-encoded micropatterns were shown to be useful templates for the growth of 3D multicellular aggregates. Thermally induced release after 5 days of incubation afforded 3D cell spheroids comprising up to 99% viable cells demonstrating that the system can be used as a 3D spheroid in vitro model for basic tumor research and anticancer drug screenings.