AbstractThe cultivation of cells requires the use of unfavorable proteolytic enzymes, which cause cell‐surface modification and also need considerable optimization. Recently, with the development of smart polymers, research has looked to using thermoresponsive polymers as cell culture substrates. These novel surfaces allow the cultivation of cells without using enzymes by utilizing the thermoresponsive phase transition property of poly(N‐isopropylacrylamide) (PNIPAAm). Copolymers of PNIPAAm and N‐tert‐butyl‐acrylamide (NtBAm) with varying ratios were synthesized and solvent cast. The copolymer films are coated with cell adhesion promoters such as collagen, poly‐L‐lysine, and laminin to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue™ and PicoGreen® assays is similar for coated copolymer films and standard tissue culture plastic controls. Deposition of cell adhesion promoters onto the copolymer films was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Cell detachment from the copolymer films is dependent on copolymer composition and is not affected by the surface coatings of extracellular matrix (ECM) proteins. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin. © 2007 Wiley Periodicals, Inc. J BiomedMater Res 2007