Poly(N-isopropylacrylamide) brushes with three different grafting densities were synthesized via surface-initiated atom-transfer radical polymerization on glass or on silicon substrates. The substrates were modified with monochlorosilane-based or trimethoxysilane-based atom-transfer radical polymerization initiators. Atomic force microscopy images showed detachment of brushes from the monochlorosilane-based system under cell culture conditions. In situ ellipsometry demonstrated the reversible swelling and collapse of the brushes as the temperature was varied across the lower critical solution temperature of poly(N-isopropylacrylamide) in pure water. The polymer brushes were evaluated as supporting substrates for MC-3T3 cell cultures. At 37°C (T>lower critical solution temperature), the seeded cells adhered, spread, and proliferated, whereas at 25°C (T<lower critical solution temperature), the cells detached from the surface. The low-density polymer brush showed the highest cell adhesion, featuring adhering cells with an elongated morphology.