Thermoresponsive Cell Culture Substrates Based on PNIPAM Brushes Functionalized with Adhesion Peptides: Theoretical Considerations of Mechanism and Design

Thermoresponsive tissue culture substrates based on PNIPAM brushes are used to harvest confluent cell sheets for tissue engineering. The prospect of clinical use imposes the utilization of culture medium free of bovine serum, thus suggesting conjugation with adhesion peptides containing the RGD minimal recognition sequence. The optimum position of the RGD along the chain should ensure both cell adhesion at 37 degrees C and cell detachment at T-L below the lower critical solution temperature of PNIPAM. Design guidelines are formulated from considerations of brush confinement by the cells: (i) Cell adhesion at 37 degrees C is controlled by the RGDs accessible without brush compression. (ii) Cell detachment at T-L is driven by a disjoining force due to confinement of the swollen brush by cells retaining integrin-RGD bonds formed at 37 degrees C. These suggest placing the RGDs at the grafting surface or its vicinity. Randomly placed RGDs do not enable efficient detachment because a large fraction of the integrin-RGD bonds are not sufficiently tensioned at T-L, in line with experimental observations (Ebara, M.; Yamato, M.; Aoyagi, T.; Kikuchi, A.; Sakai, K; Okano, T. Immobilization of celladhesive peptides to temperature-responsive surfaces facilitates both serum-free cell adhesion and noninvasive cell harvest. Tissue Eng. 2004, 10, 1125-1135). The theory framework enables analysis of culture media based on polymer brushes conjugated with adhesion peptides in general.