Intact endothelial cell sheet harvesting from thermoresponsive surfaces coated with cell adhesion promoters.

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 enzymes using the thermoresponsive phase transition property of poly(N-isopropylacrylamide) (PNIPAAm). However, this requires expensive techniques to generate a sufficiently thin film that allows cell adhesion. In this study, we looked at simple solvent cast films which normally show poor cell adhesion, but here the films are coated with cell adhesion promoters (CAPs) to improve cell growth without altering the copolymer thermoresponsive behaviour.A copolymer of PNIPAAm and N-tert-butylacrylamide (NtBAm) with a ratio of 85:15, respectively, was synthesized and solvent cast. The copolymer films were coated with CAPs, such as collagen, fibronectin and laminin, to increase their cell adhesion and growth properties. Cell activity measured by the alamarBlue assay showed similar results for coated copolymer films and standard tissue culture plastic controls. Deposition of CAPs on to the copolymer films was characterized by scanning electron microscopy and atomic force microscopy. Cell detachment from the copolymer films is not affected by the surface coatings of CAPs, and endothelial cells are recovered as an intact sheet, which has great potential for uses in tissue engineering applications. The results demonstrate a versatile method for the cultivation of cells while eliminating the need for the use of digestive enzymes such as trypsin. This study shows that cultivation on physically bonded PNIPAAm copolymers is viable and achievable by relatively simple methods.

Cell growth and detachment from protein-coated PNIPAAm-based copolymers.

The 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.