N-isopropylacrylamide-based thermoresponsive polyelectrolyte multilayer films for human mesenchymal stem cell expansion.

Human mesenchymal stem cells (hMSCs) are colony-forming unit fibroblasts (CFU-F) derived from adult bone marrow and have significant potential for many cell-based tissue-engineering applications. Their therapeutic potential, however, is restricted by their diminishing plasticity as they are expanded in culture. In this study, we used N-isopropylacrylamide (NIPAM)-based thermoresponsive polyelectrolyte multilayer (N-PEMU) films as culture substrates to support hMSC expansion and evaluated their effects on cell properties. The N-PEMU films were made via layer-by-layer adsorption of thermoresponsive monomers copolymerized with charged monomers, positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS) and compared to fetal bovine serum (FBS) coated surfaces. Surface charges were shown to alter the extracellular matrix (ECM) structure and subsequently regulate hMSC responses including adhesion, proliferation, integrin expression, detachment, and colony forming ability. The positively charged thermal responsive surfaces improved cell adhesion and growth in a range comparable to control surfaces while maintaining significantly higher CFU-F forming ability. Immunostaining and Western blot results indicate that the improved cell adhesion and growth on the positively charged surfaces resulted from the elevated adhesion of ECM proteins such as fibronectin on the positively charge surfaces. These results demonstrate that the layer-by-layer approach is an efficient way to form PNIPAM-based thermal responsive surfaces for hMSC growth and removal without enzymatic treatment. The results also show that surface charge regulates ECM adhesion, which in turn influences not only cell adhesion but also CFU-forming ability and their multi-lineage differentiation potential.