SMART POLYMERS FOR ADVANCED CELL TECHNOLOGY AND TISSUE ENGINEERING

Abstract

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Over the last couple of decades a number of innovative approaches to manufacturing thin poly-N-isopro-pylacrylamide (pNIPAm) based thermoresponsive coatings for advanced cell culture applications have been developed and refined. The development of reproducible and robust techniques for cell and cell sheet harvesting from the fabricated coatings provides a wide scope of opportunity in regenerative medicine. Unlike traditional cell culture detachment methods, detachment from thermoresponsive substrates allows for the detachment of cells without the disruption of cell-to-cell junctions. Therefore this approach can facilitate the harvesting of cell sheets, which can be applied for tissue engineering purposes. The main goal of this work is to answer the following key questions: "Why some pNIPAm surfaces are conducive to cell adhesion and proliferation while others are not? Why some pNIPAm surfaces are provide cell detachment while others are not?” EXPERIMENTAL METHODS. The polymers used in this study are pNIPAm, NIPAm-co- N-tertbutyl-acrylamide (NtBAm) with different NIPAM content , NIPAm-co-acrylamidobenzophenone (AcBzPh), NIPAm-co-NtBAm-co-AcBzPh, NIPAM-poly-(ethylpyrrolidone methacrylate) (pEPM) with different NIPAM content, NIPAm -co- octadecyl methacrylate (ODMA). LCST range from 8°C to 36°C. Four film fabrication methods were investigated: physisorption deposition, solvent casting technique, spin coating film deposition and UV-crosslinking. Film thickness varies within 10 nm- 10 mkm. Film characterisation: Characterization of the prepared films was achieved using XPS, ESM, contact angle and AFM measurements. Cell culture: The range of the cell culture lines, primary cells including tenocyte, mesenchymal stem cells and iPS cells have been used for cell detachment and cell sheet preparation. Cell proliferation and differentiation, gene expression, extracellular matrix synthesis and kinetics of cell sheet detachment have been analysed. RESULTS AND DISCUSSION. In total we investigated 14 polymers and four film fabrication methods. pNI-PAm coatings are generally poorly cell compatible and a number of complex or expensive techniques have been developed in order to overcome this issue. Results suggest that only nm -scale pNIPAm films are preferable for cell sheet fabrication. The control of pNIPAm film thickness using the spin-coating technique offers an effective tool for cell sheet-based tissue engineering. Also, we developed simple one- step method of film preparation for nonplanar surfaces. More hydrophobic NIPAm-co- NtBAM based films provide better cell growth, but hydrophobicity dramatically decreases cell detachment rate (>2 hours), which may be essential factor for cell culture operation. Copolymers NIPAM-EPM demonstrate highly cell compatibility. CONCLUSION. New thermosresponsive polymers and methods of film deposition allow to obtain "smart” coatings with the desired physical characteristics, including surface energy, roughness etc., which in turn allows to control the kinetics of cell growth and detachment. A unique potential advantage of the physical adsorption method for preparing thermoresponsive coatings is that this approach could be used to coat materials with complicated geometric profiles, which may be useful for medical devices, microfluidic systems and tissue engineering.