Development of Simplified NIPAm-based Thermoresponsive Films for Cell Preservation, Expansion and Differentiation

Abstract

Traditional cell recovery methodologies cleave cell-to-cell junctions and thus the recovery of an intact cell sheet for use in tissue engineering is rendered impossible. Additionally, these traditional cell detachment techniques can be damaging to cell surface receptors, which in turn can impair subsequent cell function. Thermoresponsive polymer film mediated cell growth and recovery, has become a popular way to recover undamaged cells, with cell to cell junctions and basally deposited ECM maintained. These cell sheets can be then used for tissue engineering purposes or tissue damage repair. Thermoresponsive polymer has a lower critical solution temperature (LCST) in aqueous solution, which phenomenon has been exploited in temperature controlled cell harvesting. It has been shown that variety of thermoresponsive surfaces are generally conducive to reasonable cell growth. Okano et al. have grafted pNIPAm onto tissue culture plastic using electron beam polymerisation to yield an ultra-thin layer of pNIPAm. Maria E Nash has demonstrated that thermoresponsive films deposited using spin coating method were able to yield cell culture delivery substrates. This issue has been focused on further to simplify the preparation techniques for fabricating thermoresponsive films with a view to cell preservation. To this end, thermoresponsive platforms were deposited using the solvent cast method to yield thin, uniform, reproducible films. Solvent casting is a basic, cheap and effective method for fabricating films in the micrometre range of thickness. It was first reported as a method to deposit a thermoresponsive film for cell culture in 1990. For comparison purposes films were prepared using solvent cast method and the spin coating method. Two types of NIPAm-based thermoresponsive polymers were used in this research; the first employed commercially sourced pNIPAm, the second a NIPAm-co-NtBAm copolymer. The advantages of using a commercially sourced polymer system paired with the operationally simple solvent cast/spin coating technique for cell sheet regeneration are that films prepared in this manner can be produced with minimal training and expense and the use of a commercially sourced product avoids the need for complex polymerisation processes. The NIPAm-co-NtBAm copolymer was selected for similar applications, it showed better cell compatibility and had a LCST lower than room temperature, which makes the biomaterial much easier and flexible for routine applications. The deposited films were characterised using a variety of analytical techniques before biological assessment. Studies have shown that there is a correlation between the thickness of the deposited pNIPAm films and successful cell adhesion and proliferation; therefore it was imperative that this parameter could be assessed. Successful cell adhesion onto a biomaterial surface is dependent on a number of physiochemical characteristics such as surface wettability, roughness and composition, therefore where a discernible difference in cell growth was observed between films of different thicknesses or deposited by different means; comparative assessments of such characteristics were made. Investigations into films prepared from commercially sourced pNIPAm show that it is the thickness determining factor for successful cell adhesion, thinner films supported more cell adhesion. Human mesenchymal stem cell and macrophage-like transformed murine cell line RAW264.7 grew on thinner solvent cast films better than that on their thicker counterparts. Films prepared from the NIPAm based thermoresponsive polymer via spin coating and solvent cast successfully hosted a wide variety of cells and cell lines to confluence and cell detachment was achieved through temperature modulation. Optimisation of the NIPAm based thermoresponsive films for cell adhesion, proliferation and differentiation allowed for the refinement of crucial parameters to thermoresponsive modifications.