Engineering Thermoresponsive Poly(N-isopropylacrylamide)-Based Films with Enhanced Stability and Reusability for Efficient Bone Marrow Mesenchymal Stem Cell Culture and Harvesting

Poly(<i>N</i>-isopropylacrylamide) (PNIPAM) offers a promising platform for non-invasive and gentle cell detachment. However, conventional PNIPAM-based substrates often suffer from limitations including limited stability and reduced reusability, which hinder their widespread adoption in biomedical applications. In this study, PNIPAM copolymer films were formed on the surfaces of glass slides or silicon wafers using a two-step film-forming method involving coating and grafting. Subsequently, a comprehensive analysis of the films' surface wettability, topography, and thickness was conducted using a variety of techniques, including contact angle analysis, atomic force microscopy (AFM), and ellipsometric measurements. Bone marrow mesenchymal stem cells (BMMSCs) were then seeded onto PNIPAM copolymer films prepared from different copolymer solution concentrations, ranging from 0.2 to 10 mg·mL^-1, to select the optimal culture substrate that allowed for good cell growth at 37 °C and effective cell detachment through temperature reduction. Furthermore, the stability and reusability of the optimal copolymer films were assessed. Finally, AFM and X-ray photoelectron spectroscopy (XPS) were employed to examine the surface morphology and elemental composition of the copolymer films after two rounds of BMMSC adhesion and detachment. The findings revealed that the surface properties and overall characteristics of PNIPAM copolymer films varied significantly with the solution concentration. Based on the selection criteria, the copolymer films derived from 1 mg·mL^-1 solution were identified as the optimal culture substrates for BMMSCs. After two rounds of cellular adhesion and detachment, some proteins remained on the film surfaces, acting as a foundation for subsequent cellular re-adhesion and growth, thereby implicitly corroborating the practicability and reusability of the copolymer films. This study not only introduces a stable and efficient platform for stem cell culture and harvesting but also represents a significant advance in the fabrication of smart materials tailored for biomedical applications.