Thermo-responsible PNIPAM-grafted polystyrene microspheres for mesenchymal stem cells culture and detachment

The preparation of cells is a critical step in cell therapy. To ensure the effectiveness of cells used for clinical treatments, it is essential to harvest adherent cells from the culture media in a way that preserves their high viability and full functionality. In this study, we developed temperature-responsive PNIPAM-grafted polystyrene microspheres using reversible addition-fragmentation chain transfer polymerization. These microspheres allow for the non-destructive harvesting of cultured cells through temperature changes. The composition and physicochemical properties of the PNIPAM-grafted polystyrene microspheres were confirmed using infrared spectroscopy, elemental analysis, dynamic light scattering, and thermogravimetric analysis. In vitro experiments demonstrated that these microspheres exhibit excellent biocompatibility, supporting the adhesion and proliferation of various cells. Moreover, the microspheres showed good temperature responsiveness in thermosensitive detachment experiments with GFP-HepG2 cells and umbilical cord mesenchymal stem cells (UC-MSCs). Additionally, through orthogonal experiments, we identified a cell detachment aid mixture that significantly improved the dispersibility of cells detached from the microspheres, enhancing the efficiency of thermosensitive cell detachment by approximately 40%. The harvested UC-MSCs retained their capacity for re-proliferation and trilineage differentiation. Consequently, the temperature-responsive microspheres developed in this study, combined with the cell detachment aid mixtures, hold great potential for large-scale culture and harvesting of therapeutic cells in clinical applications.