Non-Enzymatic Cell Expansion and Harvesting Using a Smart Thermo-Responsive Gel.

Advanced cell-based therapies, including immunotherapy, regenerative medicine, and other biotechnological applications, require large quantities of viable mammalian cells for research and clinical use. Conventional enzymatic harvesting methods, such as trypsini-zation, can compromise cell integrity and reduce viability. This study investigates an al-ternative temperature-responsive approach using alginate beads incorporated with poly(N-isopropylacrylamide) (PNIPAAm), a polymer exhibiting a lower critical solution temperature (LCST) of approximately 32 °C. This system enables temperature-controlled cell detachment while preserving cellular structure and extracellular matrix components, thereby potentially improving post-harvest viability compared to trypsin treatment. Ho-mogeneous alginate hydrogel beads were synthesized using a standard infusion pump and ionically crosslinked with calcium cations. The beads were characterized by scanning electron microscopy (SEM) for morphology and by Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and micro-computed tomography (µ-CT) for compositional and thermal analysis. Mouse fibroblast cells (L929 cell line) were cultured on the beads, and their proliferation and viability were assessed using CCK-8 and Live/Dead assays, demonstrating significant cell growth over seven days. The results suggest that PNIPAAm-modified alginate beads provide a promising, enzyme-free platform for efficient mammalian cell harvesting and delivery, with potential applications across advanced cell manufacturing and therapeutic technologies.