Gelatin Methacrylate Macroporous Cell Scaffold Fabrication via One-Pot Aqueous Two-Phase Separation.

Microgels are increasingly recognized as versatile building blocks for granular cell scaffolds, offering advantages over bulk hydrogels for a variety of biomedical applications. While existing methods for scaffold fabrication often require multistep processes involving separate microgel formation and assembly, here we introduce a streamlined, one-pot approach that achieves microgel formation and scaffold assembly in minutes. This developed method is robust, reproducible, user-friendly, and requires no specialized equipment, making it broadly accessible. Specifically, aqueous two-phase separation (ATPS) was utilized to form ~2 μm gelatin methacrylate (GelMA) microgels in sodium sulfate salt solution, which rapidly "clicked" to form macroporous scaffolds under UV light. Various parameters were modulated to observe the effect on scaffold formation including timing of UV exposure, salt concentration, photoinitiator concentration, and polymer concentration. Our results indicated a mechanically stable scaffold able to quickly imbibe water due to its interconnected macropores. U-87 glioblastoma, NIH 3T3 fibroblast, and ATDC5 chondrocyte cells were successfully encapsulated within these granular scaffolds and exhibited an elongated morphology at 24 h and > 90% viability over 14-21 days of culture. The ability to produce microgel scaffolds containing living cells in one step opens new routes to the production of cell-laden porous scaffolds.