Ethylene Glycol and Trehalose Cryopreservation of Cell-Laden Hydrogel Microspheres Enabled by Microfluidic Fabrication

Cell-laden hydrogel microspheres have gained significant attention in 3D cell culture applications, yet effective cryopreservation methods for these systems remain underexplored. This study developed a microfluidic platform for fabricating monodisperse, cell-laden microspheres and investigated a dimethyl sulfoxide (DMSO)- and fetal bovine serum (FBS)-free cryopreservation approach. The platform enabled rapid production of gelatin methacryloyl (GelMA) and calcium alginate (ALG) microspheres, demonstrating cell viability exceeding 80% for U251 cells in GelMA microspheres and 90% for both U251 cells and induced pluripotent stem cells (iPSCs) in ALG microspheres. A DMSO-/FBS-free cryoprotectant (12% ethylene glycol, 4% trehalose; E/T) was identified that maintained >90% post-thaw viability in GES, U251, HepG2, A549, and 3T6 cells, with iPSCs retaining >80% viability. Crucially, E/T effectively preserved iPSC-laden microspheres while preventing DMSO-induced apoptosis and preserving pluripotency. This work establishes a systematic protocol for cryopreserving cell-laden hydrogel microspheres without DMSO/FBS, providing a clinically translatable strategy to advance 3D cell culture technologies.