Cryogenic microcarrier-assisted stem cell storage (Cryo-MASCS): a preservation method enabling scalable culture of...

Microcarrier (µC) suspension systems enable closed, high-density manufacturing of human mesenchymal stem cells (MSCs), but current workflows remain labor-intensive because they require post-thaw cryoprotectant removal and static culture steps to allow cell attachment. To address these bottlenecks, we developed a cryogenic microcarrier-assisted stem cell storage (Cryo-MASCS) workflow that integrates cell attachment, cryopreservation, and thawing directly on surface-engineered µCs. MSCs were pre-seeded onto µCs coated with stacked heparan sulfate-collagen bilayers, cryopreserved on-carrier in either conventional dimethyl sulfoxide (DMSO)-supplemented medium or a DMSO-free, serum-free (SF) minimally supplemented medium, and then thawed and returned directly to suspension culture without intermediate processing. We optimized cell seeding density to maximize recovery. Following thaw, MSCs on engineered µCs and cryopreserved in either DMSO-containing or DMSO-free media retained viability comparable to traditional suspension cultures in DMSO-containing medium, while remaining attached to the carrier surface. Surface-engineered µCs show increased MSC yield within seven days in SF medium, significantly outperforming commercial collagen-coated µCs. Moreover, MSCs recovered metabolic activity and retained robust suppression of lipopolysaccharide-induced M1 macrophage polarization after IFN-γ priming. These findings demonstrate that direct cryopreservation of MSCs on heparan sulfate-collagen-coated µCs is compatible with both DMSO-free and DMSO-supplemented conditions and supports streamlined, scalable culture of undifferentiated MSCs for translational applications.