Alginate-Methacrylate-RGD MicroSpheres (Alg-MR-MS) for cell expansion and delivery in tissue engineering

Clinically relevant cell numbers are essential for many tissue engineering applications, requiring scalable expansion platforms. Microcarrier-based bioreactor systems address this demand; however, conventional non-dissolvable carriers require harsh enzymatic or mechanical detachment, reducing cell viability and integrity. Although dissolvable microcarriers improve recovery, most are designed only for harvesting and lack functionality for downstream tissue engineering. Here, we present a sequential dual-crosslinkable alginatemethacrylate-RGD microsphere system (Alg-MR-MS) that integrates scalable cell expansion with scaffold formation in a single platform. The workflow involves: (1) microsphere fabrication via calcium-mediated ionic crosslinking; (2) three-dimensional cell expansion in a microcarrier culture system; (3) controlled phase change through calcium chelation with sodium citrate, enabling efficient cell recovery while preserving viability and phenotype; and(4) photocrosslinking to form a stable, cell-laden hydrogel suitable for bioprinting and tissue engineering. This sequential dual-crosslinking strategy supports expansion, dissolution, and structural stabilization within one system. Human adipose-derived stem cells cultured on Alg-MR-MS showed ~5-fold increased metabolic activity over 7 days, comparable to 2D controls, with high viability and strong attachment. Compared with other dissolvable carriers, Alg-MR-MS offers efficient expansion, rapid phase transition, and simplified single-step fabrication, bridging biomanufacturing and functional tissue engineering.