An Efficient Spheroid Manufacturing Workflow for WJ-MSCs: Comparative Evaluation of Immediate 3D Aggregation and Conventional 2D Stabilization

Cryopreserved mesenchymal stem cells (MSCs) are widely used as off-the-shelf therapeutics. However, conventional manufacturing workflows typically include a post-thaw 2D monolayer recovery step before 3D spheroid formation. This study evaluated whether immediate spheroid formation following thawing (Direct-3D) could replace the conventional 2D-to-3D workflow while maintaining or improving chondrogenic potential. Using passage 5 Wharton’s jelly MSCs (WJ-MSCs), spheroid morphology, early chondrogenic priming, differentiation, and extracellular matrix (ECM) formation were systematically compared between the two strategies. The Direct-3D group exhibited faster and more uniform spheroid compaction and significantly increased SOX9 expression as early as 3 h, indicating enhanced early priming. This response was accompanied by higher COL2A1 expression and an increased COL2A1/COL1A1 ratio during early differentiation while maintaining minimal collagen type X expression. These findings demonstrate that the post-thaw monolayer recovery step is not essential for producing high-quality chondrogenic spheroids from WJ-MSCs. By simplifying the manufacturing workflow while preserving—and potentially enhancing—early chondrogenic performance, the Direct-3D strategy represents a practical and scalable approach for WJ-MSC-based cartilage tissue engineering and cell therapy.