Donor Variability and 3D Culture Models Influence Human Mesenchymal Stem Cell Differentiation

Mesenchymal stem cells (MSCs) are widely used for tissue regeneration due to their multilineage differentiation potential and ability to secrete paracrine factors with immunomodulatory and angiogenic functions. Standard MSC differentiation protocols typically rely on two-dimensional (2D) or pellet culture models that are simple to use but not well-suited for translational or clinical applications. To promote better cell survival, tissue deposition, and differentiation of MSCs, a wide variety of three-dimensional (3D) biomaterial scaffolds and platforms have been developed that provide structural support and present a carefully defined set of biochemical and biophysical cues to cells. While biomaterials can guide cell behavior and promote desirable tissue regeneration outcomes, one remaining challenge in the field is inherent donor-to-donor variability in MSC behavior, phenotype, and differentiation capacity. Although MSCs are promising tools for regeneration, the influence of donor variability on MSC differentiation across culture models remains poorly understood. Previous studies typically use cells from a single donor or rely solely on standard culture models. To address these gaps, we compared MSCs from six human donors and assessed differentiation across chondrogenic, osteogenic, and adipogenic lineages using both standard (pellet or 2D) and 3D biomaterial-based culture models. Alginate hydrogels were used to assess chondrogenesis, while gelatin microribbon (mu RB) hydrogels were used to evaluate osteogenesis and adipogenesis in 3D. Significant donor-to-donor variability was observed in differentiation outcomes across all three lineages and within both 2D and 3D culture models. By directly comparing donor variability in 2D and 3D, we provide evidence that standard 2D models cannot predict MSC differentiation capacity in 3D biomaterials. Therefore, to improve therapeutic efficacy and advance biomaterial-based strategies for tissue regeneration, it is critical to understand how donor variability affects MSC differentiation patterns across 3D biomaterial-based culture models. Impact Statement This study highlights the critical influence of donor variability and culture model on mesenchymal stem cell (MSC) differentiation outcomes. By comparing MSCs from six donors, we found that differentiation trends in standard (pellet or two-dimensional) cultures did not reliably predict outcomes in three-dimensional biomaterial-based models and that donor variability is an important variable that must be included in the evaluation of novel biomaterial-based tissue regeneration platforms.