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Human mesenchymal stromal cells (hMSCs) and their secreted products show great promise for treatment of musculoskeletal injury and inflammatory or immune diseases. However, the path to clinical utilization is hampered by donor-tissue variation and the inability to manufacture clinically relevant yields of cells or their products in a cost-effective manner. Previously we described a method to produce chemically and mechanically customizable gelatin methacryloyl (GelMA) microcarriers for culture of hMSCs. Herein, we demonstrate scalable GelMA microcarrier-mediated expansion of induced pluripotent stem cell (iPSC)-derived hMSCs (ihMSCs) in 500 mL and 3L vertical wheel bioreactors, offering several advantages over conventional microcarrier and monolayer-based expansion strategies. Human mesenchymal stromal cells derived from induced pluripotent cells were cultured on custom-made spherical gelatin methacryloyl microcarriers in single-use vertical wheel bioreactors (PBS Biotech). Cell-laden microcarriers were visualized using confocal microscopy and elastic light scattering methodologies. Cells were assayed for viability and differentiation potential in vitro by standard methods. Osteogenic cell matrix derived from cells was tested in vitro for osteogenic healing using a rodent calvarial defect assay. Immune modulation was assayed with an in vivo peritonitis model using Zymozan A. The optical properties of GelMA microcarriers permit noninvasive visualization of cells with elastic light scattering modalities, and harvest of product is streamlined by microcarrier digestion. At volumes above 500 mL, the process is significantly more cost-effective than monolayer culture. Osteogenic cell matrix derived from ihMSCs expanded on GelMA microcarriers exhibited enhanced in vivo bone regenerative capacity when compared to bone morphogenic protein 2, and the ihMSCs exhibited superior immunosuppressive properties in vivo when compared to monolayer-generated ihMSCs. These results indicate that the cell expansion strategy described here represents a superior approach for efficient generation, monitoring and harvest of therapeutic MSCs and their products. [Display omitted] • A microfluidic method to generate chemically and biomechanically tunable cell microcarriers (GelMA-M) was tested for suitability for scalable mesenchymal stem cell expansion. • GelMA-M microcarriers facilitate effective MSC expansion in vertical wheel bioreactors at 500 mL and 3 L scale. • GelMA-M microcarriers allow rapid harvest of product by digestion with trypsin. • Cell laden GelMA-M can be non-invasively imaged in 3D by light-sheet elastic light scattering. • Therapeutic extracellular matrices for repair of bone and immune-modulatory extracellular vesicles can be generated using the system. [ABSTRACT FROM AUTHOR]

The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures. [ABSTRACT FROM AUTHOR]