Mesenchymal Stem Cells, or MSC, will be key components of future therapeutics, engineered tissues, and medical devices. There are currently over 400 clinical trials (CT) investigating MSC as therapies. The CT have produced some promising results, with MSC generally deemed safe, and effective. The promise of MSC has resulted in an increased demand for research- and clinical-grade MSC. However commercially available cells for research are not well suited for translational work, and clinical-grade cells are not available to purchase. RoosterBio, Inc. was founded to reduce the costs and complexity of developing and implementing MSC technology for clinical translation, and we have recently brought to market affordable and well-characterized human bone marrow-derived MSC in translation-ready formats. Recently, exciting research from many investigators has shown that culture of MSC as 3D aggregates (3DhMSC) improves in vitro biological activity over MSC grown as a monolayer (2D-hMSC), including enhanced differentiation and increased paracrine factor secretion. 3D-MSC also have enhanced therapeutic properties in pre-clinical models. Due to the therapeutic potential of 3D-MSC, RoosterBio aims to provide researchers with affordable access to translationally-relevant 3D-hMSC aggregates that are ready for pre-clinical use. hMSC aggregates (3D-hMSC) are currently produced by expanding cells in conventional 2D culture, isolating cells and forming aggregates in a separate vessel through several non-scalable methods. For 3D-hMSC to become a commercial product, methods have to be developed that can produce 3D-hMSC using scalable, cost effective, and regulatory-friendly processes. In this Phase I SBIR we will test the feasibility of an innovative technology, developed in the T. Ma laboratory (FSU), to grow MSC and produce 3D-hMSC on microcarriers with thermo-reversible surfaces (TRM). The Ma lab has synthesized TRM and produced 3D-hMSC at small scale in culture dishes. We predict that TRM are compatible with sitrred tank bioreactors and thus are scalable to hundreds of liters in single use vessels; thus enabling commercial production of 3D-hMSC. We propose experiments to develop this innovative TRM/bioreactor technology. The milestone at the end of this Phase I SBIR will be to demonstrate that, (1) a TRM-Bioreactor can produce 3D-hMSC that (2) maintain therapeutically-relevant properties of 3D-hMSC produced by standard methods. This work will set the stage for a Phase 2 grant to optimize the production process and scale-up the TRM-Bioreactor based technology for a commercial scale, ready-to-use 3D-hMSC product that will provide translational scientists, engineers and bioprinters access to high-quality, affordable hMSC with enhanced function. Importantly, these processes will also be translatable to clinical production of 3D-hMSC.