Recent advances in cell therapy manufacturing, e.g. microcarrier-based 3D culture and perfusion, have paved the way for cost-effective large-scale expansion of mesenchymal stem cells (MSCs). In particular, perfusion and enhanced feed boosts allow the culture to reach increasingly high cell densities. Although the high cell density is favorable to customers due to the low cost and high cell dose requirements, we should not overlook its impact on cell quality. Here, we studied the correlation between 3D culture cell density with a variety of commonly used cell quality parameters for both fresh and post-thaw cells. MSC 3D cultures were carried out using different microcarriers (Cultispher-G, dissolvable Corning Synthemax, and Pall SoloHill collagen) and media in spinner flasks and stirred-tank bioreactors. MSCs were able to expand on all microcarriers (Figure1), reaching a final density of 0.6 to 1 million cells/mL. Attachment and proliferation capacities of 3D-expanded MSCs, harvested at different cell densities, were assessed by culturing them post-harvest on 2D flatware. Figure2 shows that MSCs harvested at low cell densities (day4) proliferated significantly higher than MSCs harvested at high cell densities (day5-8). Similar pattern was observed for 3D-cultured MSCs post-thaw. Cell viability was also evaluated for both fresh and post-thaw MSCs harvested at low and high cell densities. For most cases, MSCs harvested at both low and high cell densities, showed above 90% viability over 0-6 hours (Figure3). MSCs expanded using SoloHill microcarriers displayed >90% post-thaw viability for both low and high cell densities. However, dissolvable Corning-Synthemax exhibited up to 12% and 30% drops, and Cultispher-G up to 24% and 51% drops for low and high cell density groups, respectively. Figure3 also shows that the extent of decline in post-thaw vibility was higher for MSCs harvested at high cell densities versus low cell densities. In conclusions, our findings indicated that the extended 3D culture (i.e. harvesting at higher cell densities) led to compromising attachment and proliferation capacities of MSCs, regardless of the choice of microcarrier. For Cultispher-G and dissolvable Coning-Synthemax, we observed 20-30% lower cell viabilities for MSCs harvested at high densities compared to low densities. [ABSTRACT FROM AUTHOR]