Optimizing the Seeding Density of Human Mononuclear Cells to Improve the Purity of Highly Proliferative Mesenchymal...

Mesenchymal stem cells (MSCs) hold considerable promise for regenerative medicine. Optimization of the seeding density of mononuclear cells (MNCs) improves the proliferative and differentiation potential of isolated MSCs. However, the underlying mechanism is unclear. We cultured human bone marrow MNCs at various seeding densities (4.0 × 10⁴, 1.25 × 10⁵, 2.5 × 10⁵, 6.0 × 10⁵, 1.25 × 10⁶ cells/cm²) and examined MSC colony formation. At lower seeding densities (4.0 × 10⁴, 1.25 × 10⁵ cells/cm²), colonies varied in diameter and density, from dense to sparse. In these colonies, the proportion of highly proliferative MSCs increased over time. In contrast, lower proliferative MSCs enlarged more rapidly. Senescent cells were removed using a short detachment treatment. We found that these mechanisms increase the purity of highly proliferative MSCs. Thereafter, we compared MSCs isolated under optimized conditions with a higher density (1.25 × 10⁶ cells/cm²). MSCs under optimized conditions exhibited significantly higher proliferative and differentiation potential into adipocytes and chondrocytes, except for osteocytes. We propose the following conditions to improve MSC quality: (1) optimizing MNC seeding density to form single-cell colonies; (2) adjusting incubation times to increase highly proliferative MSCs; and (3) establishing a detachment processing time that excludes senescent cells.