Systematic investigation of the effects of neural stem cell spheroid size and density on fate specification in 3D culture

Abstract

A systematic understanding of how the density of neural stem/progenitor cells (NS/PCs) embedded within three-dimensional (3D) biomaterials affect cell behavior will be necessary for developing effective strategies to generate CNS tissues. Here, we investigated the effects of local and global cell density of mouse neural stem cells (mNSCs) on their viability, proliferation, and differentiation when cultured in 3D, hyaluronic acid (HA)-based hydrogel matrices. Specifically, we assessed the influence of spheroid size, which represents local cell density, (small: 100 cells per sphere, large: 200 cells per sphere) and seeding density (low: 100 000 cells per hydrogel, high: 200 000 cells per hydrogel), which represents global density, on cellular outcomes. Results reveal that these factors have both independent and interactive effects on NS/PC viability and fate. Cultures of smaller spheres at low global densities yield more glial cells, including astrocytes and oligodendrocytes. In contrast, cultures with high global densities, regardless of sphere size, better preserved stem-like mNSC phenotypes. Strikingly, cultures with 1000 total spheres per hydrogel, regardless of sphere size or overall cell concentration, best maintained viability while promoting neuronal maturation. These findings highlight the importance of controlling both local and global cell densities in 3D cultures to achieve reproducible mNSC-derived populations for use as in vitro test beds or biomanufacturing of therapeutic stem cells.