High Cell Density Three-Dimensional Neural Co-Cultures Require Continuous Medium Perfusion for Survival

Three-dimensional (3-D) models of neural cell culture may provide researchers with a more physiologically-relevant setting to study neurobiological phenomena than traditional two-dimensional (2-D) culture models. However, in the development of thick (>500mum) 3-D cultures, diffusion limited mass transport necessitated the use of cell densities much lower than those found in the central nervous system (CNS). The goal of this study was to evaluate the effects of continuous medium perfusion on the survival of thick, 3-D neuronal-astrocytic co-cultures at cell densities closer to those found in brain tissue. At the cell density and thickness used for these studies, 10⁴ cells/mm³ and 500-750mum, respectively, non-perfused cultures exhibited widespread cellular/matrix degradation and cell death. However, co-cultures perfused at relatively high rates (2.5-11.0muL/min, corresponding to 6-27 medium exchanges/day) demonstrated decreased degradation and enhanced viability compared to non-perfused co-cultures. Furthermore, the highest perfusion rate evaluated, 11.0muL/min, resulted in >90% cell viability and maintenance of culture thickness. Next generation 3-D neural cultures, with cell types and densities better approximating the CNS, may provide enhanced model fidelity and be valuable in the mechanistic study of cell growth, interactions, and the responses to chemical or mechanical perturbations