Microfluidic engineered high cell density three-dimensional neural cultures

Abstract

Three-dimensional (3D) neural cultures with cells distributed throughout a thick, bioactive protein scaffold may better represent neurobiological phenomena than planar correlates lacking matrix support. Neural cells in vivo interact within a complex, multicellular environment with tightly coupled 3D cell–cell/cell–matrix interactions; however, thick 3D neural cultures at cell densities approaching that of brain rapidly decay, presumably due to diffusion limited interstitial mass transport. To address this issue, we have developed a novel perfusion platform that utilizes forced intercellular convection to enhance mass transport. First, we demonstrated that in thick (>500 µm) 3D neural cultures supported by passive diffusion, cell densities ⩽5.0 × 103 cells mm−3 were required for survival. In 3D neuronal and neuronal–astrocytic co-cultures with increased cell density (⩾104 cells mm−3), continuous medium perfusion at 2.0–11.0 µL min−1 improved viability compared to non-perfused cultures (p < 0.01), which exhibited widespread cell death and matrix degradation. In perfused cultures, survival was dependent on proximity to the perfusion source at 2.00–6.25 µL min−1 (p < 0.05); however, at perfusion rates of 10.0–11.0 µL min−1 survival did not depend on the distance from the perfusion source, and resulted in a preservation of cell density with >90% viability in both neuronal cultures and neuronal–astrocytic co-cultures. This work demonstrates the utility of forced interstitial convection in improving the survival of high cell density 3D engineered neural constructs and may aid in the development of novel tissue-engineered systems reconstituting 3D cell–cell/cell–matrix interactions.