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 microm) 3D neural cultures supported by passive diffusion, cell densities <or=5.0 x 10(3) cells mm(-3) were required for survival. In 3D neuronal and neuronal-astrocytic co-cultures with increased cell density (10(4) cells mm(-3)), continuous medium perfusion at 2.0-11.0 microL 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 microL min(-1) (p < 0.05); however, at perfusion rates of 10.0-11.0 microL 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.