Figure 4.

Effects of perfusion on culture health. (A) Confocal z-stack through the full 3-D cell culture thickness projected onto a single plane. The color bar below the figure indicates the z-location of live stained cells across the culture thickness. The culture was 500 μm thick at plating and cell depth is shown at 6 DIV. Perfusion maintained good cellular viability and 3-D cell culture thickness. (B) A depth-colored projection of a perfused 3-D cell culture showing cells that have grown in three dimensions. (C) 3-D rendering of the confocal z-stack in (A) taken through the full culture thickness. Live cells stained with an AM dye are shown in green. The location of the pMEA with corresponding perforations and recording sites is shown for reference in gray color. (D) Live cell volume fraction for perfused (+P, N = 4) and unperfused (−P, N = 5) 3-D cell cultures as a percentage of the original plating volume. The blue bars represent the mean value and the error bar range is mean ± SEM. (E) Live cell density for perfused (+P, N = 4) and unperfused (−P, N = 4) brain slice cultures. Slices were pooled from all experiments and normalized by original thickness at plating. The gray bars represent the mean value and the error bar range is mean ± SEM. (F) Final culture thicknesses as a percentage of the thickness at plating for perfused (+P, N = 4) and unperfused (−P, N = 4) slices and 3-D cell cultures (+P, N = 4; −P, N = 5). Gray and blue bars are represent the mean and the error bar range is mean ± SEM. When perfused, both slices and 3-D cell cultures retained more of their thickness at the end of the experiment (^*P < 0.05, Wilcoxon rank-sum test). The maximum thickness was taken, which could be greater than the intended thickness at plating due to unevenness at the top surface (see Methods).