Devices were removed from the PBS bath and dried before being placed on a custom 3D-printed holder. This allowed the device to be placed in any image system compatible with standard tissue culture plates. All imaging took place on an LSM 710 (Carl Zeiss) confocal microscope. Each well was imaged sequentially with each fluorophore imaged separately. Nine uniformly distributed points throughout the bioreactor were taken to generate a focus map. Interpolation was then applied to get the X, Y, and Z positions of each of the 270 wells. For the day 3 to 8 and day 4 to 9 MBAs, Z stacks were used. The Z stacks focus was ensured to be at the bottom of each well, and subsequent images in the stacks were programmed to increment up from the bottom at a 10-μm increment.

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