For diffusion studies, we used a device previously seeded with neurons and glia and cultured for 6 weeks. Devices were placed on a heated stage (kept at 37°C). About 150 μl of fresh media was added to the central chamber, and 21.8 μl of media was added to one of the side chambers. Fluorescein was added to the side chamber (13.2 μl) to generate a final concentration of 1 mg/ml fluorescein. Time-lapse images were collected every 15 seconds on a Zeiss confocal microscope (Zeiss LSM 700, Carl Zeiss, Dublin, CA) at excitation wavelengths of 488 (for fluorescein) and 563 (for tdTomato). Images were processed in the Fiji package of ImageJ30. Diffusion distance was measured from the channel entrance to the diffusion front for every other channel in each sequential frame up to the 10th frame. The breakthrough time was determined by averaging all microchannels in which breakthrough (diffusion distance equaling 200 μm) was seen. A least-squares fit of the model ( l=4Dt) was performed to determine the diffusion coefficient of fluorescein. The diffusion was repeated in multiple side chambers and the data were pooled.
