3D Membrane-free Microfluidic BMPS Construction

The OrganoPlate (MIMETAS, Netherlands) was used to construct the 3D membrane-free microfluidic BMPS and the tissue construct method was described previously [4 (link)]. Briefly, collagen solution (Corning, Type I Rat Tail) was diluted in DMEM and the pH was neutralized to 7.0–7.4. Collagen preparation was performed on ice. N2a, C8-D1A, and BV-2 cells were re-suspended in the ECM at the following concentrations: N2a – 3.12 x 10⁶ cells/mL, C8-D1A – 3.12 x 10⁶ cells/mL, and BV-2–1.56 x 10⁶ cells/mL. The cell-ECM mixture was added to the gel lane and incubated (37°C, 5% CO₂) for 1 h for gel polymerization. After polymerization, bEnd.3 cells were dispensed into the medium lane at a concentration of 1 × 10⁷ cells/mL in DMEM. The plate was incubated against the side of the incubator at a 75° angle for 4 h to allow bEnd.3 cells to settle against the ECM. Then, 50 μL medium was added to the medium inlet and outlet. The plate was placed on an interval rocker (MIMETAS, The Netherlands) for medium perfusion inside the incubator (37°C, 5% CO₂). The rocker (switching between +7° and −7° inclination every 8 min) created a bi-directional flow with a mean flow rate of 2.02 μL/min and a mean shear rate of 0.13 Pa. Medium (50 μL each in the inlet and outlet) was refreshed every other day.