Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

1. Triple cell culture BBB model setting

1. Seeding pericytes
   1. Cultivate HBVP (Human brain vascular pericytes) in T75 culture flasks with an activated surface for cell adhesion within a 5% CO₂ incubator at 37 °C until confluent. Once confluence is reached, aspirate the old pericyte medium and wash the cells with 5 mL of warm Dulbecco's phosphate-buffered saline (DPBS). Aspirate the DPBS and detach the cells from the flask using a combination of 4 mL of warm trypsin-EDTA solution and 1 mL of DPBS.
      ​NOTE: Avoid using passages later than P7.
   2. Incubate the flask for 5 min at 37 °C in a CO₂ incubator. View under a microscope to confirm whether the cells are detached from the flask. Add 5 mL of warm pericyte medium (containing 2% fetal bovine serum [FBS]) to the flask and transfer the detached cells to a 50 mL centrifuge tube.
   3. Centrifuge the cell suspension for 3 min at 200 × g, allowing the cells to form a pellet in the bottom of the tube. Aspirate the medium from the tube, ensuring the cell pellet remains intact.
   4. Resuspend the cell pellet in pericyte medium; calculate the amount of medium depending on the confluence of the cells and the number of well-inserts needed. Take 10 µL of the resuspended cells, place them into a cell counting slide, and count the number of cells.
   5. Determine the cell density and seed 300,000 cells/insert in 1 mL of pericyte medium onto the abluminal side of the well-inserts (6-well format).
      NOTE: When seeding HBVP on the underside of inserts, it is important to flip the well-insert plates upside down. While the plate is laid flat on a surface, remove the bottom section to expose the abluminal side of the well-inserts. Cover the well-inserts with the flipped plate after adding pericyte cell suspension onto the abluminal side to prevent evaporation. Keep all plates turned upside down in a 5% CO₂ incubator at 37 °C overnight.
2. Seeding astrocytes
   1. Cultivate HA (Human astrocytes) in T75 flasks within a 5% CO₂ incubator at 37 °C until confluence is reached. Follow the above steps 1.1.1-1.1.4 using astrocyte medium (also containing 2% FBS) instead of pericyte medium.
      ​NOTE: Avoid using passages later than P9.
   2. Determine the cell density and seed 300,000 cells/well onto the bottom of the tissue culture 6-well plates. Cover the plate to prevent evaporation and keep all plates in a 5% CO₂ incubator at 37 °C overnight.
3. Seeding endothelial cells
   1. Cultivate HBMEC (Human brain microvascular endothelial cells) in tissue culture dishes within a 5% CO₂ incubator at 37 °C until confluent. Follow the above steps 1.1.1-1.1.4 using a complete classic medium (containing 10% FBS) instead of a pericyte medium.
      NOTE: Avoid using passages later than P12.
   2. Take out the tissue culture 6-well plates containing astrocytes and the well-inserts (6-well format) containing pericytes from the 5% CO₂ incubator at 37 °C. Aspirate the astrocyte medium from the tissue culture 6-well plates. Add 1 mL of pericyte medium and 1 mL of astrocyte medium to each well.
   3. Aspirate the pericyte medium from the well inserts and place it into the tissue culture 6-well plates containing the seeded astrocytes. Seed HBMEC at a density of 300,000 cells/well in 2 mL of the complete classic medium onto the apical side of the same well inserts.
      NOTE: The endothelial cells should be seeded on the apical side of the well-inserts the next day after seeding pericytes on the abluminal side of the well-inserts and astrocytes on tissue culture 6-well plates. Cells should be maintained in triple culture for six days to induce BBB-like properties. The cell culture medium should be changed in both well-insert compartments 24 hours before experiments.