2.4. BEND3 cell culture

To generate a cellular model of the BBB, BEND3 cells were obtained from American Type Culture Collection (ATCC: 2299). BEND3 cells are endothelial cells that form tight barriers through the formation of transcellular tight junctions (Montesano et al., 1990; Sikorski et al., 1993; Williams et al., 1988), and these barriers mimic the barrier properties of the BBB (He et al., 2010; Watanabe et al., 2013). Their endothelial nature has been confirmed by the expression of von Willebrand factor and the uptake of fluorescently labeled low density lipoprotein. BEND3 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% FBS and 1% (100 U/ml Penicillin and 100 μg/ml Streptomycin). The cells were quickly thawed and centrifuged at 250 ×g for 8 min in 5 ml of complete media. The cell pellets were resuspended in 1 ml of complete media and plated on T-25 flask. Cells were maintained at 5% CO₂, 37 °C, and 80% humidity in a cell-culture incubator. At confluence, the cells were washed twice with 2.5 ml DPBS, trypsinized with TrypLE and centrifuged at 250 ×g for 8 min at 4 °C. The cells were then resuspended in 1 ml of complete media and plated onto T-75 flask at appropriate densities for continuous passaging of the cells.

To characterize the membrane properties of the artificial BBB formed by these cells, the cells were seeded and cultured on permeable transwells using procedures we optimized following the literature (Wuest et al., 2013). Confluent cells from T-75 flasks the cells were washed twice with 4 ml DPBS, trypsinized with TrypLE and centrifuged at 250 ×g for 8 min at 4 °C. The cells were then resuspended in 1 ml of complete media. 15 μl of cell suspension was diluted with 15 μl of 0.4 trypan blue solution and a cell count was taken using a Countess II automated cell counting machine (MAN 0014293, Life Technologies, CA). The cells were diluted and seeded onto Costar permeable 12-well collagen coated membrane transwells with a 3 μm pore size (Corning; Corning, NY) a density of 4 × 10⁶ cells/insert in 0.5 ml of complete media. 1.5 ml of complete media was put on the basolateral side of the transwell while 0.5 ml of cell suspension was put into the apical side of the transwell insert. The media on both apical and basolateral sides of the transwell was changed every other day with complete media. All cells used for this work were below passage 33.