Notably, 21 of the 22 BBB transporters and receptors analyzed in this study tended to have higher expressions in ciBECs compared with normal ECs, which were not co-cultured with astrocytes and neurons. Of the BBB-specific transporters analyzed, six, including cationic amino acid transporter 3 (CAT3), MFSD2A, which is a transporter for the essential ω-3 fatty acid docosahexaenoic acid (DHA), and the polarized efflux transporters, ATP-binding cassette transporters ABCA1, BCRP, PGP, and MRP5, were significantly increased in ciBECs (Figure 2D). We compared the properties of the ciBECs with those of the immortalized brain microvascular endothelial cell line, hCMEC/D3, and human umbilical vein endothelial cells (HUVEC). The expressions of CAT1, PGP, and Transferrin receptor in ciBECs and hCMEC/D3 are similar. However, the expressions of efflux transporters such as ABCA1, BCRP, and MRP5 are higher in ciBECs than in hCMEC/D3 and HUVEC (Figure 2D). Immunostaining further showed that BCRP and PGP were highly expressed in ciBECs compared with ECs (Figure 2E). We next examined how these expressions changed with the culture. The co-culture of neurons (stage 3 in Figure 1I) with ECs and pericytes partially increased BBB-specific transporters and receptors. In contrast, co-culture of astrocytes (stage 5 in Figure 1I) with ECs and
