| 生长方案: |
The hiPSC line WISCi004-B/IMR90-4 was used to differentiate BCECs. a single cell suspension of hiPSCs was prepared with Accutase. The cell suspension was centrifuged at 300 x g for 5 min and cells were seeded in mTeSRTM1 supplemented with 10 µM Y-27632 onto Matrigel-coated 6-well plates. The seeding density was optimized for each hiPSC line. For the differentiation of hiPSCs to BCECs, the neurodevelopmental process in vivo has to be recapitulated in vitro. For brain-capillary endothelial-like cells, a co-differentiation of neural and endothelial cells was initiated by treatment with so-called unconditioned medium. After 2-3 days when optimal cell densities of 2-4x104 cells/cm2 were reached, medium was switched to unconditioned medium in order to initiate co-differentiation of BCECs and neuronal cells (referred to as day 0 of differentiation throughout the manuscript). Unconditioned medium was composed of 78.5% DMEM/F12 , 20% KnockOutTM serum replacement, 1% MEM NEAA, 0.5% L-glutamine, and 0.1 mM β-mercaptoethanol. A daily change of unconditioned medium for 5 days was followed by double feeding at day 6 with endothelial cell (EC) medium supplemented with 20 ng/ml hbFGF and 10 µM retinoic acid to expand the BCECs. EC medium was composed of Human Endothelial-SFM and, if not stated otherwise, 0.5% B27 Supplement as described recently (Neal et al., 2019). After 48 h without medium change, cells were dissociated with Accutase for 30 min and seeded at a cell density of 1x106 cells/cm2 onto collagen IV/fibronectin-coated transwell insert in EC medium supplemented with 20 ng/mL hFGF and 10 µM RA. This seeding step at day 8 in combination with the collagen IV/fibronectin-coating allows for an efficient purification of BCECs. At day 9, hiPS-BCECs were adapted to EC medium without hFGF and RA (in transwell systems with 200 µl of medium apical and 800 µl basolateral) for 24 h. TEER measurements were performed to evaluate the integrity of the generated in vitro BBB to be used for further applications. |
