A Human iPSC-derived Microphysiological Blood-Brain Barrier Model for Permeability Screening
A Human iPSC-derived Microphysiological Blood-Brain Barrier Model for Permeability Screening
hiPSC-derived endothelial cells were generated using a previously published protocol. hiPSCs from three different lines r-iPSC1J, ChiPS22 (TakaraBio) and C9 were seeded at 15,500 17,000 and 17,000 cells/cm2, respectively, in DEF-CS 24 hours before the start of differentiation. Differentiation was initiated by changing the medium to Essential 6 (E6) media (Gibco). Cultures were given fresh E6 daily, for four days. On day four, E6 was changed to endothelial cell media [EC-media, EC-SFM (Life Technologies), 1 % platelet-poor serum (Alfa Aesar), 10 µM retinoic acid (Sigma Aldrich) and 20 ng/ml bFGF (Peprotech)]. On day six cells were passaged at 1,000,000 cells/cm2 onto collagen (400 µg/ml, Sigma Aldrich)/fibronectin (100µg/ml Sigma Aldrich)-coated 24-well 0.4 µm pore polyester membrane Transwell inserts (Corning) or 6-well plates (Corning). After 24 hours, on day seven, the media was changed to EC-media without bFGF and retinoic acid. The next day, on day 8, cells were dissociated and cryopreserved in 90% fetal bovine serum (Gibco), 10 % DMSO (Sigma Aldrich) or seeded in OrganoPlates at 20,000 cells/µl.  Immortalized human BEC CMEC/D3 (Merck) were maintained according to the vendor's instructions on collagen I (Sigma) coated plates in EndoGro (Millipore) media.  Human umbilical vein endothelial cells (HUVECs) (Lonza) were maintained according to the vendors instructions in EGM2 growth media (Lonza). Samples for RNA sequencing were collected after 48h after confluence was reached. Fifteen samples from Transwell (2D) and fifteen from OrganoPlate (3D) were taken, each including three replicates from C9, r-iPSC1J, ChiPS22, HUVECs and CMEC, respectively. Approximately 1µg RNA per sample was collected and RNA was purified using the RNeasy Mini Kit (Qiagen) with DNase treatment according to the manufacturer's instructions. Sequencing library construction was performed using Illumina TruSeq stranded mRNA with poly-A selection.