Development of a Blood–Brain Barrier Permeability Assay Using Human Induced Pluripotent Stem Cell Derived Brain Endothelial Cells

Mouse embryonic stem cell-derived blood–brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis

“…Amniotic fluid cell (HAF) derived iPSC (HAF-iPSC) were differentiated into brain endothelial like cells (iBECs), as previously described [ 10 , 16 ]. In brief, during the initial pre-differentiation step, once the HAF-iPSCs reached 60–70% confluency the medium was switched from mTeSR1 to low osmolality KOEB medium composed of KnockOut DMEM/F12 medium (Thermo Fisher Scientific) supplemented with 20% Knock-Out serum replacement, 1 X Glutamax, 1X non-essential amino acids and 55 µM β-mercaptoethanol (all from Thermo Fisher Scientific, Waltham, Massachusetts) for 5–7 days.…”

Application of blood brain barrier models in pre-clinical assessment of glioblastoma- targeting CAR-T based immunotherapies

“…All experimental protocols using human amniotic fluid derived induced pluripotent stem cells (AF-iPSCs) were performed following the guidelines established and approved by the National Research Council Canada Research Ethics Board and the in accordance with relevant guidelines and regulations as approved by the Ottawa Hospital Research Ethics Board. AF-iPSC were generated from human amniotic fluid (AF) cells and differentiated into iBECs, as previously described [32,33]. In brief, AF-iPSC were seeded at a density 8 x 10 3 cells/cm 2 in DMEM/F12 medium (Life Technologies) supplemented with 20% KnockOut Serum Replacement, 1x Glutamax, 1x Non-Essential Amino Acids, and 0.1 mM β-mercaptoethanol (all from Life Technologies) for 6 days.…”

“…The medium was changed to EM medium (human Endothelial Serum-Free medium, Life Technologies), 20 ng/ml basic fibroblast growth factor (bFGF, Life Technologies), 10 μM retinoic acid (RA, Sigma) and 1% fetal bovine serum (FBS, Hyclone) for an additional 2 days. To establish the in vitro transwell BBB model, iBECs were dissociated with Accutase (Stem Cell Technologies) and seeded at density of 2.5 x 10 5 cells per 24 well transwell insert (3 µm pore size, 0.33 cm 2 surface area; BD Falcon) pre-coated with collagen type-IV (80 µg/ml, Sigma) and fibronectin (20 µg/ml, Sigma) in complete EM medium with 10 µM Y27362 (ROCK Inhibitor, Stem Cell Technologies), as previously described [32]. iBECs transwells were incubated overnight at 37°C in 5% CO 2 and the next day the medium was changed to EM medium without bFGF and RA for an additional 24 hours in the luminal chamber.…”