Phenotypic characterization of dhBMECs following 1-year cryopreservation. (a) Immunocytochemistry of tight junctions (claudin-5, occludin), nutrient transporters (GLUT-1), efflux pumps (P-gp), and endothelial markers (CD31, VE-cadherin). Representative images at day 2 after seeding are shown across n = 2 independent differentiations for each condition. (b) TEER values for non-frozen (n = 17) and 1-year cryopreserved (n = 7) dhBMECs. n represents the number of unique differentiations. (c) Permeability comparison of four compounds: Lucifer yellow (444 Da), 10 kDa dextran, glucose (180 Da), and rhodamine 123 (380 Da). Data represent independent differentiations for each condition, with n = 7–8 for Lucifer yellow and 10 kDa dextran, and n = 3–5 for rhodamine 123 and glucose. For independent differentiations, data represent averages across 2–6 technical replicates (individual transwells). *p < 0.05.