Design, Optimise, Standardise and Validate In Vitro Human Blood-Brain Barrier Models.

Introduction: The brain endothelial cells, pericytes, and astrocytes communicate with each other to regulate the properties of blood-brain barrier (BBB). They share a common basement membrane to mimic the anatomical, functional, and microenvironmental situation in vivo. Methods: Construction of monoculture, coculture, and triculture models were performed using human microvascular endothelial cells (hCMEC/D3), human brain vascular pericytes (HBVP), and normal human astrocytes (NHA). Human extracellular matrix (ECM) proteins combinations were optimised using collagen-IV (10 µg/ml), fibronectin (5 µg/ml), laminin (5 µg/ml), agrin (1 µg/ml), and perlecan (10 µg/ml) at the ratios (100:100:100:100:100; 50:20:20:5:5; 56:18:18:4:4; 62:16:16:3:3, v/v, %). Transepithelial/transendothelial electrical resistance (TEER), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were subsequently analysed. Results: Using 1 µm-translucent-PET in the thin-layer protocol, only at a ratio 56:18:18:4:4 is statistically significant (P<0.0016), TEER was 30±0.94 ohm*cm2. The hCMEC/D3 seeding density at 6x104 cells/ml generated the highest TEER value at day 6. The hCMEC/D3 monoculture model showed a peak TEER of 36±0.50 ohm*cm2 on day 6. TEM of endothelial cells showed close apposition to each other, with electron-dense areas at points of contact between adjacent cells, likely indicating the presence of adherent and tight junction complexes. SEM of hCMEC/D3 cells showed a confluent cell monolayer composed of closely apposed cells. HBVP seeding density at 4x104 cells/cm2 in hCMEC/D3-HBVP coculture models produced the highest TEER (41±0.97 ohm*cm2) at day 6 (p<0.0001). NHA seeding density at 4x104 cells/cm2 in hCMEC/D3-HBVP-NHA triculture-models produced the highest TEER (103±0.97 ohm*cm2) at day 6 (p<0.0001). The hCMEC/D3-HBVP-NHA triculture-model produced the highest statistically significant TEER (p<0.0001) 103±0.97 ohm*cm2 compared to the hCMEC/D3-HBVP coculture-model 41±0.49 ohm*cm2 and the hCMEC/D3 monoculture-model 35±0.97 ohm*cm2. Conclusion: The designed, optimised, standardised, and validated in vitro human BBB models were successfully constructed to achieve intact, structural, and functional BBB barrier formation. [ABSTRACT FROM AUTHOR]