An ‘all human’ 3D in vitro blood brain barrier model in nanoparticles delivery and cancer metastasis studies

Abstract:

Background: The blood brain barrier (BBB) is a dynamic functional unit consisting of endothelial cells, astrocytes and pericytes embedded in a specialised basal lamina. Most in vitro studies examine therapeutics crossing the BBB using monolayer cultures or mixed species co-cultures and rely on Trans-endothelial Electrical Resistance (TEER) measurement without verification as tight-junction (TJ) formation. More reliable high throughput in vitro models are therefore crucial in brain tumour drug delivery and cancer metastasis studies. We developed a human 3-dimensional in vitro BBB model incorporating BBB in vivo characteristics and are using it for delivery and metastasis studies.


Methods: The 3D model was constructed using human brain endothelial cells (hCMEC/D3), human cortical astrocytes (SC-1800) and human brain vascular pericytes (HBVP) in mono-, co-, and tri- cultures. TEER values were measured using a cell monitoring system- cellZscope®. TJ proteins expression was examined by Western blotting (WB) and immunocytochemistry (ICC). ECM molecules were assessed for their effects of endothelial cell adhesion and TEER changes using an Electric Cell-Substrate Impedance Sensing (ECISTM) system. Nanoparticles (NPs) and metastatic lung cancer cells were tested for effects on BBB integrity.


Results: Co-cultures of E+A resulted in significantly higher TEER values compared to endothelial cells alone (p<0.05). These results correlated with both WB and ICC data revealing higher expression of occludin in co-cultures. Perlecan pre-coated wells resulted in highest cell adhesion (%) and TEER values. Recovery time of BBB integrity following addition of NPs/ metastatic cell treatment differed in mono-, co- and tri- cultures.
Conclusions: Astrocytes contributed to BBB formation in this in vitro model. ECM had varied and profound effects on TEER values. This new model may be used to gain more reliable data on BBB penetration by novel and re-purposed drugs as well as informing on potential targets for prevention of brain metastasis.

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