Abstract W P407: Shear Stress and Co-culture with Astrocytes Determine Brain Microvascular Endothelial Cell Phenotype
Introduction: Dementia has classically been identified to be of either vascular or neural origin. These domains overlap and are complementary, thus we consider dementia a disease of the single cerebrovascular unit. Our objective was to generate a modular platform for co-culture of brain microvascular endothelial cells and astrocytes that also incorporates mechanical stresses, and to then use this model of the cerebrovascular unit microenvironment to study the unit in vitro.
Hypothesis: We assessed the hypothesis that endothelial health and blood-brain barrier integrity are modulated by shear stress and co-culture with astrocytes.
Methods: We lithographed polydimethylsiloxane substrate on Teflon negative molds with subjacent rectangular channels of 0.45 and 2 mm depth for seeding of human brain microvascular cells and astrocytes, respectively, separated by a polytetrafluoroethylene (0.45 μm pore size) membrane under no flow or physiologic flow (6.2 dynes/cm2) for one week. Immunocytochemical staining for glial fibrillary acidic protein and CD31 was simultaneously visualized by confocal microscopy. Cells from each channel were detached via trypsinization, and expression of transport proteins P-glycoprotein (P-gp) and glucose transporter-1 (GLUT-1), in addition to junction proteins zona occludens-1 (ZO-1) and CD31, was measured by Western Blot.
Results: We stably co-cultured brain microvascular endothelial cells and astrocytes with no chamber leakage or mixing. CD31 staining revealed endothelial cell alignment to direction of flow. Expression of ZO-1 by endothelial cells increased in presence of flow and co-culture independently, by 1.6-fold in combined conditions relative to static monoculture (p<0.05). For P-gp, the increase in combined conditions was 5.5-fold (p<0.05). GLUT-1 and CD31 levels did not change significantly with co-culture or flow.
Conclusion: Cell biology devoid of microenvironmental cues provides limited insight, especially when considering whole tissues, on the impact of disease. A co-culture system that introduces multiple cells, flow, controlled stress and independent visualization and sampling of each cell domain adds deeper understanding and greater value to in vitro biological models and tissue biology.
- Endothelial function
- Cerebrovascular disorders
- Cerebrovascular biology
- Cerebrovascular circulation
- Vascular disease
Author Disclosures: S. Salehi Omran: None. F. Garcia Polite: None. E. Edelman: None. M. Balcells-Camps: None.
- © 2015 by American Heart Association, Inc.