Category: Assay Development and Screening

1200-A - A High-Throughput Platform to Screen for Modulators of Endothelial Permeability

Monday, February 5, 2018
2:00 PM - 3:00 PM

Dysfunction of the endothelial barrier is a hallmark of inflammatory processes and associated with many severe diseases (e.g. atherosclerosis, rheumatoid arthritis, and asthma). The standard measurements of endothelial dysfunction in vitro (e.g. macromolecular permeability of monolayers grown on transwell inserts) are severely limited in throughput and overlook physiologically relevant ECM interactions. Using endothelial layers cultured on miniaturized protein-based 3D microtissues, we have developed a high throughput platform to quantify endothelial permeability that alleviates throughput limitations, enhances statistical power, and preserves ECM interactions. We use a microfluidic flow focusing device to rapidly generate the microtissues, which are then coated with endothelial cells and cultured in microwells that are microfabricated within multi-well plates. We verify the endothelial cell coating protocol by examining cell morphology and intercellular interactions (e.g. CD31, VE-cadherin) with immunofluorescence of endothelial-coated collagen microtissues. To quantify endothelial permeability, we incubate microtissues with fluorescently labeled macromolecules and capture high resolution optical sections, from which concentration gradients can be quantified. Using this method, we have shown that human umbilical vein endothelial cells have a permeability ~10^-8 cm/s, which is comparable to permeability of some vessels measured in vivo. We also developed a second imaging modality that relies on low-magnification wide-field imaging that allows for rapid comparison over many conditions, which is useful for high-throughput screening applications. We have demonstrated that this platform is compatible with a range of endothelial cell types, and the use of standard well plates makes the microtissues compatible with standard high throughput screening equipment. We will continue exploring the potential of this platform, measuring the effect of inflammatory cytokines, hormones, and small molecule therapeutics. Additionally, the inter-cellular interactions of stromal cells with the endothelium can be evaluated by encapsulating stromal cells in the ECM of the microtissues, transforming this into a co-culture model system with increased physiologic relevance.

Alexandra L. Crampton

Graduate Student Researcher
University of Minnesota
Minneapolis, MN

Alexandra is a graduate student researcher at the University of Minnesota in the Biomedical Engineering Department. She completed her undergraduate degree in Biomedical Engineering at the University of California, Irvine in 2013. Her research interests are using microfluidic systems to create physiologically relevant in vitro model systems that are amenable to high-throughput applications.