An official website of the United States government
Normal fibroblasts are present within the extracellular matrix (ECM). They can become activated, leading to increased proliferation and ECM protein secretion such as collagen type I to promote tissue remodeling. These cells are also involved in adult pathologies including cancer metastasis and cardiac and renal fibrosis. One source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT), in which endothelial cells lose their cell–cell and cell–ECM adhesions, gain invasive properties, and become mesenchymal cells. While EndMT is well characterized in developmental biology, the mechanisms and functional role of EndMT in adult physiology and pathology have not been fully investigated. A microfluidic device with an incorporated three-dimensional ECM chamber was developed to study the role of combined steady fluid shear stress magnitudes and transforming growth factor-βeta 1 (TGF-β1) on EndMT. Low (1 dyne per cm 2 ) steady shear stress and TGF-β1 exposure induced EndMT in endothelial cells, including upregulation of mesenchymal protein and gene expression markers. Cells exposed to TGF-β1 and high (20 dynes per cm 2 ) steady shear stress did not undergo EndMT, and protein and gene expression of mesenchymal markers was significantly downregulated. Mesenchymally transformed cells under static conditions with and without TGF-β1 showed significantly more collagen production when compared to fluidic conditions. These results confirm that both low shear stress and TGF-β1 induce EndMT in endothelial cells, but this process can be prevented by exposure to physiologically relevant high shear stress. These results also show conditions most likely to cause tissue pathology.
more »
« less
A high-throughput microtissue platform to probe endothelial function in vitro
A critical role of vascular endothelium is as a semi-permeable barrier, dynamically regulating the flux of solutes between blood and the surrounding tissue. Existing platforms that quantify endothelial function in vitro are either significantly throughput limited or overlook physiologically relevant extracellular matrix (ECM) interactions and thus do not recapitulate in vivo function. Leveraging droplet microfluidics, we developed a scalable platform to measure endothelial function in nanoliter-volume, ECM-based microtissues. In this study, we describe our high-throughput method for fabricating endothelial-coated collagen microtissues that incorporate physiologically relevant cell–ECM interactions. We showed that the endothelial cells had characteristic morphology, expressed tight junction proteins, and remodeled the ECM via compaction and deposition of basement membrane. We also measured macromolecular permeability using two optical modalities, and found the cell layers: (1) had permeability values comparable to in vivo measurements and (2) were responsive to physiologically-relevant modulators of endothelial permeability (TNF-α and TGF-β). This is the first demonstration, to the authors’ knowledge, of high-throughput assessment ( n > 150) of endothelial permeability on natural ECM. Additionally, this technology is compatible with standard cell culture equipment ( e.g. multi-well plates) and could be scaled up further to be integrated with automated liquid handling systems and automated imaging platforms. Overall, this platform recapitulates the functions of traditional transwell inserts, but extends application to high-throughput studies and introduces new possibilities for interrogating cell–cell and cell–matrix interactions.
more »
« less
- Award ID(s):
- 1704332
- PAR ID:
- 10098368
- Date Published:
- Journal Name:
- Integrative Biology
- Volume:
- 10
- Issue:
- 9
- ISSN:
- 1757-9694
- Page Range / eLocation ID:
- 555 to 565
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
null (Ed.)Drug development suffers from a lack of predictive and human-relevant in vitro models. Organ-on-chip (OOC) technology provides advanced culture capabilities to generate physiologically appropriate, human-based tissue in vitro , therefore providing a route to a predictive in vitro model. However, OOC technologies are often created at the expense of throughput, industry-standard form factors, and compatibility with state-of-the-art data collection tools. Here we present an OOC platform with advanced culture capabilities supporting a variety of human tissue models including liver, vascular, gastrointestinal, and kidney. The platform has 96 devices per industry standard plate and compatibility with contemporary high-throughput data collection tools. Specifically, we demonstrate programmable flow control over two physiologically relevant flow regimes: perfusion flow that enhances hepatic tissue function and high-shear stress flow that aligns endothelial monolayers. In addition, we integrate electrical sensors, demonstrating quantification of barrier function of primary gut colon tissue in real-time. We utilize optical access to the tissues to directly quantify renal active transport and oxygen consumption via integrated oxygen sensors. Finally, we leverage the compatibility and throughput of the platform to screen all 96 devices using high content screening (HCS) and evaluate gene expression using RNA sequencing (RNA-seq). By combining these capabilities in one platform, physiologically-relevant tissues can be generated and measured, accelerating optimization of an in vitro model, and ultimately increasing predictive accuracy of in vitro drug screening.more » « less
-
Abstract The mechanical function of the myocardium is defined by cardiomyocyte contractility and the biomechanics of the extracellular matrix (ECM). Understanding this relationship remains an important unmet challenge due to limitations in existing approaches for engineering myocardial tissue. Here, they established arrays of cardiac microtissues with tunable mechanics and architecture by integrating ECM‐mimetic synthetic, fiber matrices, and induced pluripotent stem cell‐derived cardiomyocytes (iPSC‐CMs), enabling real‐time contractility readouts, in‐depth structural assessment, and tissue‐specific computational modeling. They found that the stiffness and alignment of matrix fibers distinctly affect the structural development and contractile function of pure iPSC‐CM tissues. Further examination into the impact of fibrous matrix stiffness enabled by computational models and quantitative immunofluorescence implicates cell‐ECM interactions in myofibril assembly, myofibril maturation, and notably costamere assembly, which correlates with improved contractile function of tissues. These results highlight how iPSC‐CM tissue models with controllable architecture and mechanics can elucidate mechanisms of tissue maturation and disease.more » « less
-
null (Ed.)Abstract Purpose The endothelial glycocalyx (GCX) plays a critical role in the health of the vascular system. Degradation of the GCX has been implicated in the onset of diseases like atherosclerosis and cancer because it disrupts endothelial cell (EC) function that is meant to protect from atherosclerosis and cancer. Examples of such EC function include interendothelial cell communication via gap junctions and receptor-mediated interactions between endothelial and tumor cells. This review focuses on GCX-dependent regulation of these intercellular interactions in healthy and diseased states. The ultimate goal is to build new knowledge that can be applied to developing GCX regeneration strategies that can control intercellular interaction in order to combat the progression of diseases such as atherosclerosis and cancer. Methods In vitro and in vivo studies were conducted to determine the baseline expression of GCX in physiologically relevant conditions. Chemical and mechanical GCX degradation approaches were employed to degrade the GCX. The impact of intact versus degraded GCX on intercellular interactions was assessed using cytochemistry, histochemistry, a Lucifer yellow dye transfer assay, and confocal, intravital, and scanning electron microscopy techniques. Results Relevant to atherosclerosis, we found that GCX stability determines the expression and functionality of Cx43 in gap junction-mediated EC-to-EC communication. Relevant to cancer metastasis, we found that destabilizing the GCX through either disturbed flow-induced or enzyme induced GCX degradation results in increased E-selectin receptor-mediated EC-tumor cell interactions. Conclusion Our findings lay a foundation for future endothelial GCX-targeted therapy, to control intercellular interactions and limit the progression of atherosclerosis and cancer.more » « less
-
Microphysiological systems (MPS) incorporate physiologically relevant microanatomy, mechanics, and cells to mimic tissue function. Reproducible and standardized in vitro models of tissue barriers, such as the blood-tissue interface (BTI), are critical for next-generation MPS applications in research and industry. Many models of the BTI are limited by the need for semipermeable membranes, use of homogenous cell populations, or 2D culture. These factors limit the relevant endothelial-epithelial contact and 3D transport, which would best mimic the BTI. Current models are also difficult to assemble, requiring precise alignment and layering of components. The work reported herein details the engineering of a BTI-on-a-chip (BTI Chip) that addresses current disadvantages by demonstrating a single layer, membrane-free design. Laminar flow profiles, photocurable hydrogel scaffolds, and human cell lines were used to construct a BTI Chip that juxtaposes an endothelium in direct contact with a 3D engineered tissue. A biomaterial composite, gelatin methacryloyl and 8-arm polyethylene glycol thiol, was used for in situ fabrication of a tissue structure within a Y-shaped microfluidic device. To produce the BTI, a laminar flow profile was achieved by flowing a photocurable precursor solution alongside phosphate buffered saline. Immediately after stopping flow, the scaffold underwent polymerization through a rapid exposure to UV light (<300 mJ/cm2). After scaffold formation, blood vessel endothelial cells were introduced and allowed to adhere directly to the 3D tissue scaffold, without barriers or phase guides. Fabrication of the BTI Chip was demonstrated in both an epithelial tissue model and blood-brain barrier (BBB) model. In the epithelial model, scaffolds were seeded with human dermal fibroblasts. For the BBB models, scaffolds were seeded with the immortalized glial cell line, SVGP12. The BTI Chip microanatomy was analyzed post facto by immunohistochemistry, showing the uniform production of a patent endothelium juxtaposed with a 3D engineered tissue. Fluorescent tracer molecules were used to characterize the permeability of the BTI Chip. The BTI Chips were challenged with an efflux pump inhibitor, cyclosporine A, to assess physiological function and endothelial cell activation. Operation of physiologically relevant BTI Chips and a novel means for high-throughput MPS generation was demonstrated, enabling future development for drug candidate screening and fundamental biological investigations.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/c8ib00111a
