Abstract Background Plasma gelsolin (pGSN) is an important part of the blood actin buffer that prevents negative consequences of possible F-actin deposition in the microcirculation and has various functions during host immune response. Recent reports reveal that severe COVID-19 correlates with reduced levels of pGSN. Therefore, using an in vitro system, we investigated whether pGSN could attenuate increased permeability of the blood–brain barrier (BBB) during its exposure to the portion of the SARS-CoV-2 spike protein containing the receptor binding domain (S1 subunit). Materials and methods Two- and three-dimensional models of the human BBB were constructed using the human cerebral microvascular endothelial cell line hCMEC/D3 and exposed to physiologically relevant shear stress to mimic perfusion in the central nervous system (CNS). Trans-endothelial electrical resistance (TEER) as well as immunostaining and Western blotting of tight junction (TJ) proteins assessed barrier integrity in the presence of the SARS-CoV-2 spike protein and pGSN. The IncuCyte Live Imaging system evaluated the motility of the endothelial cells. Magnetic bead-based ELISA was used to determine cytokine secretion. Additionally, quantitative real-time PCR (qRT-PCR) revealed gene expression of proteins from signaling pathways that are associated with the immune response. Results pGSN reversed S1-induced BBB permeability in both 2D and 3D BBB models in the presence of shear stress. BBB models exposed to pGSN also exhibited attenuated pro-inflammatory signaling pathways (PI3K, AKT, MAPK, NF-κB), reduced cytokine secretion (IL-6, IL-8, TNF-α), and increased expression of proteins that form intercellular TJ (ZO-1, occludin, claudin-5). Conclusion Due to its anti-inflammatory and protective effects on the brain endothelium, pGSN has the potential to be an alternative therapeutic target for patients with severe SARS-CoV-2 infection, especially those suffering neurological complications of COVID-19.
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This content will become publicly available on December 1, 2024
Simple design for membrane-free microphysiological systems to model the blood-tissue barriers
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.
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- NSF-PAR ID:
- 10493593
- Publisher / Repository:
- Elsevier
- Date Published:
- Journal Name:
- Organs-on-a-Chip
- Volume:
- 5
- Issue:
- C
- ISSN:
- 2666-1020
- Page Range / eLocation ID:
- 100032
- Subject(s) / Keyword(s):
- ["organ-on-a-chip","microfluidics","microphysiological system"]
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Background Organ‐on‐chip technology has accelerated in vitro preclinical research of the vascular system, and a key strength of this platform is its promise to impact personalized medicine by providing a primary human cell–culture environment where endothelial cells are directly biopsied from individual tissue or differentiated through stem cell biotechniques. However, these methods are difficult to adopt in laboratories, and often result in impurity and heterogeneity of cells. This limits the power of organ‐chips in making accurate physiological predictions. In this study, we report the use of blood‐derived endothelial cells as alternatives to primary and induced pluripotent stem cell–derived endothelial cells. Methods and Results Here, the genotype, phenotype, and organ‐chip functional characteristics of blood‐derived outgrowth endothelial cells were compared against commercially available and most used primary endothelial cells and induced pluripotent stem cell–derived endothelial cells. The methods include RNA‐sequencing, as well as criterion standard assays of cell marker expression, growth kinetics, migration potential, and vasculogenesis. Finally, thromboinflammatory responses under shear using vessel‐chips engineered with blood‐derived endothelial cells were assessed. Blood‐derived endothelial cells exhibit the criterion standard hallmarks of typical endothelial cells. There are differences in gene expression profiles between different sources of endothelial cells, but blood‐derived cells are relatively closer to primary cells than induced pluripotent stem cell–derived. Furthermore, blood‐derived endothelial cells are much easier to obtain from individuals and yet, they serve as an equally effective cell source for functional studies and organ‐chips compared with primary cells or induced pluripotent stem cell–derived cells. Conclusions Blood‐derived endothelial cells may be used in preclinical research for developing more robust and personalized next‐generation disease models using organ‐on‐chips.more » « less
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