Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells

Bhatt, Aadra P.; Gunasekara, Dulan B.; Speer, Jennifer; Reed, Mark I.; Peña, Alexis N.; Midkiff, Bentley R.; Magness, Scott T.; Bultman, Scott J.; Allbritton, Nancy L.; Redinbo, Matthew R.

doi:10.1021/acsinfecdis.7b00139

Cited by 55 publications

(42 citation statements)

References 44 publications

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“…In this work, we characterize a conventional scaffold system and a new gradient cross-linked scaffold method for the measurement of drug transport in primary, human small intestinal epithelium [24, 25, 35–39]. Collagen was used as a substrate in both systems since it is the most abundant ECM in the intestine [21]; however, the two systems possessed a scaffold thickness many orders of magnitude different.…”

Section: Introductionmentioning

confidence: 99%

Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking

et al. 2019

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Background: The luminal surface of the small intestine is composed of a monolayer of cells overlying a lamina propria comprised of extracellular matrix (ECM) proteins. The ECM provides a porous substrate critical for nutrient exchange and cellular adhesion. The enterocytes within the epithelial monolayer possess proteins such as transporters, carriers, pumps and channels that participate in the movement of drugs, metabolites, ions and amino acids and whose function can be regulated or altered by the properties of the ECM. Here, we characterized expression and function of proteins involved in transport across the human small intestinal epithelium grown on two different culture platforms. One strategy employs a conventional scaffolding method comprised of a thin ECM film overlaying a porous membrane while the other utilizes a thick ECM hydrogel placed on a porous membrane. The thick hydrogel possesses a gradient of chemical cross-linking along its length to provide a softer substrate than that of the ECM film-coated membrane while maintaining mechanical stability. Results: The monolayers on both platforms possessed goblet cells and abundant enterocytes and were impermeable to Lucifer yellow and fluorescein-dextran (70 kD) indicating high barrier integrity. Multiple transporter proteins were present in both primary-cell culture formats at levels similar to those present in freshly isolated crypts/villi; however, expression of breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the monolayers on the conventional scaffold was substantially less than that on the gradient cross-linked scaffold and freshly isolated crypts/villi. Monolayers on the conventional scaffold failed to transport the BCRP substrate prazosin while cells on the gradient cross-linked scaffold successfully transported this drug to better mimic the properties of in vivo small intestine. Conclusions:The results of this comparison highlight the need to create in vitro intestinal transport platforms whose characteristics mimic the in vivo lamina propria in order to accurately recapitulate epithelial function.

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Section: Introductionmentioning

confidence: 99%

Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking

et al. 2019

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“…Changes in TEER between day 4 and day 6 were evaluated by calculating ΔTEER% as follows: ΔTEER% =100- ([(TEER day4 – TEER day6 )/TEER day4 ] × 100). 47 …”

Section: Methodsmentioning

confidence: 99%

“…Epithelial permeability was evaluated in 2D-cultured human primary IEC monolayers as we reported previously. 47 Primary IECs cultured on 48-well plates were passaged to 12-well cell culture inserts (353180; BD Falcon, San Jose, CA) coated with collagen scaffolds with a gradient of cross-linking 48 at a ratio of 1:1. Cells were expanded in EM for 4 days and stimulated with 10 ng/mL BMP9 alone or in combination with 100 ng/mL ALK1-Fc chimera protein in EM or cultured in differentiation medium (DM) as a positive control of colonocyte differentiation 44 and TEER increase for an additional 2 days.…”

Section: Epithelial Permeability Assaymentioning

confidence: 99%

Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn’s Disease

Toyonaga

Steinbach

Keith

et al. 2020

Cellular and Molecular Gastroenterology and Hepatology

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Activin receptor-like Kinase 1 (ALK1) was identified as a target of microRNA-31-5p in human colonic epithelial cells. Activation of ALK1 enhanced epithelial barrier function ex vivo. Decreased colonic ALK1 was associated with poor clinical outcomes in patients with Crohn's disease. associated with a higher risk of endoscopic relapse in CD patients. CONCLUSIONS: Decreased colonic ALK1 disrupted colonic IEC barrier integrity and was associated with poor clinical outcomes in CD patients.

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“…The monolayers display a characteristic polarized morphology (luminal/basal) and markers of mature intestinal epithelium such as brush border proteins and tight junctions (Figure 3). These monolayers typically form a contiguous, impermeable layer with a transepithelial electrical resistance (TEER) that is sufficient to support physiological assays such as IgA transcytosis [26], chemokine (CXCL10) secretion [55], inflammatory cytokine (IFN-a, TNF-a, IL-6, IL-8) synthesis [56,57], cytotoxicity, barrier function [57,58], ion transport [54,59], and hormone production (serotonin, GLP-1, FGF19) [54].…”

Section: Box 3 Support Matrixmentioning

confidence: 99%

Primary Cell-Derived Intestinal Models: Recapitulating Physiology

Dutton

Hinman

Kim

et al. 2019

Trends in Biotechnology

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The development of physiologically relevant intestinal models fueled by breakthroughs in primary cell-culture methods has enabled successful recapitulation of key features of intestinal physiology. These advances, paired with engineering methods, for example incorporating chemical gradients or physical forces across the tissues, have yielded ever more sophisticated systems that enhance our understanding of the impact of the host microbiome on human physiology as well as on the genesis of intestinal diseases such as inflammatory bowel disease and colon cancer. In this review we highlight recent advances in the development and usage of primary cell-derived intestinal models incorporating monolayers, organoids, microengineered platforms, and macrostructured systems, and discuss the expected directions of the field. Current Approaches to Modeling Intestinal Physiology The small and large intestine, located after the stomach, comprise the lower human gastrointestinal tract and play crucial roles in nutrient absorption and in housing much of the human microbiome (see Glossary) (Figure 1, Key Figure). In the past decade, model systems have attempted to recapitulate the complex, in vivo intestinal physiology using cell lines derived from intestinal tumors such as Caco-2 cells in place of primary epithelial cells. Advanced organ-on-achip systems were created by culturing Caco-2 cells on the geometrically or mechanically engineered platforms to properly mimic the structural and mechanical properties of the human intestine [1-6]. To mimic the mucosal architecture, porous scaffolds were micromolded to villus-like projections on which Caco-2 cells could be cultured [1,6]. To recapitulate the mechanically dynamic environment, microfluidic systems were developed with fluid flowing both above and below a Caco-2 cell layer growing on a rhythmically stretched flexible surface [2-5]. These systems were designed to mimic the shear forces and contractile motions occurring in the small intestine. Caco-2 cells, as well as other tumor cell lines, have also been used as surrogate intestinal epithelial cells to probe the interactions between multiple tissue types [7]. These organ-on-a-chip models incorporating tumor cells offer new abilities to emulate the structure, function, and physiology of the living human intestine that are not possible with conventional tissue-cultured monolayers. However, as our understanding of these organs progresses, it is clear that these prior tumor model systems fall short in their ability to accurately reflect in vivo physiology because the models do not possess all of the intestinal epithelial subtypes and either lack receptors, transporters, drug-metabolizing enzymes, or express these proteins at levels different from in vivo. Thus, in vitro replicas of the intestines that more accurately replicate intestinal physiology are required and will need to utilize primary cells. Accordingly, a suite of platforms employing primary cells in a variety of assay formats, including organoid [8,9], monolayer, and shape...

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Nonsteroidal Anti-Inflammatory Drug-Induced Leaky Gut Modeled Using Polarized Monolayers of Primary Human Intestinal Epithelial Cells

Cited by 55 publications

References 44 publications

Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking

Molecular transport through primary human small intestinal monolayers by culture on a collagen scaffold with a gradient of chemical cross-linking

Decreased Colonic Activin Receptor-Like Kinase 1 Disrupts Epithelial Barrier Integrity in Patients With Crohn’s Disease

Primary Cell-Derived Intestinal Models: Recapitulating Physiology

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