Transepithelial Electrical Resistance Measurements Research Articles

The barrier-protective effect of β-eudesmol against type 2-inflammatory cytokine-induced tight junction disassembly in airway epithelial cells.

Allergic inflammation, which is the pathogenesis of allergic rhinitis and asthma, is associated with disruption of the airway epithelial barrier due to the effects of type 2 inflammatory cytokines, i.e. interleukin-4 and interleukin-13 (IL-4/13). The anti-allergic inflammatory effect of β-eudesmol (BE) on the tight junction (TJ) of the airway epithelium has not previously been reported. Herein, the barrier protective effect of BE was determined by measurement of transepithelial electrical resistance and by paracellular permeability assay in an IL-4/13-treated 16HBE14o- monolayer. Pre-treatment of BE concentration- and time- dependently inhibited IL-4/13-induced TJ barrier disruption, with the most significant effect observed at 20 μM. Cytotoxicity analyses showed that BE, either alone or in combination with IL-4/13, had no effect on cell viability. Western blot and immunofluorescence analyses showed that BE inhibited IL-4/13-induced mislocalization of TJ components, including occludin and zonula occludens-1 (ZO-1), without affecting the expression of these two proteins. In addition, the mechanism of the TJ-protective effect of BE was mediated by inhibition of IL-4/13-induced STAT6 phosphorylation, in which BE might serve as an antagonist of cytokine receptors. In silico molecular docking analysis demonstrated that BE potentially interacted with the site I pocket of the type 2 IL-4 receptor, likely at Asn-126 and Tyr-127 amino acid residues. It can therefore be concluded that BE is able to prevent IL-4/13-induced TJ disassembly by interfering with cytokine-receptor interaction, leading to suppression of STAT6-induced mislocalization of occludin and ZO-1. BE is a promising candidate for a therapeutic intervention for inflammatory airway epithelial disorders driven by IL-4/13.

Cryopreservation and Rapid Recovery of Differentiated Intestinal Epithelial Barrier Cells at Complex Transwell Interfaces Is Enabled by Chemically Induced Ice Nucleation.

Cell-based models, such as organ-on-chips, can replace and inform in vivo (animal) studies for drug discovery, toxicology, and biomedical science, but most cannot be banked "ready to use" as they do not survive conventional cryopreservation with DMSO alone. Here, we demonstrate how macromolecular ice nucleators enable the successful cryopreservation of epithelial intestinal models supported upon the interface of transwells, allowing recovery of function in just 7 days post-thaw directly from the freezer, compared to 21 days from conventional suspension cryopreservation. Caco-2 cells and Caco-2/HT29-MTX cocultures are cryopreserved on transwell inserts, with chemically induced ice nucleation at warmer temperatures resulting in increased cell viability but crucially retaining the complex cellular adhesion on the transwell insert interfaces, which other cryoprotectants do not. Trans-epithelial electrical resistance measurements, confocal microscopy, histology, and whole-cell proteomics demonstrated the rapid recovery of differentiated cell function, including the formation of tight junctions. Lucifer yellow permeability assays confirmed that the barrier functions of the cells were intact. This work will help solve the long-standing problem of transwell tissue barrier model storage, facilitating access to advanced predictive cellular models. This is underpinned by precise control of the nucleation temperature, addressing a crucial biophysical mode of damage.

A 3D bioprinted hydrogel gut-on-chip with integrated electrodes for transepithelial electrical resistance (TEER) measurements

Conventional gut-on-chip (GOC) models typically represent the epithelial layer of the gut tissue, neglecting other important components such as the stromal compartment and the extracellular matrix (ECM) that play crucial roles in maintaining intestinal barrier integrity and function. These models often employ hard, flat porous membranes for cell culture, thus failing to recapitulate the soft environment and complex 3D architecture of the intestinal mucosa. Alternatively, hydrogels have been recently introduced in GOCs as ECM analogs to support the co-culture of intestinal cells in in vivo-like configurations, and thus opening new opportunities in the organ-on-chip field. In this work, we present an innovative GOC device that includes a 3D bioprinted hydrogel channel replicating the intestinal villi architecture containing both the epithelial and stromal compartments of the gut mucosa. The bioprinted hydrogels successfully support both the encapsulation of fibroblasts and their co-culture with intestinal epithelial cells under physiological flow conditions. Moreover, we successfully integrated electrodes into the microfluidic system to monitor the barrier formation in real time via transepithelial electrical resistance measurements.

Abstract 2823: Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system

Abstract Pediatric T-cell acute lymphoblastic leukemia (T-ALL), an aggressive hematological malignancy, is characterized by abnormal thymocyte proliferation, frequently with mutations of the Notch-1 gene and has an overall 10-year survival rate of ~27%. A marker of poor prognosis is the presence of T-ALL within the central nervous system (CNS), which is treated with intrathecal chemotherapy and cranial irradiation; two toxic treatments with high morbidities. Gene-expression profiling of Notch-1-induced adhesion regulators showed that C-C chemokine receptor 7 (CCR7) was highly expressed and was associated with leukemic T-cell invasion of the CNS. Normal functions of CCR7 include lymph node homing and immune cell trafficking in response to CCR7 ligands, CC-chemokine ligand 19 (CCL19) or CCL21. In several studies CCL19 has been linked to T-cell invasion of the CNS across the blood-brain barrier (BBB). We hypothesize that antagonizing CCR7 binding to CCL19 will block migration of T-ALL into the CNS and thereby alleviate the need for toxic therapies. To test our hypothesis, we are using receptor internalization assays, calcium mobilization assays, transwell migration assays and an in vitro model of an intact human BBB. We have used flow cytometry-based CCR7 internalization assays ± CCR7 antagonist and found that we can completely block CCL19-induced receptor internalization in human CCR7-expressing CCRF-CEM, and HuT-78 T-ALL cells, and chemotaxis via β1 integrins across a fibronectin-coated transwell membrane. Our BBB employs HBEC-5i, human cerebral microvascular endothelial cells, in transwell chambers coated with gelatin and fibronectin for the adhesion of endothelial cells. HBEC-5i cells are a suitable cell line to make a 3D model of the BBB, as they form an endothelial monolayer with tight junctions (TJ), which is crucial to maintaining the integrity and inhibiting permeability of the BBB. We are establishing a transwell migration assay to assess the migration of CCRF-CEM T-ALL cells across the HBEC-5i cell monolayer. The integrity of the HBEC-5i cell monolayer is assessed by trans-epithelial electrical resistance (TEER), which measures the electrical resistance across the HBEC-5i monolayer, a measure of TJ integrity. Using TEER measurements, we observe that HBEC-5i form a monolayer that reaches an intact monolayer functioning with TJs over 72-96 hours. We are using this model system to investigate the migration of CCR7-expressing CCRF-CEM cells through the BBB ± CCL19. Subsequently, we will add the CCL19 antagonist (CCL197-77) to determine if inhibiting CCR7 is sufficient to eliminate migration of the CCRF-CEM cells through the BBB. If successful, this model will allow us to test the contribution of CCR7 signaling to integrins, the contribution of other immune cell types and the overall molecular mechanisms that mediate invasion of the CNS in a 3-D model of the BBB. Citation Format: Alondra Rodriguez, Cesar I. Cardona, Colin A. Bill, Charlotte M. Vines. Development of an in vitro blood-brain barrier model of CCR7/CCL19 induced invasion of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2823.

Abstract 2125: The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity

Abstract Gastrointestinal (GI) toxicity is a common and often severe limiting factor in the development of antineoplastic drugs. Symptoms include diarrhoea, dehydration and ulceration which increase susceptibility to infection, partly due to damage of crypt and/or villus structures in the small intestine, impairing the barrier function. As novel targeted therapies are emerging, assessment of their potential GI toxicity remains crucial. Small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine. We have further developed and validated the organoid model as a screening tool to predict GI toxicity and subsequent mucosal regeneration in four species: mouse, rat, dog and human. The culture conditions have been designed to mimic the stem cell niche and allow cell differentiation and proliferation to occur. All intestinal lineages were present in the organoids derived from each species and the epithelial hierarchy closely resembled that observed in vivo. The toxic effects of antineoplastic drugs on the organoids were assessed by quantification of the total cell viability upon treatment, coupled to morphometric analysis of the organoids (size, area, perimeter, and branching efficiency). Immunofluorescence and RNAseq can be used to identify the targeted cells and the toxicity mechanisms of the drugs. To further assess the effect of drugs on barrier integrity, we have developed transwell-grown cell monolayers derived from normal human small intestinal organoids. This model allows direct access to both apical and basal surfaces, which is not possible when using organoids where their geometry prevents access to the apical surface. The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements and treatments with toxic drugs were applied after TEER reached a plateau. The direct effect on barrier integrity was evaluated by TEER reduction, increased FITC-Dextran permeability and analysis of TJ proteins by immunofluorescence. Furthermore, effects on cell death were assessed by measuring the levels of Lactate Dehydrogenase (LDH) released into the medium by dying cells. In conclusion, our models provide an innovative in vitro solution to further study the toxic effects of drugs on the normal small intestine and to analyse direct effects on barrier integrity. The monolayer assay may also be useful for developing models of transient TJ impairment to improve the bioavailability of orally administered antineoplastic drugs. The model can be considered more representative of the normal intestine than transformed cell lines such as CaCo2. Citation Format: Valentina Ubertini, Sarah Hall, Frida Ponthan, James Wilson. The use of intestinal organoids as a preclinical screen to assess gastrointestinal (GI) toxicity and barrier integrity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2125.

Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-β2 or Dexamethasone.

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-β2 or 250 nM dexamethasone (DEX). Methods: Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

Dupilumab prevents nasal epithelial function alteration by IL-4 in vitro: Evidence for its efficacy.

Chronic rhinosinusitis with nasal polyp (CRSwNP) is a typical type 2 inflammation involving interleukin (IL)-4 and IL-13. Dupilumab is a fully human monoclonal antibody targeting IL-4 receptor α subunit, thereby blocking signaling by both cytokines. Our hypothesis was that IL-4 and IL-13, by inducing a severe epithelial dysregulation, are involved in CRSwNP pathogenesis. This study aimed to evaluate the in vitro direct effect of IL-4, IL-13, and dupilumab on nasal epithelial functions. Nasal polyps and control mucosa from 28 patients, as well as human nasal epithelial cells (HNEC) from 35 patients with CRSwNP were used. Three major epithelial functions were investigated: the epithelial barrier function (characterized by transepithelial electrical resistance measurements and tight junction protein expression), the ciliary motion (characterized by the ciliary beating efficiency index), and wound healing (characterized by the wound repair rate) under various stimulations (IL-4, IL-13, and dupilumab). The main outcome was a significant change in epithelial functions following exposure to IL-4, IL-13, and dupilumab for 48h in the basal media. IL-4 (1, 10, and 100ng/mL) but not IL-13 induced a significant decrease in occludin and zonula-occludens protein expression, ciliary beating efficiency, and wound repair rate in HNEC. Dupilumab (0.04mg/mL) had no effect on HNEC and specifically restored all epithelial functions altered when cells were exposed to a 48-h IL-4 stimulation. Dupilumab, in vitro, restored epithelial integrity by counteracting the effect of IL-4 on the epithelial barrier (increased epithelial permeability, decreased ciliary beating efficiency, and decreased wound repair rate).

A181 EXPLORING THE ROLE OF KLEBSIELLA VARIICOLA AND KLEBSIELLA PNEUMONIAE IN PEDIATRIC ULCERATIVE COLITIS PATHOGENESIS

Abstract Background Recent studies indicated that Klebsiella (K) pneumoniae (isolated from the stool of IBD patients) and K. variicola (isolated from the mesenteric tissue of Crohn disease patients) have the potential to induce inflammation in epithelial and preadipocyte cells, exacerbating colitis in murine models. Aims We isolated these strains from pediatric ulcerative colitis (UC) patients' appendix (relevant to UC pathogenesis given the protective effects of appendectomy) and other non-inflamed colon sections. We hypothesized that these isolates are invasive and induce inflammation in UC. These strains have not been previously studied in UC. Methods K. variicola and K. pneumoniae were isolated from two pediatric UC patients' appendices and other non-inflamed colon sections and identified using 16S DNA Sanger sequencing. Virulence of the two strains was determined by infecting Caco2 cell lines and performing adhesion and invasion assays, quantifying biofilm formation, and assessing barrier functions using transepithelial electrical resistance (TEER) measurement. Importantly, we monitored the production of pro-inflammatory (e.g., IL-8, TNF-α) and anti-inflammatory (IL-10) cytokines using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results K. variicola and K. pneumoniae exhibited high levels of biofilm formation compared to adherent-invasive Escherichia coli (AIEC), which are commonly isolated from IBD patients. Furthermore, the Klebsiella strains adhered to epithelial cells within 2-3 hours post infection. TEER experiments showed compromised barrier integrity after 6 hours and overnight infection. Except for K. pneumoniae isolated from the ascending colon, the tested strains exhibited a significant increase in IL-8 expression. Similarly, K. variicola from the peri-appendicular region demonstrated elevated TNF-α gene expression. Conclusions K. variicola and K. pneumoniae isolated from UC patients have the potential to form biofilms, disrupt barrier integrity, and trigger inflammatory responses. These findings unravel a potential role for pathogenic Klebsiella strains in driving UC pathogenesis. Importantly, these data shed light on the role of appendix-associated bacteria in the development of UC. Future work includes using comparative genomics to map the virulence determinants of these bacteria. Funding Agencies CIHRWomen and Children's Health Research Institute (WCHRI)

P037 Modelling gut complexity and disease: development of a next-generation adult stem cell based tubular gut-on-a-chip model

Abstract Background Developing effective therapies for Inflammatory Bowel Disease (IBD) necessitates advanced in vitro models that faithfully replicate the intricacies of the gut. The current challenge is to create models that encompass the 3D morphology, heterogeneity, and boundary characteristics of intestinal tissue while also being amenable to high-throughput screening. Microfluidic techniques are emerging as vital tools to introduce physiologically relevant cues into conventional cell cultures. Methods We present an innovative in vitro model employing human adult stem cell-derived colon organoids cultivated as perfused epithelial tubules within the MIMETAS OrganoPlate®. This microfluidic platform facilitates the simultaneous culture of up to 64 independent gut tubules on an extracellular matrix, offering continuous media perfusion without artificial membrane interference. The barrier function of the epithelial tubules is assessed in real-time through transepithelial electrical resistance (TEER) measurements. Immunostaining and 3D reconstruction of confocal images evaluate the expression of relevant epithelial markers. Results The tubular epithelial model of the intestinal tract demonstrated rapid cell polarization, expression of cell-specific markers, tight junction formation, and the presence of functional transporters. TEER measurements indicated functional and reproducible barrier integrity across tubules generated from different organoid donors for at least 10 days. Incorporation of additional cell types, such as patient-derived immune cells and endothelium, marks a groundbreaking advancement in understanding disease pathogenesis. The plug-and-play modularity of these models allows for flexibility in incorporating specific cell types based on the biological pathway of interest. Conclusion Our next-generation gut-on-a-chip model faithfully mimics the IBD phenotype and facilitates screening for potential drug targets. Combining adult human stem cell-derived intestinal organoids with microfluidic technology provides a robust platform to study physiology and disease mechanisms in patient-specific gut models. The inclusion of IBD patient-derived cells promises further refinement, enabling a more profound exploration of disease phenotypes and treatment responsiveness. Moreover, organoids, as one of the most physiologically relevant cell sources, authentically represent patient biology, enhancing the translational potential of our findings.

P1238 Exploring the Role of Klebsiella variicola and Klebsiella pneumoniae in Pediatric Ulcerative Colitis Pathogenesis

Abstract Background Recent studies indicated that Klebsiella (K) pneumoniae (isolated from the stool of IBD patients) and K. variicola (isolated from the mesenteric tissue of Crohn disease patients) have the potential to induce inflammation in epithelial and preadipocyte cells, exacerbating colitis in murine models. We isolated these strains from pediatric ulcerative colitis (UC) patients' appendix (relevant to UC pathogenesis given the protective effects of appendectomy) and other non-inflamed colon sections. We hypothesized that these isolates are invasive and induce inflammation in UC. These strains have not been previously studied in UC. Methods K. variicola and K. pneumoniae were isolated from two pediatric UC patients' appendices and other non-inflamed colon sections and identified using 16S DNA Sanger sequencing. Virulence of the two strains was determined by infecting Caco2 cell lines and performing adhesion and invasion assays, quantifying biofilm formation, and assessing barrier functions using transepithelial electrical resistance (TEER) measurement. Importantly, we monitored the production of pro-inflammatory (e.g., IL-8, TNF-α) and anti-inflammatory (IL-10) cytokines using reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Results K. variicola and K. pneumoniae exhibited high levels of biofilm formation compared to adherent-invasive Escherichia coli (AIEC), which are commonly isolated from IBD patients. Furthermore, the Klebsiella strains adhered to epithelial cells within 2-3 hours post infection. TEER experiments showed compromised barrier integrity after 6 hours and overnight infection. Except for K. pneumoniae isolated from the ascending colon, the tested strains exhibited a significant increase in IL-8 expression. Similarly, K. variicola from the peri-appendicular region demonstrated elevated TNF-α gene expression. Conclusion K. variicola and K. pneumoniae isolated from UC patients have the potential to form biofilms, disrupt barrier integrity, and trigger inflammatory responses. These findings unravel a potential role for pathogenic Klebsiella strains in driving UC pathogenesis. Importantly, these data shed light on the role of appendix-associated bacteria in the development of UC. Future work includes using comparative genomics to map the virulence determinants of these bacteria.

P1208 MH002, an optimized live biotherapeutic product, for the treatment of inflammatory bowel diseases, has mode-of-actions linked to restoration of intestinal dysbiosis, mucosal integrity, and immune homeostasis

Abstract Background MH002 is a live biotherapeutic product consisting of 6 well-characterized commensal strains, that is in clinical development for the treatment of Ulcerative Colitis (UC) and acute Pouchitis. Both are multifactorial disorders which occur through a combination of dysregulated host inflammatory mechanisms and interactions with gut microbiota. Methods Faecal slurries of 6 UC patients with active disease were prepared to inoculate a standardized in vitro gut model (SHIME®) which mimics the colon microbiome of patients. A total of 24 simulators were run for 8d, with a single dose administration of MH002 (human equivalent dose [HED] ~1) or vehicle on Day0. Butyrate production in the simulators was quantified daily and host response effects were assessed in in vitro cell-based assays using samples from Day1. Caco-2 cells were co-cultured with human peripheral blood mononuclear cells and treated for 48h with Day1 samples, followed by transepithelial electrical resistance (TEER) and cytokine measurements. Wound healing was assessed by imaging of monolayers of T84 cells, scratched and treated with Day1 samples. Furthermore, the effects of MH002 (daily, 24d, HED ~30) on disease activity index (DAI; body weight [BW], stool consistency, occult blood), local inflammation, and mucosal damage were evaluated in acute dextran sulfate sodium (DSS) induced colitis, when administered to C57BL/6 mice (15/group) as from 14d prior to DSS intake, and compared to 5-aminosalicylate (5-ASA; daily, 24d, 150mg/kg) and vehicle. Animals were euthanized in the acute (Day7; 10/group) and recovery phase (Day11; 5/group). Results In vitro administration of MH002 to patient-derived colonic microbiomes restored within 1d butyrate production and increased efficiency to improve epithelial barrier integrity, modulate T-cell-mediated immune responses, and promote mucosal wound healing in in vitro cell cultures (Table 1). DSS intake induced a significant decrease in relative BW and an increase in DAI. A significant recovery (BW, DAI, stool consistency) was observed at Day11 for MH002 and 5-ASA. Moreover, at Day11, macroscopic inflammation (colon weight/length ratio) and histologic scores were lower with MH002 and 5-ASA as compared to vehicle (Figure 1). Conclusion By restoring intestinal dysbiosis and epithelial integrity, and attenuating local inflammation, MH002 treatment should lead to lower disease activity and, ultimately, to clinical remission without immune suppression. Indeed, study MH002-UC-201 in mild-to-moderate UC was completed with favourable treatment effects (endoscopic improvement, faecal calprotectin normalization, improved stool consistency) and no safety concerns (Abstract Vermeire et al.; EudraCT 2020-004355-33).

A novel “microbiota-host interaction model” to study the real-time effects of fermentation of non-digestible carbohydrate (NDCs) on gut barrier function

In this study, an in vitro co-culture model using an electric cell-substrate impedance sensing system (ECIS) for testing the impact of real-time fermentation of non-digestible carbohydrates (NDCs) by the intestinal microbiota on gut barrier function was established. We applied Lactobacillus plantarum WCFS1 as a model intestinal bacterium and alginate-pectin as immobilization polymers as well as a source of NDCs to determine the impact of pectin fermentation on the barrier function of T84 gut epithelial cells. In the first design, L. plantarum WCFS1 was encapsulated in an alginate capsule followed by embedding in an agar layer to mimic a firm mucus layer that might be present in the colon. In this experimental design, the presence of the agar layer interfered with the transepithelial electrical resistance (TEER) measurement of T84 cells. Subsequently, we removed the agar layer and used encapsulated bacteria in an alginate gel and found that the TEER measurement was adequate. The encapsulation of the L. plantarum WCFS1 does avoid direct contact with cells. Also, the encapsulation system allows higher amounts of packing densities of L. plantarum WCFS1 in a limited space which can limit the oxygen concentration within the capsule and therefore create anaerobic conditions. To test this design, T84 cells were co-incubated with L. plantarum alginate-capsules supplemented with graded loads of fermentable pectin (0, 4, and 8 mg/ml per capsule) to investigate the effect of pectin fermentation on gut barrier function. We observed that as the pectin content in the L. plantarum capsules increased, pectin showed a gradually stronger protective effect on the TEER of the gut epithelium. This could partly be explained by enhanced SCFA production as both lactate and acetate were enhanced in L. plantarum containing alginate capsules with 8 mg/ml pectin. Overall, this newly designed in vitro co-culture model allows for studying the impact of bacteria-derived fermentation products but also for studying the direct effects of NDCs on gut barrier function in a relatively high-throughput way.

Monitoring Intestinal Organoid-Derived Monolayer Barrier Functions with Electric Cell-Substrate Impedance Sensing (ECIS).

The measurement of transepithelial electrical resistance across confluent cell monolayer systems is the most commonly used technique to study intestinal barrier development and integrity. Electric cell substrate impedance sensing (ECIS) is a real-time, label-free, impedance-based method used to study various cell behaviors such as cell growth, viability, migration, and barrier function in vitro. So far, the ECIS technology has exclusively been performed on cell lines. Organoids, however, are cultured from tissue-specific stem cells, which better recapitulate cell functions and the heterogeneity of the parent tissue than cell lines and are therefore more physiologically relevant for research and modeling of human diseases. In this protocol paper, we demonstrate that ECIS technology can be successfully applied on 2D monolayers generated from patient-derived intestinal organoids. Key features • We present a protocol that allows the assessment of various cell functions, such as proliferation and barrier formation, with ECIS on organoid-derived monolayers. • The protocol facilitates intestinal barrier research on patient tissue-derived organoids, providing a valuable tool for disease modeling.

Colon-available mango (poly)phenols exhibit mitigating effects on the intestinal barrier function in human intestinal cell monolayers under inflammatory conditions.

This study investigated the impact of in vivo available colon-mango (poly)phenols on stress-induced impairment of intestinal barrier function. Caco-2/HT29-MTX cells were incubated with six extracts of ileal fluid collected pre- and 4-8 h post-mango consumption before being subjected to inflammatory stress. (Poly)phenols in ileal fluids were analysed by UHPLC-HR-MS. Epithelial barrier function was monitored by measurement of trans-epithelial electrical resistance (TEER) and the production of selected inflammatory markers (interleukin-8 (IL-8) and nitric oxide (NO)) and the major mucin of the mucosal layer (MUC2). Post-mango intake ileal fluids contained principally benzoic acids, hydroxybenzenes and galloyl derivatives. There was a high interindividual variability in the levels of these compounds, which was reflected by the degree of variability in the protective effects of individual ileal extracts on inflammatory changes in the treated cell cultures. The 24 h treatment with non-cytotoxic doses of extracts of 4-8 h post-mango intake ileal fluid significantly reduced the TEER decrease in monolayers treated with the inflammatory cytomix. This effect was not associated with changes in IL-8 expression and secretion or claudine-7 expression. The mango derived-ileal fluid extract (IFE) also mitigated cytomix-dependent nitrite secretion, as a proxy of NO production, and the MUC2 reduction observed upon the inflammatory challenge. These insights shed light on the potential protective effect of mango (poly)phenols on the intestinal barrier exposed to inflammatory conditions.

Toxicological assessment of E-cigarette flavored E-liquids aerosols using Calu-3 cells: A 3D lung model approach

Scientific progress and ethical considerations are increasingly shifting the toxicological focus from in vivo animal models to in vitro studies utilizing physiologically relevant cell cultures. Consequently, we evaluated and validated a three-dimensional (3D) model of the human lung using Calu-3 cells cultured at an air-liquid interface (ALI) for 28 days. Assessment of seven essential genes of differentiation and transepithelial electrical resistance (TEER) measurements, in conjunction with mucin (MUC5AC) staining, validated the model. We observed a time-dependent increase in TEER, genetic markers of mucus-producing cells (muc5ac, muc5b), basal cells (trp63), ciliated cells (foxj1), and tight junctions (tjp1). A decrease in basal cell marker krt5 levels was observed. Subsequently, we utilized this validated ALI-cultured Calu-3 model to investigate the adversity of the aerosols generated from three flavored electronic cigarette (EC) e-liquids: cinnamon, vanilla tobacco, and hazelnut. These aerosols were compared against traditional cigarette smoke (3R4F) to assess their relative toxicity. The aerosols generated from PG/VG vehicle control, hazelnut and cinnamon e-liquids, but not vanilla tobacco, significantly decreased TEER and increased lactate dehydrogenase (LDH) release compared to the incubator and air-only controls. Compared to 3R4F, there were no significant differences in TEER or LDH with the tested flavored EC aerosols other than vanilla tobacco. This starkly contrasted our expectations, given the common perception of e-liquids as a safer alternative to cigarettes. Our study suggests that these results depend on flavor type. Therefore, we strongly advocate for further research, increased user awareness regarding flavors in ECs, and rigorous regulatory scrutiny to protect public health.

Myrrh protects against IL-13-induced epithelial barrier breakdown in HT-29/B6 cells.

The oleoresin myrrh has been used for centuries as an anti-inflammatory remedy for a variety of diseases and is said to have a protective effect on the intestinal epithelium. An intact epithelial barrier function is the prerequisite for a healthy gut. Inflammatory and infectious diseases of the intestine, in particular, lead to barrier impairment resulting in leak-flux diarrhea and mucosal immune responses. Therefore, the aim of the present study was to investigate the protective effect of myrrh in an experimental inflammatory situation, namely, under the influence of IL-13, one of the key cytokines in ulcerative colitis. We used human intestinal epithelial HT-29/B6 cell monolayers for functional and molecular assessment of the epithelial barrier under IL-13 and myrrh treatment. IL-13 induced a loss in barrier function that was fully restored with myrrh treatment, as shown by transepithelial electrical resistance measurements. The molecular correlate of the IL-13-mediated barrier dysfunction could be assigned to an upregulation of the channel-forming tight junction (TJ) protein claudin-2 and to a subcellular redistribution of the TJ protein tricellulin, loosening the sealing of tricellular TJs. Moreover, IL-13 exposure leads to an increase in the number of apoptotic cells, contributing to the leak pathway of barrier dysfunction. Myrrh protected against changes in TJ deregulation and decreased the elevated apoptotic ratio under IL-13. The protective effects are mediated through the inhibition of the STAT3 and STAT6 pathway. In conclusion, our results demonstrate that myrrh exhibits antagonizing effects against IL-13-induced barrier impairment in a human intestinal cell model. These data suggest the use of myrrh as a promising option in the treatment of inflammatory bowel disease.

Automated measurement of transepithelial electrical resistance (TEER) in 96-well transwells using ECIS TEER96: Single and multiple time point assessments

Transepithelial electrical resistance (TEER) is a widely used technique for quantifying the permeability of epithelial and endothelial cell layers. However, traditional methods of measuring TEER are limited to single timepoint measurements and can subject cells to an altered environment during the measurement. Here, we assessed the validity of TEER measurements by the ECIS TEER96 device, which is designed to take continuous TEER measurements of a cell culture system in a standard laboratory incubator. We found that the instrument accurately measures TEER across TEER values ranging from 10 to 2050 Ω*cm2 and is more accurate than the manual epithelial voltohmmeter electrode at high TEER values. Furthermore, the high-resolution measurements provided by the device allowed for a unique insight into the mechanisms and kinetics of cells in vitro. To demonstrate the continuous measurement capability of the device, we tracked the formation of an MDCKI cell monolayer until TEER plateaued. Furthermore, we treated Caco-2 monolayers with different concentrations of DMSO and the antimicrobial and surfactant compound benzethonium chloride to measure disruptions to barrier integrity. Treatment of both compounds resulted in concentration-dependent loss of barrier integrity. Our results suggest that the ECIS TEER96 device is a reliable and convenient option for measuring TEER in cell cultures and can provide valuable insights into the behavior of cells in vitro. This technology will be especially useful for increasing throughput of drug permeability assays, inflammation studies, and gaining better understanding of disease states in a cell culture system.

Increasing hydrogel complexity from 2D towards 3D towards intestinal tissue engineering

There is a high demand for in vitro intestinal models towards studying intestinal disfunctions, such as gastrointestinal diseases (Crohn's disease, irritable bowel syndrome, colorectal cancer, etc.) which are commonly diagnosed in modern society. In parallel, in vitro models are utilized for the evaluation of novel food supplements and pharmaceuticals in the intestinal micro-environment. Gelatin-based hydrogels can offer appropriate mechanical cues (down to G’ < 10 kPa) and cell interactivity to obtain physiologically relevant soft tissue models. Therefore, the current paper focused on the development of a novel, gelatin-methacryloyl-aminoethyl-methacrylate (gel-MA-AEMA)-based in vitro intestinal model, which also provides sufficient permeability towards nutrients and drugs. Moreover, the effect of the hydrogel morphology on the cell response was assessed by comparing the formation of a functional intestinal cell monolayer on flat hydrogel films versus 3D hydrogel scaffolds maintaining a close morphological resemblance with the intestinal architecture.A gel-MA-AEMA-based biomaterial ink was formulated and processed towards flat hydrogel films, while digital light processing was exploited to replicate the intestinal microarchitecture. The mechanical assessment of the 2D films confirmed physiologically relevant scaffold stiffness (G’ = 3.30±1.07 kPa). To evaluate the permeability of the hydrogels towards a medium size marker molecule (FITC-dextran 4 kg·mol−1), a static diffusion setup was exploited with custom-made adjustable inserts, which evidenced that both the 2D films and 3D constructs exhibited sufficient permeability. Finally, the combination of the hydrogels with a Caco-2/HT29-MTX co-culture evidenced hydrogel biocompatibility, enabling the formation of a functional cell monolayer after 21 days on the 2D hydrogel films, proven by transepithelial electrical resistance measurements and immunohistochemistry, while the 3D hydrogel constructs did not achieve confluency within 35 days.

Nicotine transport across calu-3 cell monolayer: effect of nicotine salts and flavored e-liquids

Objective This study aimed to investigate the transport capability of nicotine across Calu-3 cell monolayer in various nicotine forms, including nicotine freebase, nicotine salts, and flavored e-liquids with nicotine benzoate. Significance Nicotine is rapidly absorbed from the respiratory system into systemic circulation during e-cigarettes use. However, the mechanism of nicotine transport in the lung has not been well understood yet. This study may offer critical biological evidence and have implications for the use and regulation of e-cigarettes. Methods The viability of Calu-3 cells after administration of nicotine freebase, nicotine salts and representative e-liquid were evaluated using the MTT assay, and the integrity of the Calu-3 cell monolayer was evaluated by transepithelial electrical resistance measurement and morphological analysis. Further, the nicotine transport capacity across the Calu-3 cell monolayer in various formulations of nicotine was investigated by analysis of nicotine transport amount. Results The findings indicated that nicotine transport occurred passively and was time-dependent across the Calu-3cell monolayer. In addition, the nicotine transport was influenced by the type of nicotine salts and their respective pH value. The nicotine benzoate exhibited the highest apparent permeability coefficient (Papp ), and higher nicotine-to-benzoic acid ratios led to higher Papp values. The addition of flavors to e-liquid resulted in increased Papp values, with the most significant increment being observed in tobacco-flavored e-liquid. Conclusions In summary, the transport capability of nicotine across the Calu-3 cell monolayer was influenced by the pH values of nicotine salts and flavor additives in e-liquids.

Cannabinoid receptor type 1 in the aging gut regulates the mucosal permeability via miR-191-5p.

Aging is associated with a broad loss of function throughout the body, and gastrointestinal (GI) dysfunction can occur with aging. The endocannabinoid (eCB) system plays a pivotal role in various GI diseases, and alterations in the eCB system have been observed during brain and skin aging. Therefore, we investigated the putative role of the eCB system in aging-related changes in the intestine. The expression of cannabinoid receptor type 1 (CB1) was investigated in rat intestinal tissues using quantitative real-time PCR. Cellular senescence was induced by hydrogen peroxide (H2O2) and hydroxyurea (HU) in rat and human intestinal epithelial cells. Cellular permeability was evaluated by transepithelial electrical resistance (TEER) measurement. The expression of CB1 was decreased in the small intestine of aged rats compared to that of young rats. Senescent cells showed reduced TEER values and decreased expression of ZO-1, indicating increased intestinal permeability, which is tightly regulated by the CB1 signaling. In silico miRNA analysis suggested that ZO-1 was a direct target gene of miR-191-5p. Increased expression of miR-191-5p by HU was restored by CB1 agonist ACEA co-treatment. Moreover, NF-κB p65 activation was associated with CB1-related miR-191-5p signaling. In conclusion, aging-induced CB1 reduction leads to increased intestinal permeability and decreased ZO-1 expression via upregulation of miR-191-5p and NF-κB p65 activation. Taken together, these results suggest that CB1 signaling may be a useful strategy to reduce intestinal permeability in aging-related and other inflammatory conditions in the gut.