Transepithelial Electrical Resistance Measurements Research Articles

Physiological Shear Stress Enhances Differentiation, Mucus-Formation and Structural 3D Organization of Intestinal Epithelial Cells In Vitro.

Gastrointestinal (GI) mucus plays a pivotal role in the tissue homoeostasis and functionality of the gut. However, due to the shortage of affordable, realistic in vitro GI models with a physiologically relevant mucus layer, studies with deeper insights into structural and compositional changes upon chemical or physical manipulation of the system are rare. To obtain an improved mucus-containing cell model, we developed easy-to-use, reusable culture chambers that facilitated the application of GI shear stresses (0.002–0.08 dyn∙cm−2) to cells on solid surfaces or membranes of cell culture inserts in bioreactor systems, thus making them readily accessible for subsequent analyses, e.g., by confocal microscopy or transepithelial electrical resistance (TEER) measurement. The human mucus-producing epithelial HT29-MTX cell-line exhibited superior reorganization into 3-dimensional villi-like structures with highly proliferative tips under dynamic culture conditions when compared to static culture (up to 180 vs. 80 µm in height). Additionally, the median mucus layer thickness was significantly increased under flow (50 ± 24 vs. 29 ± 14 µm (static)), with a simultaneous accelerated maturation of the cells into a goblet-like phenotype. We demonstrated the strong impact of culture conditions on the differentiation and reorganization of HT29-MTX cells. The results comprise valuable advances towards the improvement of existing GI and mucus models or the development of novel systems using our newly designed culture chambers.

Barrier-on-a-Chip with a Modular Architecture and Integrated Sensors for Real-Time Measurement of Biological Barrier Function.

Biological barriers are essential for the maintenance of organ homeostasis and their dysfunction is responsible for many prevalent diseases. Advanced in vitro models of biological barriers have been developed through the combination of 3D cell culture techniques and organ-on-chip (OoC) technology. However, real-time monitoring of tissue function inside the OoC devices has been challenging, with most approaches relying on off-chip analysis and imaging techniques. In this study, we designed and fabricated a low-cost barrier-on-chip (BoC) device with integrated electrodes for the development and real-time monitoring of biological barriers. The integrated electrodes were used to measure transepithelial electrical resistance (TEER) during tissue culture, thereby quantitatively evaluating tissue barrier function. A finite element analysis was performed to study the sensitivity of the integrated electrodes and to compare them with conventional systems. As proof-of-concept, a full-thickness human skin model (FTSm) was grown on the developed BoC, and TEER was measured on-chip during the culture. After 14 days of culture, the barrier tissue was challenged with a benchmark irritant and its impact was evaluated on-chip through TEER measurements. The developed BoC with an integrated sensing capability represents a promising tool for real-time assessment of barrier function in the context of drug testing and disease modelling.

A Design Method of Organ-on-a-Chip with Highly Accurate Measurement of Trans-Epithelial Electrical Resistance

A Design Method of Organ-on-a-Chip with Highly Accurate Measurement of Trans-Epithelial Electrical Resistance

Pigmented Full-Thickness Human Skin Model Based on a Fibroblast-Derived Matrix for Long-Term Studies.

Reconstructed human skin models are a valuable tool for drug discovery, disease modeling, and basic research. In the past decades, major progress has been made in this field leading to the development of full-thickness skin models (FTSms) better representative of the native human skin by including the cellular cross talk between the dermal and epidermal layers. However, current available FTSms still present important limitations since they are only suitable for short-term studies, include nonhuman extracellular matrix (ECM) components and have a weak skin barrier function compared with in vivo human skin. In this study, a fibroblast-derived matrix was combined with the use of an inert polystyrene scaffold for the development of a fully human dermis capable of supporting a differentiated epidermis. To produce a pigmented FTSm, a coculture with keratinocytes, melanocytes, and fibroblasts was established. The structure and functionality of the developed FTSms were studied for short- and long-term cultivation using histological and immunofluorescence staining. The integrity of the skin barrier was evaluated using transepithelial electrical resistance (TEER) measurements. It was possible to obtain a mature dermis capable of supporting an epidermis without keratinocyte infiltration in only 6 days. ECM components (collagen IV and fibrin) were secreted by the fibroblasts and accumulated in the scaffold structure, recreating the microenvironment of the native human dermis. Moreover, the use of a scaffold resulted in a structure with mechanical stability due to its noncontracting nature. The coculture of primary human keratinocytes resulted in a terminally differentiated skin equivalent that could maintain its architecture and homeostasis up to 50 days. Melanocytes were correctly integrated within the epidermal basal layer and made it possible to reproduce constitutive pigmentation. TEER levels increased during culture time, reaching values of 1.1 ± 0.2 kΩ.cm2 for the FTSm, indicative of a functional skin barrier.Impact statementThe developed fully human full-thickness skin model has the potential to reduce the dependence on animal models for long-term studies of skin diseases and safety and efficacy assessment of novel drugs. Its longevity and robustness allow the experimental testing phase to be lengthened. The presence of active melanocytes at the dermal–epidermal junction makes this model the ideal platform to study skin pigmentation disorders.

Higher Prevalence of Bacteroides fragilis in Crohn's Disease Exacerbations and Strain-Dependent Increase of Epithelial Resistance.

Bacteroides fragilis has previously been linked to Crohn’s disease (CD) exacerbations, but results are inconsistent and underlying mechanisms unknown. This study investigates the epidemiology of B. fragilis and its virulence factors bft (enterotoxin) and ubiquitin among 181 CD patients and the impact on the intestinal epithelial barrier in vitro. The prevalence of B. fragilis was significantly higher in active (n = 69/88, 78.4%) as compared to remissive (n = 58/93, 62.4%, p = 0.018) CD patients. Moreover, B. fragilis was associated with intestinal strictures. Interestingly, the intestinal barrier function, as examined by transepithelial electrical resistance (TEER) measurements of Caco-2 monolayers, increased when exposed to secretomes of bft-positive (bft-1 and bft-2 isotype; increased TEER ∼160%, p < 0.001) but not when exposed to bft-negative strains. Whole metagenome sequencing and metabolomics, respectively, identified nine coding sequences and two metabolites that discriminated TEER-increasing from non-TEER-increasing strains. This study revealed a higher B. fragilis prevalence during exacerbation. Surprisingly, bft-positive secretomes increased epithelial resistance, but we excluded Bft as the likely causative factor.

Transcriptome-wide profiling discover: PM2.5 aggravates airway dysfunction through epithelial barrier damage regulated by Stanniocalcin 2 in an OVA-induced model

BackgroundEpidemiologic evidence suggests that PM2.5 exposure aggravates asthma, but the molecular mechanisms are not fully discovered. MethodsOvalbumin (OVA)-induced mice exposed to PM2.5 were constructed. Pathological staining and immunofluorescence were performed in in vivo study. Gene set enrichment analysis (GSEA) was performed to identify the pathway involved in asthma severity by using U-BIOPRED data (human bronchial biopsies) and RNA-seq data (Beas-2B cells treated with PM2.5). Lentiviruses transfection, Real-time qPCR, immunofluorescence staining and trans-epithelial electrical resistance (TEER) measurement were performed for mechanism exploration in vitro. ResultsPM2.5 exposure aggravated airway inflammation and mucus secretion in OVA-induced mice. Based on transcriptome analysis of mild-to-severe asthma from human bronchial biopsies, gene set enrichment analysis (GSEA) showed that up-regulated reactive oxygen species (ROS) pathway gene set and down-regulated apical junction gene set correlated with asthma severity. Consistent with the analysis of mild-to-severe asthma, after PM2.5 exposure, the ROS pathway in Beas-2B cells was up-regulated with the down-regulation of apical junction. The expression levels of genes involved in the specific gene sets were validated by using qPCR. The mRNA levels of junction genes, ZO-1, E-cadherin and Occludin, were significantly decreased in cells exposed to PM2.5. Moreover, it confirmed that inhibition of ROS recovered the expression levels of E-cadherin, Occludin and ZO-1, and ameliorated inflammation and mucus secretion in airway in OVA-induced mice exposed to PM2.5. Meanwhile, ROS level was elevated by PM2.5. By checking trans-epithelial electrical resistance (TEER) value, we also found that epithelial barrier was damaged after PM2.5 exposure. Importantly, Stanniocalcin 2 (STC2) was identified as a key gene in regulation of epithelial barrier. It showed that STC2 expression was up-regulated by PM2.5, which was recovered by NAC as well. Over-expression of STC2 could decrease the expression levels of ZO-1, Occludin and E-cadherin. Contrarily, suppression of STC2 could increase the expression levels of ZO-1, Occludin and E-cadherin reduced by PM2.5. ConclusionsBy using transcriptome analysis, we revealed that STC2 played a key role in PM2.5 aggravated airway dysfunction through regulation of epithelial barrier in OVA-induced mice.

Deoxycholic Acid Modulates Cell-Junction Gene Expression and Increases Intestinal Barrier Dysfunction in Caco-2 Cell Monolayers

ObjectivesDiet-related obesity is associated with an increased risk of developing intestinal hyperpermeability. High dietary fat intake causes an increase in colonic bile acids (BAs), particularly deoxycholic acid (DCA, secondary BA), which may disrupt the intestinal epithelial barrier. To determine the potential role of bile acids in barrier dysfunction, we hypothesize that DCA modulates the gene expression in multiple cell junction pathways and increases intestinal permeability. MethodsWith a Caco-2 cell intestinal barrier model, we used cell proliferation, PCR array, biochemical, western blotting and immunofluorescent assays to examine the impact of DCA on the integrity of intestinal barrier and gene expression. ResultsHuman intestinal Caco-2 cells were grown in monolayers and challenged with DCA at physiological concentrations (sub mM levels). DCA increased transcellular and paracellular permeability (>30%) via transepithelial electrical resistance and phenol red flux measurements. Similarly, DCA increased intracellular reactive oxidative species production (>1-fold) and accompanied a modification of cellular p38 and ERK1/2 signaling pathways. Further characterization of underlying genes related to epithelial barrier with PCR array analysis identified that 23 genes (in tight junction, focal adhesion, gap junction and adhere junction pathways) were decreased at least 40% in (0.25 mM) DCA-treated Caco-2 cells when compared to untreated cells. Finally, we demonstrated that DCA decreased the protein levels of occludin gene at both cellular tight junction and nucleus in epithelial cells. ConclusionsCollectively, our data suggest that at physiological concentrations, DCA alters the gene expression of multiple pathways related to cell junctions and increases permeability in a Caco-2 intestinal barrier model. These molecular events may represent the underlying mechanistic pathways that are responsible for DCA-induced transcellular and paracellular permeation. Funding SourcesThis work was supported by U.S. Department of Agriculture, Agricultural Research Service, research project 3062-51,000-056–00D.

The role of Transcription Factor Hepatocyte Nuclear Factor 1β in a Transcriptional Network Regulating Cell Polarity in Epithelial Kidney Cells

Hepatocyte nuclear factor 1β (HNF1β) is an essential transcription factor in development and function of the kidney, the pancreas and the liver. Mutations or deletions in HNF1β cause a heterogenous phenotype including renal cysts and/or malformation, maturity-onset diabetes of the young, liver malfunction and hypomagnesemia. The spectrum of HNF1β–regulated genes and pathways is only partially deciphered. Here, we aim to identify the HNF1β target genes and pathways that explain the multifaceted role of HNF1β in the kidney. RNA-sequencing was performed in a mouse distal convoluted tubule (DCT) cell line transfected with siRNAs targeting HNF1β or non-targeting siRNAs. A total number of 989 differential expressed genes was identified. Subsequently, HNF1β binding sites in the promoter regions (defined as <5-kb from the transcription start site) of these genes were identified using ChIP-sequencing data from the same cell line. Only 9,3% (92 genes) of the differentially expressed genes contained a HNF1β-binding site within their promoter region. These genes included known targets of HNF1β including Pkhd1, Kif12 and Kcnj16 and unknown targets. GO-term pathway analysis demonstrated that regulation of cell polarity, cell-cell junction complexes and cytoskeleton organization were regulated by HNF1β. Indeed, transepithelial electrical resistance measurements were reduced upon expression of dominant negative (DN) HNF1β. Nine HNF1 β targets that are part of the apical cell polarity complex were selected for further analysis. To study the importance of HNF1β in regulating cell polarity, the integrity of this apical cell polarity complex will be analyzed in kidney cells expressing wild-type HNF1β and DN HNF1β using immunocytochemistry. Together these findings suggest a role for HNF1β in a transcriptional network regulating cell polarity in adult kidney cells by transcriptional control of the apical polarity complex in epithelial cells.

The mystery behind the nostrils – technical clues for successful nasal epithelial cell cultivation

ObjectivesResearch involving the nose reveals important information regarding the morphology and physiology of the epithelium and its molecular response to agents. The role of nasal epithelial cells and other cell subsets within the nasal epithelium play an interesting translational split between experimental and clinical research studying respiratory disorders or pathogen reactions. With an additional technical manuscript including a detailed description of important technical aspects, tips, tricks, and nuances for a successful culturing of primary, human nasal epithelial cells (NAEPCs), we here aim to improve the process of communication between experimentalists and physicians, supporting the purpose of a fruitful work for future translational projects. MethodsBased on previous work on various complex culture models of subject-derived NAEPCs, this additional manuscript harmonizes previously published facts combined with own experiences for a trouble-free implementation in laboratories. ResultsA well-designed experimental question is essential prior to the establishment of different NAEPCs culture models. The correct method of cell extraction from the nasal cavity is essential and represent an important basis for successful culture work. Prior enzymatic processing of biopsy specimens, cell culture materials, collagenization procedure, culture conditions, and choice of culture medium are some important practical notes that increase the quality of the culture. Moreover, protocols on imaging techniques including histologic and electron microscopy must be adapted for NAEPC culture. Adapted flow cytometric protocols and transepithelial electrical resistance measurements can add valuable information. OutlookA successful culturing of NAEPCs can provide an important basis for genetic studies and the implementation of omics-science, which is increasingly receiving broad attention in the scientific community. The common aim of in vitro ‘mini-noses’ will be a breakthrough in laboratories aiming to perform research under in vivo conditions. Here, organoid models are interesting models presenting a basis for translational studies.

New In Vitro Coculture Model for Evaluating Intestinal Absorption of Different Lipid Nanocapsules.

Standard models used for evaluating the absorption of nanoparticles like Caco-2 ignore the presence of vascular endothelium, which is a part of the intestinal multi-layered barrier structure. Therefore, a coculture between the Caco-2 epithelium and HMEC-1 (Human Microvascular Endothelial Cell type 1) on a Transwell® insert has been developed. The model has been validated for (a) membrane morphology by transmission electron microscope (TEM); (b) ZO-1 and β-catenin expression by immunoassay; (c) membrane integrity by trans-epithelial electrical resistance (TEER) measurement; and (d) apparent permeability of drugs from different biopharmaceutical classification system (BCS) classes. Lipid nanocapsules (LNCs) were formulated with different sizes (55 and 85 nm) and surface modifications (DSPE-mPEG (2000) and stearylamine). Nanocapsule integrity and particle concentration were monitored using the Förster resonance energy transfer (FRET) technique. The result showed that surface modification by DSPE-mPEG (2000) increased the absorption of 55-nm LNCs in the coculture model but not in the Caco-2. Summarily, the coculture model was validated as a tool for evaluating the intestinal absorption of drugs and nanoparticles. The new coculture model has a different LNCs absorption mechanism suggesting the importance of intestinal endothelium and reveals that the surface modification of LNCs can modify the in vitro oral absorption.

Transwell Insert-Embedded Microfluidic Devices for Time-Lapse Monitoring of Alveolar Epithelium Barrier Function under Various Stimulations.

This paper reports a transwell insert-embedded microfluidic device capable of culturing cells at an air-liquid interface (ALI), mimicking the in vivo alveolar epithelium microenvironment. Integration of a commercially available transwell insert makes the device fabrication straightforward and eliminates the tedious device assembly processes. The transwell insert can later be detached from the device for high-resolution imaging of the cells. In the experiments, the cells showing type-I pneumocyte markers are exploited to construct an in vitro alveolar epithelium model, and four culture conditions including conventional liquid/liquid culture (LLC) and air–liquid interface (ALI) cell culture in normal growth medium, and ALI cell culture with inflammatory cytokine (TNF-α) stimulation and ethanol vapor exposure are applied to investigate their effects on the alveolar epithelium barrier function. The barrier permeability is time-lapse monitored using trans-epithelial electrical resistance (TEER) measurement and immunofluorescence staining of the tight junction protein (ZO-1). The results demonstrate the functionalities of the device, and further show the applications and advantages of the constructed in vitro cell models for the lung studies.

Real-time quantitative monitoring of in vitro nasal drug delivery by a nasal epithelial mucosa-on-a-chip model

ABSTRACT Objectives A human nasal epithelial mucosa (NEM) on-a-chip is developed integrated with a novel carbon nanofibers-modified carbon electrode for real-time quantitative monitoring of in vitro nasal drug delivery. The integration of platinum electrodes in the chip also enables real-time measurement of transepithelial electrical resistance (TEER). Methods The air-liquid interface culture of nasal epithelial RPMI 2650 cells in the NEM-on-a-chip was optimized to mimic the key functional characteristics of the human nasal mucosa. The epithelial transport of ibuprofen in the NEM-on-a-chip was electrochemically monitored in real-time under static and physiologically realistic dynamic flow conditions. Results The NEM-on-a-chip mimics the mucus production and nasal epithelial barrier function of the human nasal mucosa. The real-time drug quantification by the NEM-on-a-chip was validated versus the high-performance liquid chromatography method. The drug transport rate monitored in the NEM-on-a-chip was influenced by the flow in the bottom compartment of the chip, highlighting the importance of emulating the dynamic in vivo condition for nasal drug transport studies. Conclusion This novel NEM-on-a-chip can be a low-cost and time-efficient alternative to the costly laborious conventional techniques for in vitro nasal drug transport assays. Importantly, its dynamic microenvironment enables conducting nasal drug transport tests under physiologically relevant dynamic conditions.

Transcellular penetration of Treponema phagedenis isolated from papillomatous digital dermatitis in polarized normal human epidermal keratinocytes in vitro.

Papillomatous digital dermatitis (PDD) is a polymicrobial infection causing lameness in dairy cattle. Culture-independent analysis has shown that Treponema phagedenis is present consistently and predominantly in the lesions. However, the pathogenesis of PDD, especially the tissue penetration pathway, has not been examined. In the present study, we investigated whether T. phagedenis strains isolated from PDD produce proteolytic enzyme (s) for disruption of the epithelial cell barrier and have the ability to translocate in polarized normal human epidermal keratinocytes (NHEK) in vitro. Ten strains of T. phagedenis isolated from lesions did not show proteolytic activity on modified skim milk agar, although a human strain of T. denticola used as a control showed such activity. The integrity of tight junctions was monitored by measurement of transepithelial electrical resistance (TER). The TER values after inoculation of the T. phagedenis strains examined did not change during the experimental period; however, apical to basolateral translocation of T. phagedenis was confirmed after 24 hr by microscopy and Treponema-specific PCR. We further confirmed that translocation of T. phagedenis was accelerated by co-inoculation with live T. denticola, but not with heat-killed organisms. Furthermore, tight junction ZO-1 protein was not lost intensity after inoculation with T. phagedenis and the organism was observed in NHEK cells using a florescence microscope. These results suggest that T. phagedenis strains may translocate via a transcellular route in vitro and that the invasion is accelerated by other bacteria, such as T. denticola, producing proteolytic activity.

Maternally expressed 3 protects the intestinal barrier from cardiac arrest-induced ischemia/reperfusion injury via miR-34a-3p/sirtuin 1/nuclear factor kappa B signaling.

BackgroundCardiac arrest (CA), a common disease with a high mortality rate, is a leading cause of ischemia/reperfusion (I/R)-induced dysfunction of the intestinal barrier. Long non-coding RNAs (lncRNAs) play crucial roles in multiple pathological processes. However, the effect of the lncRNA maternally expressed 3 (MEG3) on intestinal I/R injury and the intestinal barrier has not been fully determined. Therefore, this study aimed to investigate the function of MEG3 in CA-induced intestinal barrier dysfunction.MethodsThe oxygen and glucose deprivation (OGD) model in the human colorectal adenocarcinoma Caco-2 cells and in vivo cardiac arrest-induced intestinal barrier dysfunction model in Sprague-Dawley (SD) rats were established. The effect and underlying mechanism of MEG3 on the intestinal barrier from cardiac arrest-induced ischemia/reperfusion injury were analyzed by methyl thiazolyl tetrazolium (MTT) assays, Annexin V-FITC/PI apoptosis detection kit, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining, quantitative polymerase chain reaction (qPCR) assays, Western blot analysis, luciferase reporter gene assays, transepithelial electrical resistance (TEER) measurements, immunofluorescence analysis, and enzyme-linked immunosorbent assay (ELISA) assays.ResultsInterestingly, we found that MEG3 could protect Caco-2 cells from oxygen-glucose deprivation (OGD)/reoxygenation-induced I/R injury by modulating cell proliferation and apoptosis. Moreover, MEG3 relieved OGD-induced intestinal barrier dysfunction in vitro, as demonstrated by its significant rescue effect on transepithelial electrical resistance and the expression of tight junction proteins such as occludin and claudin-1 (CLDN1), which were impaired in OGD-treated Caco-2 cells. Mechanistically, MEG3 inhibited the expression of inflammatory factors including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon-gamma (IFN)-γ, inflammatory factors including interleukin (IL)-10, and transforming growth factor beta (TGFb)-1, as well as nuclear factor-kappa B (NF-κB) signaling. In response to OGD treatment in vitro, MEG3 also activated the expression of sirtuin 1 (SIRT1) by Caco-2 cells via sponging miR-34a-3p. Furthermore, MEG3 relieved CA-induced intestinal barrier dysfunction through NF-κB signaling in vivo.ConclusionsLncRNA MEG3 can protect the intestinal barrier from cardiac arrest-induced I/R injury via miR-34a-3p/SIRT1/NF-κB signaling. This finding provides new insight into the mechanism by which MEG3 restores intestinal barrier function following I/R injury, presenting it as a potential therapeutic candidate or strategy in intestinal injury.

High throughput transepithelial electrical resistance (TEER) measurements on perfused membrane-free epithelia.

Assessment of epithelial barrier function is critically important for studying healthy and diseased biological models. Here we introduce an instrument that measures transepithelial electrical resistance (TEER) of perfused epithelial tubes in the microfluidic OrganoPlate platform. The tubules are grown in microfluidic channels directly against an extracellular matrix, obviating the need for artificial filter membranes. We present TEER measurements on Caco-2 intestinal and renal proximal tubule epithelium. Forty tubules on one single plate were interrogated in less than a minute. We show that TEER measurement is significantly more sensitive than a fluorescent reporter leakage assay in response to staurosporine. We demonstrate a 40-channel time-lapse data acquisition over a 25 hour time period under flow conditions. We furthermore observed a 50% reduction in Caco-2 TEER values following exposure to a cocktail of inflammatory cytokines. To our best knowledge, this is the first instrument of its kind that allows routine TEER studies in perfused organ-on-a-chip systems without interference by artificial filter membranes. We believe the apparatus will contribute to accelerating routine adoption of perfused organ-on-a-chip systems in academic research and in industrial drug development.

Alginate- and Hyaluronic Acid-Based Hydrogels as Vitreous Substitutes: An In Vitro Evaluation.

PurposeTo study alginate- and hyaluronic acid–based hydrogels in vitro as vitreous substitutes.MethodsBiopolymeric hydrogels based on high-molecular alginate (0.5% and 1.0%) and hyaluronic acid (1.0% and Healaflow) were compared with extracted human vitreous bodies and silicone oil (SIL-5000) regarding their optical properties (refractive index, transmission) and viscoelastic characteristics (storage modulus G′, loss modulus G″). The cytotoxic (metabolic activity, apoptosis) and antiproliferative profiles were determined using cultured human fibroblasts, ARPE-19, and photoreceptor cells. The hydrogel systems were applied to human fetal retinal pigment epithelial cells cultured for two months until maximum transepithelial electrical resistance (TEER) to investigate the effect of the gel matrices on tight junctions using TEER measurements and immunostainings against the tight junction protein ZO-1.ResultsTested alginate- and hyaluronic acid–based hydrogels resembled the natural refractive index of human vitreous bodies (1.3356–1.3360) in contrast to SIL-5000 (1.4034) and showed high optical transparency (>90%) within the visible light region. The biopolymeric hydrogels exhibited viscoelastic properties similar to juvenile vitreous bodies with G′>G″ adjustable via different gelation times, contrary to SIL-5000 (G′<G″). The metabolic activity, apoptosis and tight junctions of all tested ocular cells were unaffected by the alginate- and hyaluronic acid–based vitreous substitutes.ConclusionsThe present in vitro study demonstrates good optical, viscoelastic, and biocompatible properties of alginate- and hyaluronic acid–based hydrogels required for their use as vitreous substitutes.Translational RelevanceBiopolymer-based hydrogels represent a promising vitreous replacement strategy to treat vitreoretinal diseases.

Zinc deficiency induces epithelial barrier dysfunction and altered intestinal ion transport in novel murine model of Shigella flexneri diarrhea

Background: Zinc (Zn) deficiency has been linked with etiology for wide range of gastrointestinal diseases. Although low serum Zn has been associated with diarrhea and growth retardation, however the underlying mechanism of Zn deficiency aggravating the diarrheal manifestation remains elusive. Here, we have studied the physiological cascade elicited during Zn deficiency in both colonic cell lines and murine model leading to altered intestinal barrier function and ion transport. Methods and materials: T84 were grown onto transwell inserts in Zn deficient conditions for measurements of transepithelial electrical resistance (TER) and short-circuit current (Isc). Adult BALB/c mice fed with Zn deficient diet for 14 days and were intraperitoneally challenged with virulent S.flexneri 2a for following assessments of electrophysiological properties of the colonized colon in ussing chamber. Colonization evaluation from feces and colon homogenate were further analyzed for bacterial translocation assay. Results: Zn deficient cells infected with Shigella flexneri 2a showed significant increase in transmigration of the pathogens (6.58 ± 0.13 vs 7.98 ± 0.23 log10 CFU/ml) for 6 h post infection along with poor formation of transepithelial resistance (1014 + 165 Ωcm2 vs 3663 + 293 Ωcm2). Ultrastructure of T84 cells grown in Zn deficient medium showed widened TJ complex and due to few membrane fusion proteins showing distorted TJ morphology and shorthened microvilli. Immunoblotting analysis further showed increased expression of Claudin-2 leaky tight junction protein in Zn deficient cells by modulating ERK dependent pathways. Following studies with Ussing chambers further demonstrated increase in Ca2+ dependent electrogenic Cl− secretion in Zn deficient cells. In vivo studies showed Zn deficient condition in mice increased bacterial translocation, colonization, bacterial shedding and pronounced disruption in intestinal permeability. Conclusion: Our results suggest that in T84 cells and murine of Shigellosis, Zn deficiency causes (1) pronounced barrier dysfunction and (2) altered Cl− secretion leading to heightened diarrheal pathogenesis thus linking micronutrient deficiency with bacterial infection and inflammation.

Design strategy of electrode patterns based on finite element analysis in microfluidic device for Trans‐Epithelial Electrical Resistance (TEER) measurement

AbstractTrans‐epithelial electrical resistance (TEER) is a widely used non‐invasive and label‐free measurement to evaluate the barrier function of mono‐layered cells in real‐time. However, TEER values depend on distribution of electric current densities generated by a combination of shapes and arrangements of plane electrodes for performing TEER, but until now, there is no systematical analysis of their effects on TEER values, causing less reproducibility. Here, we report a new strategy to design the shapes and arrangements of electrodes to perform TEER in a microfluidic device. To investigate the effects of the electrodes design on TEER values, we analyze the distribution of electric current densities using finite element method and evaluate their uniformity of sensitivity. Our strategy allowed designing several electrode patterns of electrodes combinations, capable of simultaneous cell observation with a microscope and TEER measurements.

Differential effects of Hsp90 inhibition on corneal cells in vitro and in vivo

The transformation of quiescent keratocytes to activated fibroblasts and myofibroblasts (KFM transformation) largely depends on transforming growth factor beta (TGFβ) signaling. Initiation of the TGFβ signaling cascade results from binding of TGFβ to the labile type I TGFβ receptor (TGFβRI), which is stabilized by the 90 kDa heat shock protein (Hsp90). Since myofibroblast persistence within the corneal stroma can result in stromal haze and corneal fibrosis in patients undergoing keratorefractive therapy, modulation of TGFβ signaling through Hsp90 inhibition would represent a novel approach to prevent myofibroblast persistence.In vitro, rabbit corneal fibroblasts (RCFs) or stratified immortalized human corneal epithelial cells (hTCEpi) were treated with a Hsp90 inhibitor (17AAG) in the presence/absence of TGFβ1. RCFs were cultured either on tissue culture plastic, anisotropically patterned substrates, and hydrogels of varying stiffness. Cellular responses to both cytoactive and variable substrates were assessed by morphologic changes to the cells, and alterations in expression patterns of key keratocyte and myofibroblast proteins using PCR, Western blotting and immunocytochemistry. Transepithelial electrical resistance (TEER) measurements were performed to establish epithelial barrier integrity. In vivo, the corneas of New Zealand White rabbits were wounded by phototherapeutic keratectomy (PTK) and treated with 17AAG (3× or 6× daily) either immediately or 7 days after wounding for 28 days. Rabbits underwent clinical ophthalmic examinations, SPOTS scoring and advanced imaging on days 0, 1, 3, 7, 10, 14, 21 and 28. On day 28, rabbits were euthanized and histopathology/immunohistochemistry was performed.In vitro data demonstrated that 17AAG inhibited KFM transformation with the de-differentiation of spindle shaped myofibroblasts to dendritic keratocyte-like cells accompanied by significant upregulation of corneal crystallins and suppression of myofibroblast markers regardless of TGFβ1 treatment. RCFs cultured on soft hydrogels or patterned substrates exhibited elevated expression of α-smooth muscle actin (αSMA) in the presence of 17AAG. Treatment of hTCEpi cells disrupted zonula occludens 1 (ZO-1) adherens junction formation. In vivo, there were no differences detected in nearly all clinical parameters assessed between treatment groups. However, rabbits treated with 17AAG developed greater stromal haze formation compared with controls, irrespective of frequency of administration. Lastly, there was increased αSMA positive myofibroblasts in the stroma of 17AAG treated animals when compared with controls.Hsp90 inhibition promoted reversion of the myofibroblast to keratocyte phenotype, although this only occurred on rigid substrates. By contrast, in vivo Hsp90 inhibition was detrimental to corneal wound healing likely due to impairment in corneal epithelial closure and barrier function restoration. Collectively, our data demonstrated a strong interplay in vitro between biophysical cues and soluble signaling molecules in determining corneal stromal cell phenotype.

Cutibacterium acnes Phylotype I and II Strains Interact Differently With Human Skin Cells.

Acne vulgaris is one of the most common skin disorders and affects the pilosebaceous units. Although the exact pathogenesis of acne is still unknown, Cutibacterium acnes (formerly known as Propionibacterium acnes) is considered one of the key contributing factors. In fact, a significant association exists between C. acnes strains belonging to phylotype I and acne. However, there is still heavy debate on the exact role of C. acnes in acne and its behavior in the pilosebaceous unit, and more specifically its interactions with the human skin cells. In this study, key elements of the host-pathogen interaction were studied for a collection of C. acnes strains, belonging to phylotype I and II, including association with HaCaT keratinocytes and SZ95 sebocytes, the effect of C. acnes on keratinocyte tight junctions in a HaCaT monoculture and in an additional keratinocyte-sebocyte co-culture model, and C. acnes invasion through the keratinocyte cell layer. Our data showed association of all C. acnes strains to both skin cell lines, with a significantly higher association of type I strains compared to type II strains. Microscopic imaging and western blot analysis of the tight junction protein ZO-1, together with transepithelial electrical resistance (TEER) measurements revealed an initial induction of keratinocyte tight junctions after 24 h infection but a degradation after 48 h, demonstrating a decline in cell lining integrity during infection. Subsequently, C. acnes was able to invade after 48 h of infection, although invasion frequency was significantly higher for type II strains compared to type I strains.