scholarly journals Measurements of transepithelial electrical resistance (TEER) are affected by junctional length in immature epithelial monolayers

2021 ◽  
Author(s):  
Kannapin Felix ◽  
Schmitz Tobias ◽  
Hansmann Jan ◽  
Schlegel Nicolas ◽  
Meir Michael
Keyword(s):  
Epithelial Cell ◽  
Electrical Resistance ◽  
Fluorescein Isothiocyanate ◽  
Cell Types ◽  
Transepithelial Electrical Resistance ◽  
Cell Junctions ◽  
Junctional Complex ◽  
Additional Increase ◽  
Cell Monolayers ◽  
Epithelial Cell Monolayers

AbstractThe measurement of transepithelial electrical resistance (TEER) is a common technique to determine the barrier integrity of epithelial cell monolayers. However, it is remarkable that absolute TEER values of similar cell types cultured under comparable conditions show an immense heterogeneity. Based on previous observations, we hypothesized that the heterogeneity of absolute TEER measurements can not only be explained by maturation of junctional proteins but rather by dynamics in the absolute length of cell junctions within monolayers. Therefore, we analyzed TEER in epithelial cell monolayers of Caco2 cells during their differentiation, with special emphasis on both changes in the junctional complex and overall cell morphology within monolayers. We found that in epithelial Caco2 monolayers TEER increased until confluency, then decreased for some time, which was then followed by an additional increase during junctional differentiation. In contrast, permeability of macromolecules measured at different time points as 4 kDA fluorescein isothiocyanate (FITC)-dextran flux across monolayers steadily decreased during this time. Detailed analysis suggested that this observation could be explained by alterations of junctional length along the cell borders within monolayers during differentiation. In conclusion, these observations confirmed that changes in cell numbers and consecutive increase of junctional length have a critical impact on TEER values, especially at stages of early confluency when junctions are immature.

scholarly journals Adherence to and Penetration of Human Intestinal Caco-2 Epithelial Cell Monolayers by Pseudomonas aeruginosa

1998 ◽  
Vol 66 (4) ◽  
pp. 1748-1751 ◽  
Author(s):  
Yoichi Hirakata ◽  
Kohichi Izumikawa ◽  
Toshiyuki Yamaguchi ◽  
Shizunobu Igimi ◽  
Nobuhiko Furuya ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Epithelial Cell ◽  
Virulence Factor ◽  
Electrical Resistance ◽  
Transepithelial Electrical Resistance ◽  
Clinical Isolates ◽  
Exotoxin A ◽  
Cell Monolayers ◽  
Drastic Decrease ◽  
Epithelial Cell Monolayers

ABSTRACT Clinical isolates of Pseudomonas aeruginosa from blood adhered to and penetrated intestinal Caco-2 cell monolayers to a greater degree than did isolates from sputum, with a concomitant drastic decrease in transepithelial electrical resistance. PAO-PR1, an avirulent exotoxin A mutant of PAO1, did not cause a decrease in the resistance. The Caco-2 monolayer system may be useful for the evaluation of certain P. aeruginosa virulence factor activities.

Effect of stretch on structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin

2006 ◽  
Vol 290 (6) ◽  
pp. L1104-L1110 ◽  
Author(s):  
Xavier Trepat ◽  
Ferranda Puig ◽  
Nuria Gavara ◽  
Jeffrey J. Fredberg ◽  
Ramon Farre ◽  
...  
Keyword(s):  
Mechanical Ventilation ◽  
Epithelial Cell ◽  
Structural Integrity ◽  
Alveolar Epithelial Cell ◽  
Compressive Strain ◽  
Cell Junctions ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers ◽  
Human Alveolar Epithelial Cell

Alveolar epithelial cells in patients with acute lung injury subjected to mechanical ventilation are exposed to increased procoagulant activity and mechanical strain. Thrombin induces epithelial cell stiffening, contraction, and cytoskeletal remodeling, potentially compromising the balance of forces at the alveolar epithelium during cell stretching. This balance can be further compromised by the loss of integrity of cell-cell junctions in the injured epithelium. The aim of this work was to study the effect of stretch on the structural integrity and micromechanics of human alveolar epithelial cell monolayers exposed to thrombin. Confluent and subconfluent cells (A549) were cultured on collagen-coated elastic substrates. After exposure to thrombin (0.5 U/ml), a stepwise cell stretch (20%) was applied with a vacuum-driven system mounted on an inverted microscope. The structural integrity of the cell monolayers was assessed by comparing intercellular and intracellular strains within the monolayer. Strain was measured by tracking beads tightly bound to the cell surface. Simultaneously, cell viscoelasticity was measured using optical magnetic twisting cytometry. In confluent cells, thrombin did not induce significant changes in transmission of strain from the substrate to overlying cells. By contrast, thrombin dramatically impaired the ability of subconfluent cells to follow imposed substrate deformation. Upon substrate unstretching, thrombin-treated subconfluent cells exhibited compressive strain (9%). Stretch increased stiffness (56–62%) and decreased cell hysteresivity (13–22%) of vehicle cells. By contrast, stretch did not increase stiffness of thrombin-treated cells, suggesting disruption of cytoskeletal structures. Our findings suggest that thrombin could exacerbate epithelial barrier dysfunction in injured lungs subjected to mechanical ventilation.

scholarly journals Enterococcus faecalis Gelatinase Mediates Intestinal Permeability via Protease-Activated Receptor 2

2015 ◽  
Vol 83 (7) ◽  
pp. 2762-2770 ◽  
Author(s):  
Nitsan Maharshak ◽  
Eun Young Huh ◽  
Chorlada Paiboonrungruang ◽  
Michael Shanahan ◽  
Lance Thurlow ◽  
...  
Keyword(s):  
Ulcerative Colitis ◽  
Epithelial Cell ◽  
Enterococcus Faecalis ◽  
Fluorescein Isothiocyanate ◽  
Cell Permeability ◽  
Content Type ◽  
Cell Monolayers ◽  
Microbial Protease ◽  
Epithelial Cell Monolayers ◽  
Protease Activated Receptor 2

Microbial protease-mediated disruption of the intestinal epithelium is a potential mechanism whereby a dysbiotic enteric microbiota can lead to disease. This mechanism was investigated using the colitogenic, protease-secreting enteric microbeEnterococcus faecalis. Caco-2 and T-84 epithelial cell monolayers and the mouse colonic epithelium were exposed to concentrated conditioned media (CCM) fromE. faecalisV583 andE. faecalislacking the gelatinase gene (gelE). The flux of fluorescein isothiocyanate (FITC)-labeled dextran across monolayers or the mouse epithelium following exposure to CCM from parental or mutantE. faecalisstrains indicated paracellular permeability. A protease-activated receptor 2 (PAR2) antagonist and PAR2-deficient (PAR2−/−) mice were used to investigate the role of this receptor inE. faecalis-induced permeability. Gelatinase (GelE) purified fromE. faecalisV583 was used to confirm the ability of this protease to induce epithelial cell permeability and activate PAR2. The protease-mediated permeability of colonic epithelia from wild-type (WT) and PAR2−/−mice by fecal supernatants from ulcerative colitis patients was assessed. SecretedE. faecalisproteins induced permeability in epithelial cell monolayers, which was reduced in the absence ofgelEor by blocking PAR2 activity. SecretedE. faecalisproteins induced permeability in the colonic epithelia of WT mice that was absent in tissues from PAR2−/−mice. Purified GelE confirmed the ability of this protease to induce epithelial cell permeability via PAR2 activation. Fecal supernatants from ulcerative colitis patients induced permeability in the colonic epithelia of WT mice that was reduced in tissues from PAR2−/−mice. Our investigations demonstrate that GelE fromE. faecaliscan regulate enteric epithelial permeability via PAR2.

scholarly journals Application of Measurements of Transepithelial Electrical Resistance of Intestinal Epithelial Cell Monolayers To Evaluate Probiotic Activity

2005 ◽  
Vol 71 (11) ◽  
pp. 7528-7530 ◽  
Author(s):  
Trine Danø Klingberg ◽  
Maja Herold Pedersen ◽  
Avrelija Cencic ◽  
Birgitte Bjørn Budde
Keyword(s):  
Electrical Resistance ◽  
Intestinal Epithelial Cell ◽  
Transepithelial Electrical Resistance ◽  
Intestinal Epithelial ◽  
Lactobacillus Salivarius ◽  
Cell Monolayers ◽  
Epithelial Cell Monolayers ◽  
Probiotic Lactobacilli ◽  
Dose Dependent ◽  
Probiotic Activity

ABSTRACT Among five potentially probiotic lactobacilli investigated, Lactobacillus plantarum MF1298 and Lactobacillus salivarius DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by Listeria monocytogenes.

Absorption of intact albumin across rat alveolar epithelial cell monolayers

2003 ◽  
Vol 284 (3) ◽  
pp. L458-L465 ◽  
Author(s):  
Kwang-Jin Kim ◽  
Yasuhisa Matsukawa ◽  
Hiroshi Yamahara ◽  
Vijay K. Kalra ◽  
Vincent H. L. Lee ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Alveolar Epithelial Cell ◽  
Apical Cell ◽  
Fluorescein Isothiocyanate ◽  
Transport Rate ◽  
Maximal Concentration ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Mammalian Lung ◽  
Epithelial Cell Monolayers

Transport characteristics of intact albumin were investigated using primary cultured rat alveolar epithelial cell monolayers. The apical-to-basolateral ( ab) flux of intact fluorescein isothiocyanate (FITC)-labeled albumin (F-Alb) is greater than basolateral-to-apical ( ba) flux at the same upstream [F-Alb]. Net absorption of intact F-Alb occurs with half-maximal concentration of ∼1.6 μM and maximal transport rate of ∼0.15 fmol · cm−2 · s−1. At 15 and 4°C, both ab and ba F-Alb fluxes are not different from zero, collapsing net absorption. The presence of excess unlabeled albumin (but not other macromolecule species) in either the apical or basolateral fluid significantly reduces both ab and ba unidirectional F-Alb fluxes. Photoaffinity labeling of apical cell membranes revealed an ∼60-kDa protein that exhibits specificity for albumin. These data indicate that net absorption of intact albumin takes place via saturable receptor-mediated transcellular endocytotic processes recognizing albumin, but not other macromolecules, that may play an important role in alveolar homeostasis in the mammalian lung.

scholarly journals Epithelial Barrier Integrity Profiling: Combined Approach Using Cellular Junctional Complex Imaging and Transepithelial Electrical Resistance

2021 ◽  
pp. 247255522110130
Author(s):  
Theresa J. Pell ◽  
Mike B. Gray ◽  
Sarah J. Hopkins ◽  
Richard Kasprowicz ◽  
James D. Porter ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Electrical Resistance ◽  
Cell Function ◽  
Transepithelial Electrical Resistance ◽  
Junctional Complex ◽  
Chronic Obstructive ◽  
Vitro Assay ◽  
High Content Imaging ◽  
Barrier Integrity

A core aspect of epithelial cell function is barrier integrity. A loss of barrier integrity is a feature of a number of respiratory diseases, including asthma, allergic rhinitis, and chronic obstructive pulmonary disease. Restoration of barrier integrity is a target for respiratory disease drug discovery. Traditional methods for assessing barrier integrity have their limitations. Transepithelial electrical resistance (TEER) and dextran permeability methods can give poor in vitro assay robustness. Traditional junctional complex imaging approaches are labor-intensive and tend to be qualitative but not quantitative. To provide a robust and quantitative assessment of barrier integrity, high-content imaging of junctional complexes was combined with TEER. A scalable immunofluorescent high-content imaging technique, with automated quantification of junctional complex proteins zonula occludens-1 and occludin, was established in 3D pseudostratified primary human bronchial epithelial cells cultured at an air–liquid interface. Ionic permeability was measured using TEER on the same culture wells. The improvements to current technologies include the design of a novel 24-well holder to enable scalable in situ confocal cell imaging without Transwell membrane excision, the development of image analysis pipelines to quantify in-focus junctional complex structures in each plane of a Z stack, and the enhancement of the TEER data analysis process to enable statistical evaluation of treatment effects on barrier integrity. This novel approach was validated by demonstrating measurable changes in barrier integrity in cells grown under conditions known to perturb epithelial cell function.

scholarly journals Characteristics of Passive Solute Transport across Primary Rat Alveolar Epithelial Cell Monolayers

Membranes ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 331
Author(s):  
Yong Ho Kim ◽  
Kwang-Jin Kim ◽  
David Z. D’Argenio ◽  
Edward D. Crandall
Keyword(s):  
Epithelial Cell ◽  
Tight Junctions ◽  
Pore Radius ◽  
Alveolar Epithelial Cell ◽  
Alveolar Epithelial Cells ◽  
Type I ◽  
Cell Monolayers ◽  
Alveolar Epithelial ◽  
Epithelial Cell Monolayers ◽  
Hydrophilic Solutes

Primary rat alveolar epithelial cell monolayers (RAECM) were grown without (type I cell-like phenotype, RAECM-I) or with (type II cell-like phenotype, RAECM-II) keratinocyte growth factor to assess passive transport of 11 hydrophilic solutes. We estimated apparent permeability (Papp) in the absence/presence of calcium chelator EGTA to determine the effects of perturbing tight junctions on “equivalent” pores. Papp across RAECM-I and -II in the absence of EGTA are similar and decrease as solute size increases. We modeled Papp of the hydrophilic solutes across RAECM-I/-II as taking place via heterogeneous populations of equivalent pores comprised of small (0.41/0.32 nm radius) and large (9.88/11.56 nm radius) pores, respectively. Total equivalent pore area is dominated by small equivalent pores (99.92–99.97%). The number of small and large equivalent pores in RAECM-I was 8.55 and 1.29 times greater, respectively, than those in RAECM-II. With EGTA, the large pore radius in RAECM-I/-II increased by 1.58/4.34 times and the small equivalent pore radius increased by 1.84/1.90 times, respectively. These results indicate that passive diffusion of hydrophilic solutes across an alveolar epithelium occurs via small and large equivalent pores, reflecting interactions of transmembrane proteins expressed in intercellular tight junctions of alveolar epithelial cells.

scholarly journals Energetics of mesoscale cell turbulence in two-dimensional monolayers

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shao-Zhen Lin ◽  
Wu-Yang Zhang ◽  
Dapeng Bi ◽  
Bo Li ◽  
Xi-Qiao Feng
Keyword(s):  
Kinetic Energy ◽  
Tumor Metastasis ◽  
Cell Types ◽  
Boltzmann Distribution ◽  
Scaling Exponent ◽  
Biological Processes ◽  
Two Dimensional ◽  
Cell Monolayers ◽  
Wide Range ◽  
Epithelial Cell Monolayers

AbstractInvestigation of energy mechanisms at the collective cell scale is a challenge for understanding various biological processes, such as embryonic development and tumor metastasis. Here we investigate the energetics of self-sustained mesoscale turbulence in confluent two-dimensional (2D) cell monolayers. We find that the kinetic energy and enstrophy of collective cell flows in both epithelial and non-epithelial cell monolayers collapse to a family of probability density functions, which follow the q-Gaussian distribution rather than the Maxwell–Boltzmann distribution. The enstrophy scales linearly with the kinetic energy as the monolayer matures. The energy spectra exhibit a power-decaying law at large wavenumbers, with a scaling exponent markedly different from that in the classical 2D Kolmogorov–Kraichnan turbulence. These energetic features are demonstrated to be common for all cell types on various substrates with a wide range of stiffness. This study provides unique clues to understand active natures of cell population and tissues.

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