Improved micro-impedance spectroscopy to determine cell barrier properties

2020 ◽  
Vol 4 (3) ◽  
pp. 150-155 ◽  
Author(s):  
Md. Mehadi Hasan Sohag ◽  
Olivier Nicoud ◽  
Racha Amine ◽  
Abir Khalil-Mgharbel ◽  
Jean-Pierre Alcaraz ◽  
...  
Keyword(s):  
Impedance Spectroscopy ◽  
Epithelial Cells ◽  
Lipid Bilayers ◽  
Cultured Cells ◽  
Cell Model ◽  
Measurement Techniques ◽  
Cell Monolayer ◽  
Microfluidic Channel ◽  
Small Surface ◽  
Resistance Pathway

AbstractThe goal of this study was to determine whether the Tethapod system, which was designed to determine the impedance properties of lipid bilayers, could be used for cell culture in order to utilise micro-impedance spectroscopy to examine further biological applications. To that purpose we have used normal epithelial cells from kidney (RPTEC) and a kidney cancer cell model (786-O). We demonstrate that the Tethapod system is compatible with the culture of 10,000 cells seeded to grow on a small area gold measurement electrode for several days without affecting the cell viability. Furthermore, the range of frequencies for EIS measurements were tuned to examine easily the characteristics of the cell monolayer. We demonstrate significant differences in the paracellular resistance pathway between normal and cancer kidney epithelial cells. Thus, we conclude that this device has advantages for the study of cultured cells that include (i) the configuration of measurement and reference electrodes across a microfluidic channel, and (ii) the small surface area of 6 parallel measurement electrodes (2.1 mm2) integrated in a microfluidic system. These characteristics might improve micro-impedance spectroscopy measurement techniques to provide a simple tool for further studies in the field of the patho-physiology of biological barriers.

scholarly journals Improving Membrane Staining of Cultured Cells Using Ferrocyanide as a Post-Fixative

2007 ◽  
Vol 15 (2) ◽  
pp. 38-39
Author(s):  
Stéphane Nizet
Keyword(s):  
Ultrathin Section ◽  
Cultured Cells ◽  
Cell Model ◽  
Cell Monolayer ◽  
Cancer Cell Line ◽  
Colon Cancer Cell Line ◽  
Colon Cancer Cell ◽  
Human Intestine ◽  
A Cell ◽  
Permeability Studies

Lack of contrast is a common problem encountered when doing TEM of cultured cells, especially of membranes. Using ferrocyanide as a post-fixative can greatly improve membrane fixation and staining. This protocol has been used to study Caco-2 cells grown on PET membranes (the “Caco model”). Caco-2 is a colon cancer cell line that differentiates upon reaching confluency. This allows permeability studies on a cell model, which is reasonably similar to the human intestine.Basically, the protocol is classical, the only peculiarity consisting in including ferrocyanide in post-fixation. I describe how I prepare and embed the membrane in order to obtain transverse sections of a cell monolayer because I find this is the only way to obtain regular sections with the cells sticking to the membrane (otherwise the ultrathin section splits between the cell and membrane).

scholarly journals Cytopathogenic Effect of Trichomonas vaginalis on Human Vaginal Epithelial Cells Cultured In Vitro

2000 ◽  
Vol 68 (7) ◽  
pp. 4200-4206 ◽  
Author(s):  
R. O. Gilbert ◽  
G. Elia ◽  
D. H. Beach ◽  
Suzanne Klaessig ◽  
B. N. Singh
Keyword(s):  
Epithelial Cells ◽  
Trichomonas Vaginalis ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Cell Monolayer ◽  
Cytotoxic Effects ◽  
Permeable Membrane ◽  
Vaginal Epithelial Cells ◽  
Vaginal Fibroblasts

ABSTRACT In this study we established human vaginal epithelial cells (hVECs) in culture and evaluated their interaction with Trichomonas vaginalis parasites to complement previous studies using other cell types. Primary cultures of hVECs were established. Contaminating fibroblasts were separated from epithelial cells by differential trypsinization. Specific antibody staining revealed that over 92% of cells in hVEC monolayers were epithelial cells. T. vaginalis adhered to hVECs and produced severe cytotoxic effects resulting in obliteration of the monolayer within 24 h. Adherence and cytotoxicity were not observed when T. vaginalis was exposed to human vaginal fibroblasts or bovine vaginal epithelial cells. Likewise, the bovine parasite Tritrichomonas foetushad no cytotoxic effects on hVECs. We concluded that the interaction between T. vaginalis and hVECs is both cell specific (limited to epithelial cells and not vaginal fibroblasts) and species specific (limited to human vaginal cells and not bovine cells). Pretreatment of T. vaginalis with metronidazole or periodate abolished the adhesion of parasites to cell monolayers and the cytotoxic effect, suggesting involvement of carbohydrate-containing molecules in these processes. Different clinical isolates of T. vaginalis caused damage to cultured cells at different rates. Parasites separated from the vaginal cell monolayer by a permeable membrane did not produce a cytopathic effect, suggesting contact-dependent cytotoxicity.

Analysis of the Dynamics of the Electric Capacitance of a Cell Monolayer at the Late Stages of Caco-2 cell Differentiation

2019 ◽  
Vol 35 (6) ◽  
pp. 87-90
Author(s):  
S.V. Nikulin ◽  
V.A. Petrov ◽  
D.A. Sakharov
Keyword(s):  
Impedance Spectroscopy ◽  
Cell Monolayer ◽  
Microfluidic Chips ◽  
Russian Science ◽  
Electric Capacitance ◽  
A Cell ◽  
Static Conditions ◽  
Late Stages

The real-time monitoring of electric capacitance (impedance spectroscopy) allowed obtaining evidence that structures which look like intestinal villi can be formed during the cultivation under static conditions as well as during the cultivation in microfluidic chips. It was shown in this work via transcriptome analysis that the Hh signaling pathway is involved in the formation of villus-like structures in vitro, which was previously shown for their formation in vivo. impedance spectroscopy, intestine, villi, electric capacitance, Hh The study was funded by the Russian Science Foundation (Project 16-19-10597).

Stimulation of both human bronchial epithelium and neutrophils is needed for maximal interactive adhesion

1996 ◽  
Vol 270 (1) ◽  
pp. L80-L87 ◽  
Author(s):  
P. G. Bloemen ◽  
M. C. Van den Tweel ◽  
P. A. Henricks ◽  
F. Engels ◽  
M. J. Van de Velde ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cultured Cells ◽  
Bronchial Epithelium ◽  
Bronchial Epithelial Cells ◽  
Intercellular Adhesion Molecule ◽  
Tnf Alpha ◽  
Epithelial Cell Line ◽  
Ifn Gamma ◽  
Bronchial Epithelial ◽  
Stimulation Of

It has become clear that the bronchial epithelium is not just a passive barrier but plays an active role in inflammation. It can produce several inflammatory mediators and does express cell adhesion molecules of which intercellular adhesion molecule (ICAM)-1 can be upregulated by cytokines like interferon (IFN)-gamma. In the present study, we analyzed in detail the interaction of neutrophils with human bronchial epithelial cells, both primary cultured cells and the bronchial epithelial cell line BEAS-2B. Confluent monolayers of epithelial cells were incubated with freshly isolated 51Cr-labeled neutrophils for 30 min at 37 degrees C; then the nonadherent cells were removed by washing gently. Stimulation of the epithelial cells with IFN-gamma or the combination of IFN-gamma and tumor necrosis factor-alpha (TNF-alpha) (which doubles the ICAM-1 expression) increased neutrophil adhesion. Activation of the neutrophils themselves with N-formylmethionyl-leucyl-phenylalanine (fMLP), platelet-activating factor, or TNF-alpha also caused a profound enhancement of the adhesion. A significant additional increase was found when the epithelial cells had been exposed to IFN-gamma and the neutrophils were stimulated with fMLP simultaneously. This effect was even more pronounced with epithelium preincubated with IFN-gamma and TNF-alpha. With the use of monoclonal antibodies against CD18 and ICAM-1, it was demonstrated that the increased adhesion was mainly mediated by the ICAM-1/beta 2-integrin interaction. This study highlights that both the activation state of the bronchial epithelial cells and the activation state of the neutrophils are critical for their interactive adhesion.

scholarly journals Effect of a Berry Polyphenolic Fraction on Biofilm Formation, Adherence Properties and Gene Expression of Streptococcus mutans and Its Biocompatibility with Oral Epithelial Cells

Antibiotics ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 46
Author(s):  
Mariem Souissi ◽  
Amel Ben Lagha ◽  
Kamel Chaieb ◽  
Daniel Grenier
Keyword(s):  
Epithelial Cells ◽  
Streptococcus Mutans ◽  
Biofilm Formation ◽  
Cell Model ◽  
Pcr Analysis ◽  
Adhesion Properties ◽  
Oral Epithelial Cells ◽  
Polymerase Chain ◽  
Oral Epithelial ◽  
Wild Blueberry

The ability of Streptococcus mutans to adhere to oral surfaces and form biofilm is a key step in the tooth decay process. The aim of this study was to investigate a berry (wild blueberry, cranberry, and strawberry) polyphenolic fraction, commercialized as Orophenol®, for its antibacterial, anti-biofilm, and anti-adhesion properties on S. mutans. Moreover, the biocompatibility of the fraction with human oral epithelial cells was assessed. Phenolic acids, flavonoids (flavonols, anthocyanins, flavan-3-ols), and procyanidins made up 10.71%, 19.76%, and 5.29% of the berry polyphenolic fraction, respectively, as determined by chromatography and mass spectrometry. The berry polyphenolic preparation dose-dependently inhibited S. mutans biofilm formation while not reducing bacterial growth. At concentrations ranging from 250 to 1000 µg/mL, the fraction inhibited the adhesion of S. mutans to both saliva-coated hydroxyapatite and saliva-coated nickel–chrome alloy. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis showed that incubating S. mutans with the berry polyphenolic fraction was associated with a reduced expression of luxS gene, which regulates quorum sensing in S. mutans. The berry fraction did not show any significant cytotoxicity in an oral epithelial cell model. In conclusion, Orophenol®, which is a mixture of polyphenols from wild blueberry, cranberry and strawberry, possesses interesting anti-caries properties while being compatible with oral epithelial cells.

scholarly journals Impact of Heterogeneity within Cultured Cells on Bacterial Invasion: Analysis of Pseudomonas aeruginosa andSalmonella enterica Serovar Typhi Entry into MDCK cells by Using a Green Fluorescent Protein-Labelled Cystic Fibrosis Transmembrane Conductance Regulator Receptor

2000 ◽  
Vol 68 (2) ◽  
pp. 861-870 ◽  
Author(s):  
A. Alev Gerçeker ◽  
Tanweer Zaidi ◽  
Peter Marks ◽  
David E. Golan ◽  
Gerald B. Pier
Keyword(s):  
Epithelial Cells ◽  
Protein Expression ◽  
Fluorescent Protein ◽  
Cultured Cells ◽  
Mdck Cells ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Bacterial Uptake ◽  
Cftr Protein ◽  
Green Fluorescent

ABSTRACT The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel that also serves as a receptor for entry ofPseudomonas aeruginosa and Salmonella entericaserovar Typhi into epithelial cells. To evaluate heterogeneity in CFTR protein expression in cultured cells and the effect of heterogeneity on internalization of different P. aeruginosa and serovar Typhi strains, we used two-color flow cytometry and confocal laser microscopy to study bacterial uptake by Madin-Darby canine kidney (MDCK) type I epithelial cells stably expressing a green fluorescent protein (GFP)-CFTR fusion construct (MDCK–GFP-CFTR cells). We found a strong correlation between cell size and GFP-CFTR protein expression, with 60 to 70% of cells expressing low levels of GFP-CFTR protein, 20 to 30% expressing intermediate levels, and <10% expressing high levels. The cells were sorted into low-, intermediate-, or high-level producers of CFTR protein; in vitro growth of each sorted population yielded the same distribution of CFTR protein expression as that in the original population. Cells expressing either low or high levels of CFTR protein internalized bacteria poorly; maximal bacterial uptake occurred in the cells expressing intermediate levels of CFTR protein. Treatment of MDCK cells with sodium butyrate markedly enhanced the production of CFTR protein without increasing cell size; butyrate treatment also increased the proportion of cells with internalized bacteria. However, there were fewer bacteria per butyrate-treated cell and, for P. aeruginosa, there was an overall decrease in the total level of bacterial uptake. The most highly ingested bacterial strains were internalized by fewer total MDCK–GFP-CFTR cells, indicating preferential bacterial uptake by a minority of epithelial cells within a given culture. Confocal fluorescence microscopy showed that P. aeruginosa and serovar Typhi induced cytoplasmic accumulation of CFTR protein close to the plasma membrane where the bacteria were adherent. These results show that within a population of MDCK–GFP-CFTR cells, there are cells with markedly different abilities to ingest bacteria via CFTR, the majority of the P. aeruginosa and serovar Typhi cells are ingested by the one-fourth to one-third of the cells that exhibit an intermediate size and level of CFTR protein expression, and overexpression of the CFTR receptor does not increase total bacterial uptake but rather allows more epithelial cells to ingest fewer total bacteria.

Cultured epithelial cells derived from human foetal pancreas as a model for the study of cystic fibrosis: further analyses on the origins and nature of the cell types

1988 ◽  
Vol 90 (1) ◽  
pp. 73-77
Author(s):  
A. Harris ◽  
L. Coleman
Keyword(s):  
Cystic Fibrosis ◽  
Tissue Culture ◽  
Epithelial Cells ◽  
Culture System ◽  
Cultured Cells ◽  
Cell Types ◽  
Cultured Epithelial Cells ◽  
Different Cell Types ◽  
Foetal Pancreas

The establishment of a tissue-culture system for epithelial cells derived from human foetal pancreas has recently been reported. Further analyses have now been made on these cells in vitro, together with parallel investigation of the distribution of different cell types within the intact foetal pancreas. Results support the view that the cultured cells are ductal in origin and nature. Pancreatic epithelial cell cultures have also been established from foetuses with cystic fibrosis.

scholarly journals Glass beads load macromolecules into living cells

1987 ◽  
Vol 88 (5) ◽  
pp. 669-678
Author(s):  
P.L. McNeil ◽  
E. Warder
Keyword(s):  
Culture Medium ◽  
Small Volume ◽  
Cultured Cells ◽  
Cell Monolayer ◽  
Cell Loss ◽  
Living Cells ◽  
Glass Beads ◽  
Cell Type ◽  
Large Numbers ◽  
Micron Diameter

We describe and characterize an exceptionally rapid and simple new technique for loading large numbers of cultured cells with large macromolecules. The culture medium of the cell monolayer is replaced by a small volume of the macromolecule to be loaded. Glass beads (75–500 micron diameter) are then sprinkled onto the cells, the cells are washed free of beads and exogenous macromolecules, and ‘bead-loading’ is completed. The conditions for bead-loading can readily be modified to accommodate cell type and loading objectives: for example, the amount of loading per cell increases if bead size is increased or if beads are agitated after sprinkling onto the monolayer, but at the expense of increased cell loss. As many as 97% of a population of bovine aortic endothelial (BAE) cells were loaded with a 10,000 Mr dextran; and 79% with a 150,000 Mr dextran using bead-loading. Various cell lines have been loaded using glass beads. Moreover, bead-loading has the advantage of producing loaded cells that remain adherent and well-spread, thus minimizing recovery time and permitting immediate microscopic examination.

scholarly journals Cold-induced microtubule disruption and relocalization of membrane proteins in kidney epithelial cells.

1998 ◽  
Vol 9 (2) ◽  
pp. 155-166
Author(s):  
S Breton ◽  
D Brown
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cultured Cells ◽  
Cold Treatment ◽  
Partial Recovery ◽  
Solution Ph ◽  
Microtubule Network ◽  
Cold Preservation ◽  
Aquaporin 2 ◽  
Microtubule Disruption

Cold preservation of kidneys is commonly used in human transplantation and in vitro studies. However, although disruption of the cytoskeleton by cold has been demonstrated in cultured cells, the effect of cold treatment on intact kidney is poorly understood. In this study, specific antibodies were used to examine the effect of hypothermia on the cytoskeletal network and the trafficking of some membrane proteins in the urinary tubule. Rat kidneys were cut into thin slices (approximately 0.5 mm) that were divided into several groups: (1) some were immediately fixed in paraformaldehyde, sodium periodate, and lysine (PLP); (2) some were stored at 4 degrees C for 15 min or 4 h before being fixed in cold PLP; or (3) after 4 h cold treatment, some slices were rewarmed to 37 degrees C for 15, 30, and 60 min in a physiologic solution, pH 7.4, and were then fixed in warm PLP. Immunofluorescence staining revealed an almost complete disruption of the microtubule network in proximal tubules after 15 min cold treatment, whereas microtubules in other segments were affected after 4 h. A partial recovery of the microtubule network was observed after 60 min rewarming. In contrast, actin filaments seemed to be resistant to cold treatment. gp330, aquaporin-2, H+ ATPase, and the AE1 anion exchanger were all relocated into numerous vesicles that were distributed throughout the cytoplasm after hypothermia followed by rewarming, whereas Na-K-ATPase retained its basolateral localization. The vasopressin-stimulated insertion of aquaporin-2 water channels into the apical membrane was inhibited during the initial rewarming period after cold exposure. Thus, cold preservation of tissues might impair, at least transiently, the polarized membrane expression and function of some transport proteins in renal epithelial cells.

scholarly journals A Miniaturized Pump Out Method for Characterizing Molecule Interaction with ABC Transporters

2019 ◽  
Vol 20 (22) ◽  
pp. 5529 ◽  
Author(s):  
Emmanuel Sevin ◽  
Lucie Dehouck ◽  
Romain Versele ◽  
Maxime Culot ◽  
Fabien Gosselet
Keyword(s):  
Abc Transporters ◽  
Detection Method ◽  
Cell Model ◽  
Early Stage ◽  
Cell Monolayer ◽  
Efflux Pumps ◽  
Apparent Permeability ◽  
Traditional Use ◽  
Molecule Design ◽  
Molecule Interaction

Characterizing interaction of newly synthetized molecules with efflux pumps remains essential to improve their efficacy and safety. Caco-2 cell line cultivated on inserts is widely used for measuring apparent permeability of drugs across biological barriers, and for estimating their interaction with efflux pumps such as P-gp, BCRP and MRPs. However, this method remains time consuming and expensive. In addition, detection method is required for measuring molecule passage across cell monolayer and false results can be generated if drugs concentrations used are too high as demonstrated with quinidine. For this reason, we developed a new protocol based on the use of Caco-2 cell directly seeded on 96- or 384-well plates and the use of fluorescent substrates for efflux pumps. We clearly observed that the new method reduces costs for molecule screening and leads to higher throughput compared to traditional use of Caco-2 cell model. This accelerated model could provide quick feedback regarding the molecule design during the early stage of drug discovery and therefore reduce the number of compounds to be further evaluated using the traditional Caco-2 insert method.

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