This work is supported by 17K00860 (to HH) and 19K20152 (to NI). WH would like to acknowledge the Otsuka Toshimi Scholarship Foundation for their financial support from 2018-2021.

<ol><li>Ussing, H. H., Zerahn, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Active+transport+of+sodium+as+the+source+of+electric+current+in+the+short-circuited+isolated+frog+skin%5BTitle%5D)+AND+%22Acta+Physiologica+Scandinavica%22%5BJournal%5D)">Active transport of sodium as the source of electric current in the short-circuited isolated frog skin.</a> Acta Physiologica Scandinavica. 23, 110-127 (1951).</li>
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<li>Östh, K. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22The+horizontal+Ussing+chamber+method+in+studies+of+nasal+drug+delivery+-+Method+Delopment+and+Applications+Using+Different+Formulations%22">The horizontal Ussing chamber method in studies of nasal drug delivery - Method Delopment and Applications Using Different Formulations</a>. , Uppsala University. Dissertation thesis (2002).</li>
<li>Guo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Study+of+penetration+mechanism+of+labrasol+on+rabbit+cornea+by+Ussing+chamber%2C+RT-PCR+assay%2C+Western+blot+and+immunohistochemistry%5BTitle%5D)+AND+%22Asian+Journal+of+Pharmaceutical+Sciences%22%5BJournal%5D)">Study of penetration mechanism of labrasol on rabbit cornea by Ussing chamber, RT-PCR assay, Western blot and immunohistochemistry.</a> Asian Journal of Pharmaceutical Sciences. 14, 329-339 (2019).</li>
<li>Okabe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+Benzalkonium+Chloride+on+transscleral+drug+delivery%5BTitle%5D)+AND+%22Investigative+Opthalmology+%26+Visual+Science%22%5BJournal%5D)">Effect of Benzalkonium Chloride on transscleral drug delivery.</a> Investigative Opthalmology & Visual Science. 46, 703(2005).</li>
<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+expansion+of+epithelial+organoids+from+human+colon%2C+adenoma%2C+adenocarcinoma%2C+and+Barrett%27s+epithelium%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141, 1762-1772 (2011).</li>
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<li>Tamura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Megaintestine+in+claudin-15-deficient+mice%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Megaintestine in claudin-15-deficient mice.</a> Gastroenterology. 134, 523-534 (2008).</li>
<li>Nakayama, M., Ishizuka, N., Hempstock, W., Ikari, A., Hayashi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Na%2B-coupled+nutrient+cotransport+induced+luminal+negative+potential+and+Claudin-15+play+an+important+role+in+paracellular+Na%2B+recycling+in+mouse+small+intestine%5BTitle%5D)+AND+%22International+Journal+of+Molecular+Sciences%22%5BJournal%5D)">Na+-coupled nutrient cotransport induced luminal negative potential and Claudin-15 play an important role in paracellular Na+ recycling in mouse small intestine.</a> International Journal of Molecular Sciences. 21, 376(2020).</li>
<li>Tamura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Loss+of+claudin-15%2C+but+not+claudin-2%2C+causes+Na%2B+deficiency+and+glucose+malabsorption+in+mouse+small+intestine%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Loss of claudin-15, but not claudin-2, causes Na+ deficiency and glucose malabsorption in mouse small intestine.</a> Gastroenterology. 140, 913-923 (2011).</li>
<li>Clarke, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+guide+to+Ussing+chamber+studies+of+mouse+intestine%5BTitle%5D)+AND+%22American+Journal+of+Physiology+-+Gastrointestinal+and+Liver+Physiology%22%5BJournal%5D)">A guide to Ussing chamber studies of mouse intestine.</a> American Journal of Physiology - Gastrointestinal and Liver Physiology. 296, (2009).</li>
<li>Dobson, J. G., Kidder, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Edge+damage+effect+in+in+vitro+frog+skin+preparations%5BTitle%5D)+AND+%22American+Journal+of+Physiology%22%5BJournal%5D)">Edge damage effect in in vitro frog skin preparations.</a> American Journal of Physiology. 214, 719-724 (1968).</li>
<li>Corman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Streaming+potentials+and+diffusion+potentials+across+rabbit+proximal+convoluted+tubule%5BTitle%5D)+AND+%22Pfl%C3%BCgers+Archiv%3A+European+Journal+of+Physiology%22%5BJournal%5D)">Streaming potentials and diffusion potentials across rabbit proximal convoluted tubule.</a> Pflügers Archiv: European Journal of Physiology. 403, 156-163 (1985).</li>
<li>Shen, L., Weber, C. R., Raleigh, D. R., Yu, D., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tight+junction+pore+and+leak+pathways%3A+A+dynamic+duo%5BTitle%5D)+AND+%22Annual+Review+of+Physiology%22%5BJournal%5D)">Tight junction pore and leak pathways: A dynamic duo.</a> Annual Review of Physiology. 73, 283-309 (2011).</li>
<li>Frizzell, R. A., Schultz, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ionic+conductances+of+extracellular+shunt+pathway+in+rabbit+ileum%5BTitle%5D)+AND+%22Journal+of+General+Physiology%22%5BJournal%5D)">Ionic conductances of extracellular shunt pathway in rabbit ileum.</a> Journal of General Physiology. 59, 318-346 (1972).</li>
<li>Otani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Claudins+and+JAM-A+coordinately+regulate+tight+junction+formation+and+epithelial+polarity%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Claudins and JAM-A coordinately regulate tight junction formation and epithelial polarity.</a> Journal of Cell Biology. 218, 3372-3396 (2019).</li>
</ol>

The authors have no potential conflicts of interest to disclose.

In this experiment, Ussing chambers were used to measure the baseline electrical parameters and the dilution potential of NaCl in the small intestine of Cldn15-/- and WT mice. It is very important when doing Ussing chamber experiments to verify that the membrane preparation used in the experiments is viable. This is usually done by adding glucose or the adenylate cyclase activator forskolin and seeing whether there is an appropriate rise in Isc (100-300 &#181;A/cm2 in mi...

The Ussing chamber method was first developed by the Danish scientist Hans Ussing. Ussing first used it to measure the short-circuit current of sodium transport across frog skin after it was observed that NaCl could be transported across the skin against a steep concentration gradient1. His system consisted of the frog skin mounted between two chambers with access to either side of the skin. Each chamber contained Ringer&#39;s solution which was circulated and aerated. Two narrow agar ringer bridges situated near the skin and connected to saturated KCl-calomel electrodes measured the potential difference as read by a potentiator. A second pair of agar ringer bridges were situated at the opposite end of each chamber connected to beakers with saturated KCl saturated with AgCl to apply an electromotive force provided by a battery. A potential divider was used to adjust the voltage so that the potential difference across the skin remained zero, thus creating short-circuit conditions. A microampere meter was also connected to read the current passing through the skin (see the figure in ref.1 for original chamber design).
Over the past 70 years, this technique has been applied to many different tissues, particularly intestinal tissue, to study nutrient and ion transport. For example, the mechanism of cholera-induced diarrhea was studied by mounting rabbit ileum in these chambers, and it was found that cholera toxin-induced diarrhea is mediated by cAMP2. In addition, these chambers were also used to study the mechanism underlying glucose transport via Na+-Glucose cotransporter 1 (SGLT1)3. Our lab focuses on transcellular and paracellular transport in intestinal epithelial cells. Using the Ussing chamber method, peptide transport was assessed in Claudin 15 knockout mice, which have impaired paracellular sodium transport, using Ussing chambers to measure the absorption of the nonhydrolyzable dipeptide glycylsarcosine. It was found that luminal Na+ homeostasis is important for proton-coupled peptide transport4. In addition, these chambers were also used to investigate anion secretion in the murine cecum in response to submucosal activation of proteinase activated receptor 1 by the serine protease trypsin5.
Ussing chambers have also recently been used to assess the paracellular pathways in epithelial tissue. Paracellular pathways are regulated by tight junctions, which are complexes of proteins that form at the point where two or more cells meet6. The barrier function and ion selectivity (whether anions or cations are selectively able to pass through the tight junction) is determined by the presence of claudin family proteins; some of which act as barriers (claudin 3 and 7), anion pores (claudin 10a), or cation pores (claudin 2, 10b, and 15)7. Other methods have been used to assess the paracellular pathway, such as oral gavage of FITC accompanied by blood plasma FITC concentration8, or EDTA-Cr9; however, these techniques are of lower resolution and cannot assess ion selectivity or a specific section of the sections of the intestinal tract. Ussing chambers, however, can be used to assess the dilution potential of target ions, and, therefore, determine the ion selectivity of the tight junctions. For example, with NaCl, the selectivity of the tight junctions for Na+ and Cl- can be calculated by diluting one side of the membrane (usually the mucosal side) and measuring the change in transepithelial potential difference. The relative permeabilities of Na+ and Cl- can be estimated by the Goldman-Hodgkin-Katz equation10 and the selectivity of the tight junction can be estimated using the Kimizuka-Koketsu equation11. These chambers, therefore, have the advantage of measuring the electrophysiological parameters of tissue and as a result provide more information about the passage of ions through the tight junctions than other lower resolution methods.
The Ussing chamber method is not only limited to the intestinal tract, although it is widely used in studies concerning the intestine, it has many other applications as well. For example, these chambers have been used to study Cystic Fibrosis, and specifically the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR)12. Cystic Fibrosis is caused by a mutation in CFTR13, which results in impaired chloride secretion and fluid transport by respiratory epithelial cells, and a resulting thicker, drier mucous layer14. Study of airway epithelial CFTR has been performed with these chambers to not only understand the disease, but to discover ways to treat the disease. For example, in patients with rare mutations causing Cystic Fibrosis, analysis of patient respiratory epithelial cells has been used to test therapies such as Orkambi and an amplifier co-therapy15.
Ussing chambers have also been used to study routes of drug delivery, such as with human biopsy tissue to study drug uptake and pharmacokinetics16. Intestinal uptake is not the only route of drug delivery. These chambers have also been used to study nasal drug delivery systems17. Drug delivery studies with Ussing chambers have also been performed for the eye. In the rabbit cornea, permeability and uptake studies were conducted with Labrasol, a drug that is designed to increase the absorption of drugs across tissues18. Another study examined the effect of benzylalkonium chloride on transscleral drug delivery in the rabbit sclera19.
The Ussing chamber method is useful because native tissue can be used. As such, it is preferable over in vitro models such as Caco-2 cell lines. However, the technique requires skill and time to prepare specimens, so it is not suitable for high throughput applications. The electrophysiological properties of cell monolayers can be studied using cell culture inserts in these chambers. Recent discoveries have allowed for the culture of organoids which are mini-organs grown in culture from the harvest of epithelial or endothelial stem cells20. Organoid culture can be manipulated to be grown in a monolayer, thereby making it possible to mount organoids in an Ussing chamber21. Organoids of various epithelial and endothelial tissues can be studied, lowering the number of animals required, as organoid culture can be maintained long term. This will also increase the throughput since time consuming and laborious tissue dissection and preparation steps will not be needed. In the future, Ussing chamber studies will continue to be very useful for studying tissue transport and they will be especially important in the field of personalized medicine.
The following protocol demonstrates the application of the Ussing chamber method to assess the permselectivity and barrier function of the tight junctions in the small intestine of Claudin 15 knockout (Cldn15-/-) mice and wild type (WT) controls by measuring the dilution potential of NaCl. Tight junctions (TJ) are formed at the point where two or more cells meet in epithelial and endothelial tissue. Bicellular tight junctions (bTJ), particularly the claudin family proteins found within the bTJ, are thought to determine the barrier function and permselectivity of TJ7. Cldn15-/- mice have a mega small intestine22 and reduced nutrient uptake capability due to the loss of intestinal Na+ recycling that occurs via claudin 154,23,24. Cldn15-/-&#160;mice have impaired Na+ homeostasis, which makes them an interesting model for studying the permselectivity of the TJ. The following protocol assesses the permeability of the TJ to NaCl by measuring the dilution potential of NaCl (PNa/PCl) in the middle small intestine. Briefly, the change in membrane potential difference that occurs by diluting one side of the membrane (M side or S side, both are measured in the below protocol) can be used to calculate the permeability of Na+ (PNa) and Cl- (PCl), and the dilution potential (PNa/PCl) will show whether the tight junction has a cationic or anionic selectivity.
The experiments in this protocol were conducted using a customized Ussing chamber (Figure 1A), which consists of two halves, between which the intestinal preparation is mounted vertically, voltage clamp amplifier, electrical recorder, electrodes, salt bridges, Ringer&#39;s solution, HEPES buffer (150 mM NaCl), diluted HEPES buffer (75 mM NaCl), intestinal preparation (for details about equipment see the Table of Materials).

<table><tbody><tr><td>#3 polyethyl tubing</td><td>Hibiki</td><td></td><td>outer diameter 1.0 mm; inner diameter 0.5 mm</td></tr><tr><td>#7 polyethyl tubing</td><td>Hibiki</td><td></td><td>outer diameter 2.3 mm; inner diameter 1.3 mm</td></tr><tr><td>10 mL locking syringe</td><td>Terumo</td><td>SS-10LZ</td><td>Locking syringes are necessary to prevent the needle from dislodging during filling</td></tr><tr><td>19 g needle</td><td>Terumo</td><td>NN-1938R</td><td>Please use caution when working with needles and dispose of in sharps container</td></tr><tr><td>23 g needle</td><td>Terumo</td><td>NN-2332R</td><td>Please use caution when working with needles and dispose of in sharps container</td></tr><tr><td>5 mm punch</td><td>NA</td><td>NA</td><td>Use to punch holes in filter paper and parafilm</td></tr><tr><td>acupuncture needles</td><td>Seirin</td><td>NS</td><td>Used as dissection pins to pin tissue to dissection plate</td></tr><tr><td>Agar</td><td>Fujifilm Wako</td><td>010-15815</td><td></td></tr><tr><td>Alligator clips</td><td>NA</td><td>NA</td><td>Connects the electrode to the amplifier</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fujifilm Wako</td><td>038-00445</td><td></td></tr><tr><td>D(-)-Mannitol</td><td>Fujifilm Wako</td><td>133-00845</td><td>This is used to correct for the osmolality difference in dilution HEPES buffer</td></tr><tr><td>D(+)-Glucose</td><td>Fujifilm Wako</td><td>049-31165</td><td></td></tr><tr><td>Dissection kit</td><td></td><td></td><td>You will need, scissors and curved forceps</td></tr><tr><td>Dissection plates</td><td></td><td></td><td>We used 10 cm cell culture plates and covered with silicon rubber</td></tr><tr><td>DMSO</td><td>Sigma</td><td>472301-500ML</td><td>For making forskolin stock</td></tr><tr><td>Electrical recorder</td><td>TOA Electronics</td><td>PRR-5041</td><td>Other equivalent electrical recorders are available commercially</td></tr><tr><td>Epithelial voltage clamp amplifier</td><td>Nihon Kohden</td><td>CEZ9100</td><td>Other equivalent amplifiers are available commerically</td></tr><tr><td>filter paper, cut into squares</td><td>NA</td><td>NA</td><td>Punched with a 5 mm punch, used to hold intestinal preparation</td></tr><tr><td>fine forceps</td><td>Fast Gene</td><td>FG-B50476</td><td>For blunt dissection of the muscle layer</td></tr><tr><td>Forskolin</td><td>Alomone Labs</td><td>F-500</td><td>Make 10 mM stock in DMSO, final concentration will be 10 &amp;micro;M</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H4034-1KG</td><td></td></tr><tr><td>Indomethacin</td><td>Sigma</td><td>I7338-5G</td><td>Make a 1 mM stock in 21 mM NaHCO3, final concentration is 10 &amp;micro;M</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fujifilm Wako</td><td>164-04295</td><td></td></tr><tr><td>KCl</td><td>Fujifilm Wako</td><td>163-03545</td><td></td></tr><tr><td>KCl/calomel electrode</td><td>Asch Japan Co.</td><td>SCE-100</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Kanto chemical</td><td>32379-00</td><td></td></tr><tr><td>L(+)-Glutamine</td><td>Fujifilm Wako</td><td>074-00522</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fujifilm Wako</td><td>135-00165</td><td></td></tr><tr><td>Mixed Gas (95% O&lt;sub&gt;2&lt;/sub&gt;/5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Shizuoka Oxygen Company</td><td></td><td>Used for bubbling Ringer solution and chambers when using Ringer solution</td></tr><tr><td>NaCl</td><td>Fujifilm Wako</td><td>191-01665</td><td></td></tr><tr><td>NaCl electrode</td><td>NA</td><td>NA</td><td>Handmade electrodes which require concentrated NaCl and Silver wire</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fujifilm Wako</td><td>191-01305</td><td></td></tr><tr><td>O&lt;sub&gt;2&lt;/sub&gt; Gas</td><td>Shizuoka Oxygen Company</td><td></td><td>Used for bubbling chambers when using HEPES buffer</td></tr><tr><td>parafilm</td><td>Bemis</td><td>PM-996</td><td>Used to help seal Ussing chambers</td></tr><tr><td>pH meter</td><td>DKK-TOA Corp</td><td>HM-305</td><td>HEPES buffer needs to be adjusted to pH 7.4 at 37 &amp;deg;C</td></tr><tr><td>pH meter electrode</td><td>DKK-TOA Corp</td><td>GST-5311C</td><td></td></tr><tr><td>silicone rubber</td><td>Shinetsu Chemical</td><td>KE-12</td><td>Used to fill dissection plates</td></tr><tr><td>silver wire</td><td></td><td></td><td>Used for making NaCl electrodes</td></tr><tr><td>Small jars w/ plastic lids</td><td>NA</td><td>NA</td><td>Use for NaCl electrodes</td></tr><tr><td>stereomicroscope</td><td>Zeiss</td><td>Stemi 305</td><td>A stereomicroscope allows you to see depth, so you can dissect the tissue more easily</td></tr><tr><td>Tris (Trizma base)</td><td>Sigma</td><td>T1503-1KG</td><td>Make a 1M solution to adjust pH of HEPES buffers</td></tr><tr><td>Ussing chambers</td><td>Sanki Kagaku Kougei</td><td></td><td>These chambers are custom made continuous perfusion Ussing chambers with a window diameter of 5 mm</td></tr><tr><td>Water pump and heating system</td><td>Tokyo Rikakikai Co. Ltd.</td><td>NTT-110</td><td></td></tr></tbody></table>

functional assessment of intestinal tight junction barrier and ion permeability in native tissue by ussing chamber technique

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog skin. Since then, this technique has been applied to many different tissues to study the physiological parameters of transport across membranes. The Ussing chamber method is preferable to other methods because native tissue can be used, making it more applicable to what is happening in vivo. However, because native tissue is used, throughput is low, time is limited, and tissue preparation requires skill and training. These chambers have been used to study specific transporter proteins in various tissues, understand disease pathophysiology such as in Cystic Fibrosis, study drug transport and uptake, and especially contributed to the understanding of nutrient transport in the intestine. Given the whole epithelial transport process of a tissue, not only transepithelial pathways, but also paracellular pathways are important. Tight junctions are a key determinant of tissue specific paracellular permeability across the intestine. In this article, the Ussing chamber technique will be used to assess paracellular permselectivity of ions by measuring transepithelial conductance and dilution potentials.

The results shown in this paper are results that were part of larger project that has been completed (see ref.4,23,24).
Transepithelial electrical conductance of the small intestine is decreased in Cldn15-/- mice. 
The baseline transmucosal conductance (under short circuit conditions) of the middle small intesti...

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Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

Intestinal epithelium confers not only nutrient absorption but protection against noxious substances. The apical-most epithelial intercellular junction, i.e., the tight junction, regulates paracellular solute and ion permeability. Here, a protocol for the preparation of mucosal sheets and assessment of the ion selectivity of tight junctions using Ussing chamber technique is described.

The Ussing chamber technique was first invented by the Danish scientist Hans Ussing in 1951 to study the transcellular transport of sodium across frog ...

functional-assessment-intestinal-tight-junction-barrier-ion

Research

JoVE Journal

Biology

5.4K Views.  University of Shizuoka. Dit protocol wordt gebruikt om de transportkwaliteiten van tight junctions te bestuderen. Met behulp van verdunningspotentialen kunt u de permselectiviteit meten en inzicht krijgen in tight junctions in een weefsel. Deze techniek is specifiek en maakt gebruik van inheemse weefsels voor functionele studies van epithelia.Bovendien meet deze techniek de real-time fysiologische eigenschappen van een weefsel, wat erg handig is. De grootste uitdaging is de weefselvoorbereiding. Het oefenen v...

Video: Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

Bioengineering

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Medicine

An Intravital Microscopy-Based Approach to Assess Intestinal Permeability and Epithelial Cell Shedding Performance

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. Therefore, the intestinal mucosa of anesthetized mice is topically stained with unspecific staining (acriflavine) and a fluorescent tracer (rhodamine-B dextran), mounted on a saline solution-rinsed plate and directly imaged using a confocal microscope. This technique can complement other non-invasive techniques to identify leakage of intestinal permeability, such as transmucosal passage of orally administered tracers. Besides this, the approach presented here allows the direct observation of cell shedding events at real-time. In combination with appropriate fluorescent reporter mice, this approach is suitable for shedding light into cellular and molecular mechanisms controlling intestinal epithelial cell extrusion, as well as to other biological processes. In the last decades, interesting studies using intravital microscopy have contributed to knowledge on endothelial permeability, immune cell gut homing, immune-epithelial communication and invasion of luminal components, among others. Together, the protocol presented here would not only help increase the understanding of mechanisms controlling epithelial cell extrusion, but could also be the basis for the developmental of other approaches to be used as instruments to visualize other highly dynamic cellular process, even in other tissues. Among technical limitations, optical properties of the specific tissue, as well as the selected imaging technology and microscope configuration, would in turn, determine the imaging working distance, and resolution of acquired images.

Taking advantage of intravital microscopy, the method presented here enables real-time visualization of intestinal epithelial cell shedding in living animals. Therefore, topically stained intestinal mucosa (acriflavine and rhodamineB-dextran) of anesthetized mice is imaged up to single-cell resolution using confocal microscopy.

Intravital microscopy of the gut using confocal imaging allows real time observation of epithelial cell shedding and barrier leakage in living animals. ...

Immunology and Infection

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). 
Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers.
We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. 
The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Dysregulated intestinal epithelial barrier function and immune responses are hallmarks of inflammatory bowel disease that remain poorly investigated due to a lack of physiological models. Here, we describe a mouse intestinal loop model that employs a well-vascularized and exteriorized bowel segment to study mucosal permeability and leukocyte recruitment in vivo.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water ...

Investigating Intestinal Barrier Breakdown in Living Organoids

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was not possible in classical cell culture monolayers. Moreover, monolayer cell cultures are good in vitro model systems but do not represent the complex cellular differentiation processes and functions that rely on 3D structure. This has so far only been possible in animal experiments, which are laborious, time consuming, and hard to assess by optical techniques. Here we describe an assay to quantitatively determine the barrier integrity over time in living small intestinal mouse organoids. To validate our model, we applied interferon gamma (IFN-&#947;) as a positive control for barrier destruction and organoids derived from IFN-&#947; receptor 2 knock out mice as a negative control. The assay allowed us to determine the impact of IFN-&#947; on the intestinal barrier integrity and the IFN-&#947; induced degradation of the tight junction proteins claudin-2, -7, and -15. This assay could also be used to investigate the impact of chemical compounds, proteins, toxins, bacteria, or patient-derived probes on the intestinal barrier integrity.

Here we describe a technique to quantify the barrier integrity of small intestinal organoids. The fact that the method is based on living organoids enables the sequential investigation of different barrier integrity modulating substances or combinations thereof in a time-resolved manner.

Organoids and three-dimensional (3D) cell cultures allow the investigation of complex biological mechanisms and regulations in vitro, which previously was ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Evaluation and Quantification of Micro Epithelial Gaps in the Colonic Mucosa using Immunofluorescence Staining

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier impairment has been associated with the development of various pathologies such as inflammatory bowel diseases (IBD). In the inflamed mucosa, superficial erosions or micro-erosions that corrupt epithelial monolayers correspond to sites of high permeability. Several mechanisms have been implicated in the formation of micro-erosions including cell shedding and apoptosis. These micro-erosions often represent microscopic epithelial gaps randomly distributed in the colon. Visualization and quantification of those epithelial gaps has emerged as an important tool to investigate intestinal epithelial barrier function. Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa. Additional use of cell death markers revealed that HPR encompass apoptotic foci where epithelial extrusion/shedding occurs. The protocol described here provides a simple but effective approach to visualize and quantify micro-erosions in the intestine, which is a very useful tool in disease models, in which the intestinal epithelial barrier is compromised.

Here, we describe a new method to visualize the specific location of where transcellular and paracellular permeability is enhanced in the inflamed colonic mucosa. In this assay, we apply a 10 kDa fluorescent dye conjugated to a lysine fixable dextran to visualize high permeability regions (HPR) in the colonic mucosa.

Epithelial cells lining the intestinal mucosa create a physical barrier that separates the luminal content from the interstitium. Epithelial barrier ...

Developing 2D Monolayer Cultures from Bovine Intestinal Organoids

Advancements in Bovine Organoid Technology Using Small and Large Intestinal Monolayer Interfaces

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models that faithfully mimic in vivo intestinal tissues. This is particularly vital for investigating host-pathogen interactions in bovines, which are significant reservoirs for pathogens that pose serious public health risks. Traditional 3D organoids offer limited access to the intestinal epithelium's apical surface, a hurdle overcome by the advent of 2D monolayer cultures. These cultures, derived from organoid cells, provide an exposed luminal surface for more accessible study. In this research, a detailed protocol is introduced for creating and sustaining 2D monolayer cultures from cells of bovine small and large intestinal organoids. This method includes protocols for assessing membrane integrity through transepithelial electrical resistance and paracellular permeability alongside immunocytochemistry staining techniques. These protocols lay the groundwork for establishing and characterizing a 2D bovine monolayer culture system, pushing the boundaries of these method applications in biomedical and translational research of public health importance. Employing this innovative approach enables the development of physiologically pertinent in vitro models for exploring both normal and diseased states of cattle intestinal physiology. The implications for biomedical and agricultural advancements are profound, paving the way for more effective treatments for intestinal ailments in cattle, thereby enhancing both animal welfare and food safety.

Author Spotlight: Development and Characterization of 2D Intestinal Monolayer Models from Bovine Organoids for Pathogen Interaction Studies

This study presents a protocol for generating bovine intestinal 2D monolayers from organoids, offering improved access for studying host-pathogen interactions. It includes methods for assessing membrane integrity and functionality, advancing in vitro models that mimic cattle's gastrointestinal physiology. This approach promises significant biomedical and agricultural benefits, including enhanced treatment strategies.

Advancing knowledge of gastrointestinal physiology and its diseases critically depends on the development of precise, species-specific in vitro models ...

Functional Assessment of Intestinal Tight Junction Barrier and Ion Permeability in Native Tissue by Ussing Chamber Technique

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