This study has been supported by the Department of Biochemistry, Sri Venkateswara College, University of Delhi. The authors thank the laboratory staff for their support.

The entire protocol with all steps is depicted as a schematic diagram in Figure 4. The method is adapted from a previous study using rat intestines8. The experiment was performed in compliance with institutional guidelines. The samples used in this study were procured from a commercial vendor.
CAUTION: Wear gloves during this experiment.
1. Preparation of inverted Jejunum sacs
<ol>
	<li>Pre-treat and clean goat jejunum.
	<ol>
		<li>Procure the entrails of a freshly sacrificed healthy goat from a licensed vendor.</li>
		<li>Place the entrails in enough 1x phosphate buffer saline (PBS) so that they are immersed completely for cleaning.</li>
		<li>Separate the small intestine from the large intestine, and make sure that the external connective tissue is removed using scissors.</li>
		<li>Gently flush the small intestine with 1x PBS using a 10 mL syringe, ensuring the removal of undigested food material to obtain a hollow bag of small intestine. Repeat this process at least thrice for effective cleaning. 
		NOTE: Avoid using excessive force or needles while flushing. The use of force can result in the puncturing of the intestinal walls and thus damage the intestinal enterocytes.</li>
		<li>Measure the complete length of the small intestine using a ruler to obtain the three parts: Duodenum (1/4th of the small intestine) and the remainder split evenly into Jejunum and Ileum.</li>
		<li>Proceed with the experiment using the Jejunum part.</li>
	</ol>
	</li>
	<li>Prepare inverted Jejunum.
	<ol>
		<li>Clean the jejunum part by removing all the connective tissue using a blade.</li>
		<li>Cut the jejunum into 12-15 cm long portions, wash with 1x PBS, and place in a Petri dish containing PBS.</li>
		<li>Invert the jejunum portions using a glass rod to allow the villi portion to be exposed on the outside. 
		CAUTION: Avoid using a glass rod with sharp edges. Moreover, the diameter of the glass rod should not be greater than the diameter of the sac. 
		NOTE: At this point, one can visualize the villi under a light microscope (Figure 4).</li>
		<li>Gently flush the inverted jejunum portions with 1x PBS.</li>
		<li>Check for any leaks from the sides of the inverted jejunum.</li>
		<li>Cut the inverted jejunum portions into 5 cm long sacs.</li>
		<li>Tie one end with twine and recheck for visual leaks at the tied end by filling the sacs with PBS.</li>
		<li>Tie the other end to create an empty inverted sac with villi on the external surface.</li>
		<li>Recheck for external leaks by patting the inverted sac on filter paper.</li>
	</ol>
	</li>
	<li>Equilibrate the inverted sacs in 1x PBS.
	<ol>
		<li>Proceed with the experiment on the same day or store the sacs overnight in 1x PBS at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Experimental setup for Histidine transport
<ol>
	<li>Assemble two sets of three 50 mL tubes according to different concentrations of sodium chloride as given in Table 1.</li>
	<li>Dip the prepared intestinal sacs in the specified tubes for 30 min and 60 min for each of the sets.</li>
	<li>After the designated time, empty the solution inside the sac in a separate 1.5 mL microcentrifuge tube and determine the volume.</li>
	<li>Aspirate the liquid inside the sac using a 2 mL syringe. Alternatively, untie one end of the sac and collect the contents in a fresh 1.5 mL tube.</li>
	<li>Assay 0.1 mL of the collected samples for histidine estimation and calculate the concentration using the standard graph.</li>
</ol>
3. Estimation of Histidine using Pauly&#39;s reaction
<ol>
	<li>Prepare a standard curve for histidine.
	<ol>
		<li>Prepare solutions with varying concentrations of histidine as indicated in Table 2 using stock solutions of histidine (5 mM) and water.</li>
		<li>Add sulphanilic acid (0.5 mL) and sodium nitrate (0.5 mL) to each tube.</li>
		<li>Incubate the tubes for 5 min at room temperature, then add 1 mL each of sodium carbonate and ethanol to each tube.</li>
		<li>Incubate the tubes for 20 min at room temperature (~20 &#176;C) and note the absorbance at wavelength 490 nm.</li>
		<li>Plot a standard curve of absorbance versus Histidine concentration. 
		NOTE: This test for histidine is based on the property of histidine to form a diazo compound (Figure 5).</li>
	</ol>
	</li>
</ol>

Sodium-Dependent Histidine Absorption in Goat Jejunal Inverted Sacs

<ol><li>Nelson, D. L., Cox, M. M. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNelson%2C+DL+%22Lehninger+Principles+of+Biochemistry.+7th+Edition%22">Lehninger Principles of Biochemistry. 7th Edition</a>. , W.H. Freeman and Company. (2017).</li>
<li>Stillwell, W. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aStillwell%2C+W+%22Membrane+Transport.+An+Introduction+to+Biological+Membranes%22">Membrane Transport. An Introduction to Biological Membranes</a>. , Elsevier Science. (2013).</li>
<li>Fick, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+liquid+diffusion%5BTitle%5D)+AND+%22The+London%2C+Edinburgh%2C+and+Dublin+Philosophical+Magazine+and+Journal+of+Science%22%5BJournal%5D)">On liquid diffusion.</a> The London, Edinburgh, and Dublin Philosophical Magazine and Journal of Science. 10 (63), 30-39 (1855).</li>
<li>Sherwood, L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aSherwood%2C+L+%22Introduction+to+Human+Physiology%22">Introduction to Human Physiology</a>. , Brooks/Cole Cengage Learning. (2013).</li>
<li>Bröer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+amino+acid+transport+and+metabolic+health%5BTitle%5D)+AND+%22Annu+Rev+Nutr%22%5BJournal%5D)">Intestinal amino acid transport and metabolic health.</a> Annu Rev Nutr. 43, 73-99 (2023).</li>
<li>Avissar, N. E., Ryan, C. K., Ganapathy, V., Sax, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Na%2B-dependent+neutral+amino+acid+transporter+ATB0+is+a+rabbit+epithelial+cell+brush-border+protein%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Na+-dependent neutral amino acid transporter ATB0 is a rabbit epithelial cell brush-border protein.</a> Am J Physiol Cell Physiol. 281 (3), C963-C971 (2001).</li>
<li>Bröer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amino+acid+transport+across+mammalian+intestinal+and+renal+epithelia%5BTitle%5D)+AND+%22Physiol+Rev%22%5BJournal%5D)">Amino acid transport across mammalian intestinal and renal epithelia.</a> Physiol Rev. 88 (1), 249-286 (2008).</li>
<li>Agar, W. T., Hird, F. J. R., Sidhu, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+uptake+of+amino+acids+by+the+intestine%5BTitle%5D)+AND+%22BBA+-+Biochim+Biophys+Acta%22%5BJournal%5D)">The uptake of amino acids by the intestine.</a> BBA - Biochim Biophys Acta. 14 (1), 80-84 (1954).</li>
<li>Pauly, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%C3%9Cber+die+Konstitution+des+Histidins%5BTitle%5D)+AND+%22I.+Mitteilung.+Biological+Chemistry%22%5BJournal%5D)">Über die Konstitution des Histidins.</a> I. Mitteilung. Biological Chemistry. 42 (5-6), 508-518 (1904).</li>
<li>Wilson, T. H., Wiseman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+use+of+sacs+of+everted+small+intestine+for+the+study+of+the+transference+of+substances+from+the+mucosal+to+the+serosal+surface%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">The use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface.</a> J Physiol. 123 (1), 116-125 (1954).</li>
<li>Barthe, L., Woodley, J. F., Kenworthy, S., Houin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+everted+gut+sac+as+a+simple+and+accurate+technique+to+measure+paracellular+transport+across+the+small+intestine%5BTitle%5D)+AND+%22Eur+J+Drug+Metab+Pharmacokinet%22%5BJournal%5D)">An improved everted gut sac as a simple and accurate technique to measure paracellular transport across the small intestine.</a> Eur J Drug Metab Pharmacokinet. 23 (2), 313-323 (1998).</li>
<li>Alam, M. A., Al-Jenoobi, F. I., Al-Mohizea, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Everted+gut+sac+model+as+a+tool+in+pharmaceutical+research%3A+Limitations+and+applications%5BTitle%5D)+AND+%22J+Pharm+Pharmacol%22%5BJournal%5D)">Everted gut sac model as a tool in pharmaceutical research: Limitations and applications.</a> J Pharm Pharmacol. 64 (3), 326-336 (2012).</li>
<li>Pento, J. T., Mousissian, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Time-dependent+deterioration+of+active+transport+in+duodenal+segments+of+rat+intestine%5BTitle%5D)+AND+%22J+Pharmacol+Methods%22%5BJournal%5D)">Time-dependent deterioration of active transport in duodenal segments of rat intestine.</a> J Pharmacol Methods. 20 (1), 9-14 (1988).</li>
<li>Williams, L., Sembiante, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Experiential+learning+in+U.S.+undergraduate+teacher+preparation+programs%3A+A+review+of+the+literature%5BTitle%5D)+AND+%22Teach+Teach+Educ%22%5BJournal%5D)">Experiential learning in U.S. undergraduate teacher preparation programs: A review of the literature.</a> Teach Teach Educ. 112, 103630(2022).</li>
</ol>

The authors have no competing financial interests or other conflicts of interest.

Membrane transport is one of the most fundamental concepts taught to undergraduate students of all major biological science disciplines, basic or applied. Traditionally, movement across membranes has been visualized using metabolites labeled with radioactive isotopes. However, these methods are extremely hazardous and not feasible for teaching or learning. While experiential learning is the best pedagogical technique to understand such complex concepts, it is a challenge that is further compounded by the lack of infrastr...

Biological cells are surrounded by a membrane lipid bilayer that separates the intracellular cytosol from the extracellular content. The membrane serves as a semipermeable barrier that regulates the movement of solutes1. Transport across biological membranes is affected by a solute&#39;s permeability coefficient, which depends on several factors, including the concentration and charge of the solute. In general, solutes move through the membrane using three mechanisms (Figure 1): Passive diffusion, facilitated diffusion, and active transport2. Simple diffusion is the process by which soluble, uncharged, and non-polar solutes pass down their concentration gradient through a semipermeable membrane (Figure 1A). Membrane proteins do not assist in this process since it involves the movement of solutes from a region of higher concentration to a region of lower concentration. The rate of diffusion is based on Fick&#39;s Law3. On the other hand, facilitated diffusion is a protein-dependent transport wherein the membrane allows only selective solutes to pass along a concentration gradient without the expenditure of energy (Figure 1B). This kind of transport is specific and differs from simple diffusion in exhibiting saturation kinetics.
Active transport is a protein-dependent transport of molecules against their concentration gradient, i.e., from the region of lower concentration to a region of higher concentration with the use of ATP or ion gradients (Figure 1C). When the transporter hydrolyses ATP, the transport is termed primary active transport (Figure 1C; left panel). Another form of active transport is secondary active transport (Figure 1C; right panel). In secondary active transport, solutes are moved based on an electrochemical gradient. It takes place when a transporter protein couples the movement of an ion (typically Na+) down its concentration gradient with the movement of another molecule or an ion against its concentration gradient. This kind of solute movement can be a co-transport (Symport) wherein both the solute and the ion move in the same direction or an exchange (Antiport), in which case the solute and the ion move in opposite directions.
The dietary amino acids and monosaccharides from food sources are absorbed in the small intestine. The small intestine can be functionally divided into three segments: Duodenum, Jejunum, and Ileum (Figure 2). Absorption of solute occurs throughout the small intestine, with maximum absorption occurring at the jejunum and at the proximal end of the ileum. The intestinal enterocytes are polarized cells, and the tight junctions connecting two adjacent cells create two distinct membrane sites - the basolateral and the apical membrane site (Figure 2). The absorption of luminal solutes generated by digestion occurs on the apical membrane site4.
Histidine transport in the intestinal enterocytes at the apical membrane is an example of a secondary active symport that is sodium-dependent. At the basolateral end, the histidine entering the enterocyte moves down the concentration gradient into the hepatic portal circulation. The intracellular concentrations of sodium within the enterocyte are maintained at 12 mmoles/L1, which is lower than the extracellular/luminal concentrations, due to the active pumping of sodium out of the cell by Na+ K+ATPase located on the basolateral membrane (Figure 3). At the apical membrane of enterocytes, the B0AT and the sodium neutral amino acid transporter (SNAT) 5 are the major transporters that not only transport histidine but also amino acids such as asparagine and glutamine in a sodium-dependent cotransport5,6. Another transport protein called Large Amino acid Transporter (LAT)1 present at the basolateral membrane of enterocytes transports large neutral amino acids such as leucine, tryptophan, tyrosine, and phenylalanine across the membrane7.
With the objective of teaching the concept of membrane transport integrated with techniques such as spectrophotometry and routine biochemical assays through experiential learning pedagogy, it is imperative to develop methodologies that can not only demonstrate the concept in easy-to-understand terms but also enable participatory learning for undergraduate students. Currently, there are limited resources available to the students for hands-on engagement to learn such concepts of biochemistry. Here, we report a simple protocol for demonstrating Histidine transport across goat intestinal membrane that is easy to reproduce in undergraduate laboratories and can be adapted for evaluating the transport of other metabolites as well. More importantly, the method utilizes inexpensive materials in an undergraduate laboratory, thereby enabling experiential learning in even the simplest of laboratory settings.

<table><tbody><tr><td>1.5 mL Microcentrifuge Tubes</td><td>TARSONS</td><td>500020</td><td></td></tr><tr><td>10 mL Test Tubes</td><td>BOROSIL</td><td>9800U04</td><td></td></tr><tr><td>50 mL Sterile Falcon Tubes</td><td>TARSONS</td><td>546041</td><td></td></tr><tr><td>500 mL Beaker</td><td>BOROSIL</td><td>10044977</td><td></td></tr><tr><td>500 mL Conical Flask</td><td>BOROSIL</td><td>691467</td><td></td></tr><tr><td>D-Glucose</td><td>SRL</td><td>42738</td><td></td></tr><tr><td>Digital Spectrophotometer</td><td>SYSTRONICS</td><td>2710</td><td></td></tr><tr><td>Ethanol</td><td>EMSURE</td><td>1009831000</td><td></td></tr><tr><td>Finpipettes</td><td>THERMOFISHER</td><td>4642090</td><td></td></tr><tr><td>Glass Stirrer Rod</td><td>BOROSIL</td><td>9850107</td><td></td></tr><tr><td>L-Histidine&amp;nbsp;</td><td>SRL</td><td>17849</td><td></td></tr><tr><td>NaCl</td><td>SRL</td><td>41721</td><td></td></tr><tr><td>Nitrile Gloves</td><td>KIMTECH</td><td>112-4847</td><td></td></tr><tr><td>Petri Dish&amp;nbsp;</td><td>TARSONS</td><td>460090</td><td></td></tr><tr><td>Phosphate Buffered Saline (ph 7.4)</td><td>SRL</td><td>95131</td><td></td></tr><tr><td>Pipette Tips</td><td>ABDOS</td><td>P10102</td><td></td></tr><tr><td>Sodium Carbonate</td><td>SRL</td><td>89382</td><td></td></tr><tr><td>Sodium Nitrate&amp;nbsp;</td><td>SRL</td><td>44618</td><td></td></tr><tr><td>Sodium Phosphate Dibasic (anhydrous)</td><td>SRL</td><td>53046</td><td></td></tr><tr><td>Sodium Phosphate Monobasic (anhydrous)</td><td>SRL</td><td>22249</td><td></td></tr><tr><td>Sulphanilic Acid&amp;nbsp;</td><td>SRL</td><td>15354</td><td></td></tr></tbody></table>

demonstration of membrane transport of histidine using goat intestinal inverted sacs: an experiential pedagogical tool for undergraduates

Histidine is an essential amino acid that is also a precursor for metabolites implicated in the immune system, pulmonary ventilation, and vascular circulation. Absorption of dietary histidine relies largely on the sodium-coupled neutral amino acid transport by the Broad neutral amino acid transporter (B0AT) present on the apical membrane of the enterocyte. Here, we demonstrate the absorption of histidine by the intestinal villus enterocytes from the lumen using goat jejunal inverted sacs. The jejunal sacs exposed to varying concentrations of sodium and histidine were assayed to determine the concentration of histidine inside the sacs as a function of time. The results show active histidine absorption. Increasing the concentration of salt resulted in higher absorption of histidine, suggesting a symport of sodium and histidine absorption in goat intestinal inverted sacs. This protocol may be applied to visualize the intestinal mobility of amino acids or other metabolites with appropriate modifications. We propose this experiment as an experiential pedagogical tool that can help undergraduate students comprehend the concept of membrane transport.

The experimental workflow for demonstrating intestinal mobility of histidine through absorption of histidine by intestinal villi into the lumen of the inverted sacs is illustrated in Figure 4, Table 1 and Table 2. Three independent experimental setups were performed, and representative data are presented in Figure 6.
Under the given experimental conditions, Histidine estimation using Pauly&#39;s react...

Author Spotlight: Experiential Tool for Teaching Active Transport Using Ex Vivo Histidine Uptake

Here, we report an inexpensive and reproducible method demonstrating the membrane transport of histidine in a goat intestine. This process occurs by co-transporting histidine and sodium ions enabled by the sodium gradient across the enterocyte membrane. This method exploits experiential learning pedagogy to better understand solute movement across biological membranes.

Demonstration of Membrane Transport of Histidine using Goat Intestinal Inverted Sacs: An Experiential Pedagogical Tool for Undergraduates

Histidine is an essential amino acid that is also a precursor for metabolites implicated in the immune system, pulmonary ventilation, and vascular ...

demonstration-membrane-transport-histidine-using-goat-intestinal

Research

JoVE Journal

Biochemistry

1.6K Views.  University of Delhi. Here, we report an inexpensive and reproducible method demonstrating the membrane transport of histidine in a goat intestine. This process occurs by co-transporting histidine and sodium ions enabled by the sodium gradient across the enterocyte membrane. This method exploits experiential learning pedagogy to better understand solute movement across biological membranes.

Video: Author Spotlight: Experiential Tool for Teaching Active Transport Using Ex Vivo Histidine Uptake

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

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

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

Biology

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral permeability of new therapeutic drug candidates. However, the use of these conventional cell lines has limitations, such as altered expression of tight junctions, partial cell differentiation, and the absence of key nuclear receptors. Despite these shortcomings, the Caco-2 and MDCK models are widely accepted and validated for the prediction of human in vivo oral permeability.
Dogs are a relevant translational model for biomedical research due to their similarities in gastrointestinal anatomy and intestinal microflora with humans. Accordingly, and in support of parallel drug development, the elaboration of an efficient and accurate in vitro tool for predicting in vivo drug permeability characteristics both in dogs and humans is highly desirable. Such a tool could be the canine intestinal organoid system, characterized by three-dimensional (3D), self-assembled epithelial structures derived from adult stem cells.
The (1) Permeable Support Seeding Protocol describes the experimental methods for dissociating and seeding canine organoids in the inserts. Canine organoid isolation, culture, and harvest have been previously described in a separate set of protocols in this special issue. Methods for general upkeep of the canine intestinal organoid monolayer are discussed thoroughly in the (2) Monolayer Maintenance Protocol. Additionally, this protocol describes methods to assess the structural integrity of the monolayer via transepithelial electrical resistance (TEER) measurements and light microscopy. Finally, the (3) Permeability Experimental Protocol&#160;describes the tasks directly preceding an experiment, including in vitro validation of experimental results.
Overall, the canine organoid model, combined with a dual-chamber cell culture technology, overcomes limitations associated with 2D experimental models, thereby improving the reliability of predictions of the apparent oral permeability of therapeutic drug candidates both in the canine and human patient.

Here, we present a protocol describing the culture of canine intestinal organoids in a dual-chamber, permeable support system. Organoid seeding in the permeable supports, the monolayer maintenance, and subsequent drug permeability experiments are described.

The permeable support system is typically used in conjunction with traditional two-dimensional (2D) cell lines as an in vitro tool for evaluating the oral ...

Immunology and Infection

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown in the basolateral-out (BO) conformation, where the apical surface of the epithelial cell faces the inner lumen. In this model, access to the luminal surface of enteroids for treatment and experimentation is challenging, which limits the ability to study host-pathogen interactions. To circumvent this, a neonatal apical-out (AO) model for necrotizing enterocolitis was created. Since intestinal epithelial cell permeability changes are pathognomonic for NEC, this protocol outlines using lucifer yellow (LY) as a marker of paracellular permeability. LY traverses the intestinal epithelial barrier via all three major paracellular pathways: pore, leak, and unrestricted. Using LY in an AO model allows for a broader study of permeability in NEC. Following IRB approval and parental consent, surgical samples of intestinal tissue were collected from human preterm neonates. Intestinal stem cells were harvested via crypt isolation and used to grow enteroids. Enteroids were grown to maturity and then transformed AO or left in BO conformation. These were either not treated (control) or were treated with lipopolysaccharide (LPS) and subjected to hypoxic conditions for the induction of in vitro NEC. LY was used to assess for permeability. Immunofluorescent staining of the apical protein zonula occludens-1 and basolateral protein &#946;-catenin confirmed AO conformation. Both AO and BO enteroids treated with LPS and hypoxia demonstrated significantly increased paracellular permeability compared to controls. Both AO and BO enteroids showed increased uptake of LY into the lumen of the treated enteroids compared to controls. The utilization of LY in an AO enteroid model allows for the investigation of all three major pathways of paracellular permeability. It additionally allows for the investigation of host-pathogen interactions and how this may affect permeability compared to the BO enteroid model.

The present protocol outlines a method that utilizes lucifer yellow in an apical-out enteroid model to determine intestinal permeability. This method can be used to determine paracellular permeability in enteroids that model inflammatory bowel diseases such as necrotizing enterocolitis.

Enteroids are an emerging research tool in the study of inflammatory bowel diseases such as necrotizing enterocolitis (NEC). They are traditionally grown ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

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 ...

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

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.

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 ...

Demonstration of Membrane Transport of Histidine using Goat Intestinal Inverted Sacs: An Experiential Pedagogical Tool for Undergraduates

Summary

Explore More Videos

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

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

Introduction to Solid Supported Membrane Based Electrophysiology

Canine Intestinal Organoids in a Dual-Chamber Permeable Support System

Determining Intestinal Permeability Using Lucifer Yellow in an Apical-Out Enteroid Model

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

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

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

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