We thank Mr. Christopher Manzella, MD/PhD student in the RKG laboratory, for helping to edit and proofread our manuscript.

1. Study of Intestinal Transporters in a 3D Culture System of Caco-2: Growing 3D Caco-2 Cell Culture on a Gelatinous Protein Mixture
<ol>
	<li>Thaw growth factor reduced gelatinous protein mixture on ice for 8 h (or O/N) at 4 &#176;C. Once thawed, make 1 mL or 500 &#181;L aliquots for use or store at -20 &#176;C for later use.</li>
	<li>On the day of culture, precool the culture plates (for RNA and protein extraction) or chambered slides (for immunostaining) on ice. Add 30 &#181;L of gelatinous protein mixture to each well of an eight-well chambered-glass slide (or 120 &#181;L per well of a 6-well plate) and spread evenly. Take care not to generate air bubbles during the process.</li>
	<li>Place the slides/plates inside a cell culture incubator at 37 &#176;C for 15 - 30 min and allow the gelatinous protein mixture to solidify.</li>
	<li>While the gelatinous protein mixture is solidifying, trypsinize a confluent flask of Caco-2 cells.</li>
	<li>Count the number of cells and spin them at 500 x g in a tabletop centrifuge for 5 min. Re-suspend the cell pellet in 3D Caco-2 Medium as needed.</li>
	<li>Seed the glass chamber slides at 4,000 cells per well (for plates, 20,000 per well). Allow the cells to grow in a 5% CO2 humidified incubator at 37 &#176;C. Change medium every 3 - 4 d.</li>
</ol>
2. Immunofluorescence Staining for Epithelial Transporters in 3D Caco-2 Cells: Day 1
<ol>
	<li>Aspirate the medium and fix the cells with 400 &#181;L of 2% paraformaldehyde (PFA) (in PBS with the pH adjusted to 8.5 with NaOH) for 30 min at RT. 
	NOTE: PFA solution should not be stored for more than 1 week at 4 &#176;C, and freshly made solution can be used. CAUTION: PFA is highly toxic and a potential carcinogen. Always handle it with caution in a chemical hood and using personal protective equipment.</li>
	<li>Rinse the wells twice with 1x PBS and store the slides at 4 &#176;C in PBS (400 &#181;L/well). Once fixed, store them at 4 &#176;C for up to one week.</li>
	<li>Warm the chamber slides to RT (this will harden the gelatinous protein mixture bed and make it easy to aspirate during the washing steps).</li>
	<li>Permeabilize the cells with 0.5% Triton in PBS for no longer than 15 min.</li>
	<li>Prepare PBS-glycine buffer (130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, and 100 mM Glycine). Rinse 3x with 1x PBS-glycine for 10 min each at RT.</li>
	<li>Prepare immunofluorescence buffer (IF): 130 mM NaCl, 7 mM Na2HPO4, 3.5 mM NaH2PO4, 100 mM Glycine, 7.7 mM NaN3, 0.1% BSA, 0.2% TritonX-100, and 0.05% Tween-20.</li>
	<li>Wash 3x in IF buffer for 10 min each at RT. Block in 5% Normal Goat Serum (NGS) in IF buffer. Incubate with primary antibody diluted in IF + 1% goat serum, 200 &#181;L/well, for 1 - 2 h at RT. Wash with IF buffer 3x for 10 min each.</li>
	<li>Incubate with secondary antibody (1:200) diluted in IF + 1% NGS, 200 &#181;L/well, for 1 h at RT.</li>
	<li>Wash with IF buffer three times for 10 min each. Keep the slides in the dark from this step on. Lift the plastic chambers from the glass slides by placing the chamber slide in a black holder and sliding a white lifter through the holder until its edge contacts the edge of the wells. Gently pull up the chambers (the holder and lifter should come with the culture slides).</li>
	<li>Mount with slow anti-fade medium and allow it to dry for 10 min at RT.</li>
	<li>Once dried completely, seal the slides with nail polish and image them with confocal microscopy. Store the slides at 4 &#176;C for two weeks or at -20 &#176;C for several months.</li>
</ol>
3. RNA and Protein Extraction from 3D Caco-2 Cells
<ol>
	<li>Treat the cells cultured on gelatinous protein mixture for 12 - 14 d&#160;with a test agent, such as TGF- &#946;1 (10 ng/mL, 1 h).</li>
	<li>After treatment, wash the cells with 1x PBS. Keep the cells on ice for 30 min to liquefy the gelatinous protein mixture.</li>
	<li>Wash the wells with chilled PBS and collect the cells in individual centrifuge tubes. Collect the cells using low-speed centrifugation at 500 x g and 4 &#176;C for 10 min. Extract the RNA using standard procedures and use real time RT-PCR.</li>
	<li>For the protein extraction, lyse the cells using 1x cell lysis buffer supplemented with 1x protease cocktail inhibitor. After adding cell lysis buffer to the pellet, lyse the cells by sonication and remove the cell debris by centrifugation at 7,500 x g and 4 &#176;C for 10 min.</li>
</ol>
4. Measurement of 5-HT Uptake in Mouse Ileum Utilizing an Ussing Chamber: Intestinal Preparation and Seromuscular Stripping
<ol>
	<li>Following euthanasia, dissect the mice and remove the small intestines (after the stomach and before the cecum). Divide the small intestine into three parts. Discard the middle third, use the proximal third as jejunum, and use the distal third as ileum. Avoid pulling the intestine from its mesenteric attachment to prevent damage to the epithelium. Keep the tissue section cold by covering it with ice-cold Krebs-Ringer bicarbonate (KBR) buffer.</li>
	<li>Open the intestine longitudinally using scissors. Incubate freshly excised intestinal sections in ice-cold, gassing KBR containing 1 &#181;M indomethacin for 10 min before the seromuscular stripping.</li>
	<li>Pin the intestinal section (~1 cm in length) mucosal-side down to a plate containing 7% agarose or cured silicone elastomer (0.5 cm thick).</li>
	<li>Strip the seromuscular layers under a dissection stereomicroscope with bottom illumination. Cut the seromuscular layer using a feather scalpel blade. Use fine forceps to obtain the edge of the layer along the longitudinal axis of the intestine. 
	NOTE: During the stripping procedure, the bottom lighting of the stereomicroscope helps to identify and avoid areas with Peyer&#39;s patches.</li>
	<li>After the completion of the seromuscular stripping, mount the mucosa carefully on the pins of an Ussing half-chamber. During the whole process, take care to hold the stripped mucosal tissue by the edges to avoid tearing it.</li>
</ol>
5. Equilibration and Tissue Treatment
<ol>
	<li>Prepare Kreb&#39;s solution: 115 mM NaCl, 25 mM NaHCO3, 2.4 mM K2HPO4, 1.2 mM CaCl2, 1.2 mM MgCl2, and 0.4 mM KH2PO4 at pH 7.4, gassed with 95% O2/5% CO2 at 37 &#176;C. Add 10 mM glucose to the serosal bath to serve as an energy substrate and 10 mM mannitol to the mucosal bath to maintain the osmotic balance. 
	NOTE: Kreb&#39;s solution is supplemented with L-ascorbic acid (100 &#181;M) and pargyline (100 &#181;M, monoamine oxidase inhibitor) to prevent intracellular 5-HT degradation.</li>
	<li>Insert the slider into the chamber to expose both the apical (luminal) side and the basolateral (serosal) side of the tissue to Kreb&#39;s solution.</li>
	<li>After an equilibration period of 10 min, pretreat the ileal tissue with fluoxetine (10 &#181;M) for 30 min on the apical side for Jm-s (mucosal-to-serosal flux).</li>
	<li>Treat the tissue with TGF-&#946;1 (10 ng/mL) for 1 h on basolateral side and then incubate it with 3[H]-5-HT (20 nM) for 30 min on the apical side.</li>
</ol>
6. Quantification of Mucosal to Serosal Flux (Jm-s) and 3[H]-5-HT Accumulation in the Tissue
<ol>
	<li>Collect 0.75 mL aliquots from the serosal reservoir (total of 5 mL) to measure the radioactivity in a liquid scintillation counter and to calculate mucosal-to-serosal (Jm-s) flux rates; fluoxetine-sensitive flux represents SERT-mediated 3[H]-5-HT uptake.</li>
	<li>To avoid hydrostatic pressure differences across the mucosa during a flux period, take samples from the serosal side and replace them with identical volumes of bath medium. 
	NOTE: The flux calculation corrects for the dilution of the end sample (4.25 mL / 5 mL = 0.85).</li>
	<li>For the quantification of 3[H]-5-HT accumulated in the tissue, dismount the mucosa from the slider, wash it once with ice-cold KRB buffer, and place it in glass culture tubes.</li>
	<li>Incubate the mucosa in 0.5 mL of 10% KOH overnight at 37 &#176;C. Measure the radioactivity in 150 &#181;L aliquots of the lysates (in triplicates) using a liquid scintillation counter.</li>
	<li>Use aliquots (3 - 5 &#181;L) of the lysates to measure protein concentration using the Bradford method. 
	NOTE: 10 mL of incubation medium containing radioactive serotonin is used as a standard. 5-HT retained in the tissue is expressed as pmol/mg protein/min.</li>
</ol>

<ol><li>Simon-Assmann, P., Turck, N., Sidhoum-Jenny, M., Gradwohl, G., Kedinger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+models+of+intestinal+epithelial+cell+differentiation%5BTitle%5D)+AND+%22Cell+Biol+Toxicol%22%5BJournal%5D)">In vitro models of intestinal epithelial cell differentiation.</a> Cell Biol Toxicol. 23 (4), 241-256 (2007).</li>
<li>Shen, C., Meng, Q., Zhang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Design+of+3D+printed+insert+for+hanging+culture+of+Caco-2+cells%5BTitle%5D)+AND+%22Biofabrication%22%5BJournal%5D)">Design of 3D printed insert for hanging culture of Caco-2 cells.</a> Biofabrication. 7 (1), 015003(2014).</li>
<li>Jaffe, A. B., Kaji, N., Durgan, J., Hall, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cdc42+controls+spindle+orientation+to+position+the+apical+surface+during+epithelial+morphogenesis%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis.</a> J Cell Biol. 183 (4), 625-633 (2008).</li>
<li>Martel, F., Monteiro, R., Lemos, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Uptake+of+serotonin+at+the+apical+and+basolateral+membranes+of+human+intestinal+epithelial+%28Caco-2%29+cells+occurs+through+the+neuronal+serotonin+transporter+%28SERT%29%5BTitle%5D)+AND+%22J+Pharmacol+Exp+Ther%22%5BJournal%5D)">Uptake of serotonin at the apical and basolateral membranes of human intestinal epithelial (Caco-2) cells occurs through the neuronal serotonin transporter (SERT).</a> J Pharmacol Exp Ther. 306 (1), 355-362 (2003).</li>
<li>Gill, R. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Function%2C+expression%2C+and+characterization+of+the+serotonin+transporter+in+the+native+human+intestine%5BTitle%5D)+AND+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">Function, expression, and characterization of the serotonin transporter in the native human intestine.</a> Am J Physiol Gastrointest Liver Physiol. 294 (1), G254-G262 (2008).</li>
<li>Esmaili, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enteropathogenic+Escherichia+coli+infection+inhibits+intestinal+serotonin+transporter+function+and+expression%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Enteropathogenic Escherichia coli infection inhibits intestinal serotonin transporter function and expression.</a> Gastroenterology. 137 (6), 2074-2083 (2009).</li>
<li>Wade, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Localization+and+function+of+a+5-HT+transporter+in+crypt+epithelia+of+the+gastrointestinal+tract%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Localization and function of a 5-HT transporter in crypt epithelia of the gastrointestinal tract.</a> J Neurosci. 16 (7), 2352-2364 (1996).</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+%22Am+J+Physiol+Gastrointest+Liver+Physiol%22%5BJournal%5D)">A guide to Ussing chamber studies of mouse intestine.</a> Am J Physiol Gastrointest Liver Physiol. 296 (6), G1151-G1166 (2009).</li>
<li>Debnath, J., Muthuswamy, S. K., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphogenesis+and+oncogenesis+of+MCF-10A+mammary+epithelial+acini+grown+in+three-dimensional+basement+membrane+cultures%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.</a> Methods. 30 (3), 256-268 (2003).</li>
<li>Vedeler, A., Pryme, I. F., Hesketh, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insulin+induces+changes+in+the+subcellular+distribution+of+actin+and+5%27-nucleotidase%5BTitle%5D)+AND+%22Mol+Cell+Biochem%22%5BJournal%5D)">Insulin induces changes in the subcellular distribution of actin and 5'-nucleotidase.</a> Mol Cell Biochem. 108 (1), 67-74 (1991).</li>
<li>Botvinkin, A. D., Selimov, M. A., Zgurskaia, G. N., Kulikov, L. G., Aksenova, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%5BDetection+of+rabies+virus+antibodies+in+human+blood+serum+by+an+immunoenzyme+method%5D%5BTitle%5D)+AND+%22Vopr+Virusol%22%5BJournal%5D)">[Detection of rabies virus antibodies in human blood serum by an immunoenzyme method].</a> Vopr Virusol. 31 (3), 333-334 (1986).</li>
<li>Woods, G. L., Mills, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effect+of+dexamethasone+on+detection+of+herpes+simplex+virus+in+clinical+specimens+by+conventional+cell+culture+and+rapid+24-well+plate+centrifugation%5BTitle%5D)+AND+%22J+Clin+Microbiol%22%5BJournal%5D)">Effect of dexamethasone on detection of herpes simplex virus in clinical specimens by conventional cell culture and rapid 24-well plate centrifugation.</a> J Clin Microbiol. 26 (6), 1233-1235 (1988).</li>
<li>Chase, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Acyclovir+for+shingles%5BTitle%5D)+AND+%22Br+Med+J+%28Clin+Res+Ed%22%5BJournal%5D)">Acyclovir for shingles.</a> Br Med J (Clin Res Ed. 295 (6603), 926-927 (1987).</li>
<li>Churchill, P. F., Churchill, S. A., Martin, M. V., Guengerich, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+a+rat+liver+cytochrome+P-450UT-H+cDNA+clone+and+comparison+of+mRNA+levels+with+catalytic+activity%5BTitle%5D)+AND+%22Mol+Pharmacol%22%5BJournal%5D)">Characterization of a rat liver cytochrome P-450UT-H cDNA clone and comparison of mRNA levels with catalytic activity.</a> Mol Pharmacol. 31 (2), 152-158 (1987).</li>
<li>Steiner, M., Tateishi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distribution+and+transport+of+calcium+in+human+platelets%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Distribution and transport of calcium in human platelets.</a> Biochim Biophys Acta. 367 (2), 232-246 (1974).</li>
<li>McClelland, M., Nelson, M., Cantor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purification+of+Mbo+II+methylase+%28GAAGmA%29+from+Moraxella+bovis%3A+site+specific+cleavage+of+DNA+at+nine+and+ten+base+pair+sequences%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Purification of Mbo II methylase (GAAGmA) from Moraxella bovis: site specific cleavage of DNA at nine and ten base pair sequences.</a> Nucleic Acids Res. 13 (20), 7171-7182 (1985).</li>
<li>Chatterjee, I. shita, K, A., Gill, R. avinder K., Alrefai, W. addah A., Dudeja, P. radeep K. <a target="_blank" href="http://www.gastrojournal.org/article/S0016-5085(14)62381-7/abstract">3D Cell Culture of CaCo2: A Better Model to Study the Functionality and Regulation of Intestinal Ion Transporters.</a> Gastroenterology. 146 (5, Supplement 1), S-654(2014).</li>
<li>Fagg, R. H., Hyslop, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+a+variant+strain+of+foot-and-mouth+disease+virus+%28type+O%29+during+passage+in+partly+immunized+cattle%5BTitle%5D)+AND+%22J+Hyg+%28Lond%29%22%5BJournal%5D)">Isolation of a variant strain of foot-and-mouth disease virus (type O) during passage in partly immunized cattle.</a> J Hyg (Lond). 64 (4), 397-404 (1966).</li>
<li>Shilkina, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((%5BThe+aspects+of+the+problem+of+systemic+vasculitis+open+to+discussion%5D%29%5BTitle%5D)+AND+%22Ter+Arkh%22%5BJournal%5D)">[The aspects of the problem of systemic vasculitis open to discussion]).</a> Ter Arkh. 61 (12), 102-106 (1989).</li>
<li>Derichs, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+current+measurement+for+diagnostic+classification+of+patients+with+questionable+cystic+fibrosis%3A+validation+and+reference+data%5BTitle%5D)+AND+%22Thorax%22%5BJournal%5D)">Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data.</a> Thorax. 65 (7), 594-599 (2010).</li>
</ol>

The authors have nothing to disclose.

Fully differentiated Caco-2 cell monolayers have been used extensively as polarized epithelial monolayers to study intestinal transport10,11,12,13,14,15. However, to mimic the physiological organization of intestinal epithelial cells, there has been considerable interest in the development of a Caco-2 culture in 3D. A number ...

A correction was made to the Authors section in: <a href="http://www.jove.com/video/55304">Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D</a>.
One of the authors&#39; names was corrected from:
Arivarasu N. Anabazhagan
to:
Arivarasu N. Anbazhagan

A correction was made to the Authors section in: <a href="http://www.jove.com/video/55304">Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D</a>.

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

The intestinal epithelium is equipped with various transport proteins (channels, ATPases, co-transporters and exchangers) that perform numerous functions ranging from the absorption of nutrients, electrolytes, and drugs to the secretion of fluid and ions in the lumen. Transport proteins generate electrochemical gradients that permit the movement of ions or molecules in a vectorial manner. This is achieved by the asymmetric distributions of the transport systems in the apical and basolateral membranes of polarized epithelial cells. In addition, tight junctions, which tether adjacent epithelial cells, play an important role in this process by serving as a barrier to the intramembrane diffusion of components between the apical and basolateral membrane domains. Appropriate model systems mimicking these characteristic features of the native intestine (i.e.&#160;polarity, differentiation, and tight junction integrity) are critical for the study of the functionality of epithelial transport systems.
With respect to models, the typical cell lines used currently in intestinal epithelial transport research are Caco-2, a model of fully differentiated, absorptive small intestinal epithelial cells; and T84 cells or HT-29 subclones, models of crypt-derived large intestinal epithelial cells1. Conventionally, these cell lines are grown as monolayers in plastic surfaces or in coated transwell inserts. Trans-well cell culture inserts to some extent resemble the in vivo environment by allowing polarized cells to feed basolaterally. However, a limitation in conventional 2D&#160;culture systems is that the cells are forced to adapt to an artificial, flat, and rigid surface. Thus, the physiological complexity of the native epithelia is not accurately reflected in a 2D system. This limitation has been overcome by methods to grow cells in 3D&#160;in a specific microenvironment, such as gelatinous protein mixture, containing a variety of extracellular matrix components2,3. Nevertheless, the in vitro cultures cannot simulate the complexity of the intestinal epithelium, which has multiple cell types and region-specific architecture in the intestine. Thus, the studies using cell culture models require further validation in the native intestine. Using the in vitro 3D cell culture and mouse intestinal mucosa, we describe here simple methods to study the regulation of the intestinal serotonin transporter (SLC6A4, SERT).
The SERT transporter regulates the extracellular availability of an important hormone and neurotransmitter, 5-hydroxytryptamine (5-HT), by rapidly transporting it through a Na+/Cl--dependent process. SERT is a known target of anti-depressants and has recently emerged as a novel therapeutic target of GI disorders, such as diarrhea and intestinal inflammation. Methods to investigate 5-HT uptake in intestinal epithelial cells have been previously described. For example, Caco-2 cells grown on plastic supports or permeable inserts have been shown to exhibit fluoxetine-sensitive 3H-5-HT uptake at both apical and basolateral domains4. The measurement of SERT function in isolated Brush Border Membrane Vesicles (BBMVs) prepared from human organ donor small intestines has also been described by us5. 3H-5-HT uptake in human BBVMs was shown to be fluoxetine-sensitive and Na+/Cl--dependent, and it exhibited saturation kinetics with a Km of 300 nM5. Utilizing similar methods, we also previously measured SERT function as 3H-5-HT uptake in mouse intestinal BBMVs6. However, the preparation of pure plasma membrane vesicles requires a large amount of tissue mucosa. Other methods, such as the radiographic visualization of 3H-5-HT uptake sites, have also been shown previously in guinea pig and rat small intestines7.
The Ussing chamber provides a more physiological system to measure the transport of ions, nutrients, and drugs across various epithelial tissues. The main advantage of the Ussing chamber technique is that it enables the precise measurement of the electrical and transport parameters of intact, polarized intestinal epithelium. Further, to minimize the influence of the intrinsic neuromuscular system, seromuscular stripping of intestinal mucosa can be performed to investigate the regulation of transporters in the epithelium8.
We demonstrate that Caco-2 cells grown in 3D on gelatinous protein form a hollow lumen expressing distinct apical and basolateral markers. These cells show higher expression of SERT than 2D Caco-2 cells. Methods to grow 3D cells and to perform immunostaining or RNA and protein extraction are described. In addition, we describe methods to study SERT function and regulation by TGF-&#946;1, a pleiotropic cytokine, in small intestinal mucosa by utilizing an Ussing chamber technique.

<table><tbody><tr><td>10x PBS</td><td>ATCC</td><td>ATCC&amp;reg; HTB&amp;shy;37&amp;trade;</td><td>Cells</td></tr><tr><td>10x PBS</td><td>Carl Zeiss</td><td></td><td>Microsope for confocal Imaging</td></tr><tr><td>&lt;sup&gt;3&lt;/sup&gt;[H]-5-HT&amp;nbsp;</td><td>LabtekII</td><td>152453</td><td>Culturing cells for microscopy</td></tr><tr><td>5-HT&amp;nbsp;</td><td>Gibco</td><td>70013-032</td><td>Not a hazardous substance</td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;- 5% CO&lt;sub&gt;2&lt;/sub&gt; cylinder</td><td>KPL</td><td>71-00-27</td><td>Not a hazardous substance</td></tr><tr><td>Agar (for Agar plates)</td><td>Fischer</td><td>BP151-100</td><td>Acute toxicity, Ora</td></tr><tr><td>Agar (for electrode)</td><td>Sigma</td><td>G7126-1KG</td><td>Not a hazardous substance</td></tr><tr><td>Alexa Fluor Phalloidin</td><td>Sigma</td><td>C3867-1VL</td><td>Not a hazardous substance</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>P6148-500G</td><td>Harmful if swallowed or if inhaled</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>LifeTechnologies</td><td>A12380</td><td>Not a hazardous substance</td></tr><tr><td>Caco-2 Cells</td><td>Sigma</td><td>A8806-5G</td><td>Not a hazardous substance</td></tr><tr><td>Cell lysis Buffer</td><td>Fischer</td><td>BP337-100</td><td>Not a hazardous substance</td></tr><tr><td>Chabered slides</td><td>Sigma</td><td>S5886-1KG</td><td>Not a hazardous substance</td></tr><tr><td>Collagen Type-1 Solution</td><td>Sigma</td><td>S8045-500G</td><td></td></tr><tr><td>Electrodes</td><td>Sigma</td><td>S-0876</td><td></td></tr><tr><td>EMEM</td><td>Gibco by Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Corning</td><td>356234</td><td>Used as Extracellular matrix</td></tr><tr><td>Fluoxetine</td><td>Qiagen</td><td>74104</td><td></td></tr><tr><td>Gentamicin</td><td>ATCC</td><td>30-2003</td><td>Cell culture Media</td></tr><tr><td>Glycin</td><td>Cell Signaling, Danvers, MA</td><td></td><td></td></tr><tr><td>Hepes&amp;nbsp;</td><td>Roche</td><td>11836145001</td><td></td></tr><tr><td>Indomethacin</td><td>Gibco by Life Technologies</td><td>15070-063</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Gibco by Life Technologies</td><td>15710-064</td><td></td></tr><tr><td>KOH</td><td>Gibco by Life Technologies</td><td>15630-080</td><td></td></tr><tr><td>L-ascorbic acid&amp;nbsp;</td><td>Gibco by Life Technologies</td><td>10082147</td><td></td></tr><tr><td>Liquid scintillation counter&amp;nbsp;</td><td>Gibco by Life Technologies</td><td>70013--032</td><td></td></tr><tr><td>LSM710 Meta</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>EM-CSYS-8</td><td></td></tr><tr><td>Mannitol</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>P2304</td><td></td></tr><tr><td>Matrigel</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>VCC MC8</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>DM MC6</td><td></td></tr><tr><td>Multichannel Voltage/current Clamp</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>P2300</td><td></td></tr><tr><td>NaCl</td><td>Physiologic Instruments, San Diego, CA&amp;nbsp;</td><td>P2023-100</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific, Hampton, NH</td><td>BP1423</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich, St Louis, MO</td><td>S5886</td><td></td></tr><tr><td>Normal Goat Serum (NGS, 10%)</td><td>Fisher Scientific, Hampton, NH</td><td>S233</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich, St Louis, MO</td><td>P288</td><td></td></tr><tr><td>Pen-Strp</td><td>Sigma-Aldrich, St Louis, MO</td><td>C3881</td><td></td></tr><tr><td>Protein assay reagent</td><td>Sigma-Aldrich, St Louis, MO</td><td>10420</td><td></td></tr><tr><td>Proteinase Inhibitor Cocktail</td><td>MEDOX, Chicago, IL&amp;nbsp;</td><td>style G- 12300</td><td></td></tr><tr><td>RNA easy Mini kit</td><td>Sigma-Aldrich, St Louis, MO</td><td>M4125</td><td></td></tr><tr><td>Single Channel Electrode Input Module&amp;nbsp;</td><td>Sigma-Aldrich, St Louis, MO</td><td>A92902</td><td></td></tr><tr><td>Sliders for snapwell Chambers</td><td>Sigma-Aldrich, St Louis, MO</td><td>H9523</td><td></td></tr><tr><td>Sodium Phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich, St Louis, MO</td><td>F132</td><td></td></tr><tr><td>Sodium-Hydroxide (NaOH)</td><td>Sigma-Aldrich, St Louis, MO</td><td>T7039</td><td></td></tr><tr><td>TGF-&amp;beta;1</td><td>Perkin-Elmer,Waltham, MA</td><td>NFT1167250UC</td><td></td></tr><tr><td>Triton X-100</td><td>Packard Instruments, Downers Grove, IL</td><td>B1600</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Sigma-Aldrich, St Louis, MO</td><td>221473</td><td></td></tr><tr><td>Tween-20</td><td>Bio Rad Laboratories, Hercules, CA</td><td>5000006</td><td></td></tr><tr><td>Ussing Chamber (chamber only)</td><td>Sigma-Aldrich, St Louis, MO</td><td>I7378</td><td></td></tr><tr><td>Ussing Chamber Systems</td><td>Sigma-Aldrich, St Louis, MO</td><td>A1296</td><td></td></tr></tbody></table>

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.

Immunostaining in 3D Caco-2 cysts is shown in Figure 1. A representative image of an XY-plane showing well-demarcated lumen in the 3D cyst stained with phalloidin actin is depicted in Figure 1A. The side facing the lumen denotes the apical side. Epithelial cells cultured in a 3D environment result in a robust epithelial phenotype. Different Caco-2 cysts on day 12, when actin and SERT were co-stained, are shown in Figure 1B. In this repres...

Watch this Scientific Journal Video about Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D at JoVE.com

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

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 ...

methods-to-study-epithelial-transport-protein-function-expression

Research

JoVE Journal

Biology

16.3K Views.  University of Illinois at Chicago.  We beschrijven eenvoudige methoden de regulering van intestinale serotonine transporter (SERT) functie en expressie onder toepassing van een in vitro celkweek model van Caco-2-cellen gekweekt in 3D en een ex vivo model van muis darmen bestuderen. Deze methoden zijn geschikt voor de studie van andere epitheliale transporters. 

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

Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200&#160;nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. ...

Bioengineering

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological questions in cell and developmental biology. In addition, organoids together with recent technical advances in gene editing and viral gene delivery promises to advance medical research and development of new drugs for treatment of diseases. Organoids grown in vitro in basement matrix provide powerful model systems for studying the behavior and function of various proteins and are well suited for live-imaging of fluorescent-tagged proteins. However, establishing the expression and localization of the endogenous proteins in ex vivo tissue and in in vitro organoids is important to verify the behavior of the tagged proteins. To this end we have developed and modified tissue isolation, fixation, and immuno-labeling protocols for localization of microtubules, centrosomal, and associated proteins in ex vivo intestinal tissue and in in vitro intestinal organoids. The aim was for the fixative to preserve the 3D architecture of the organoids/tissue while also preserving antibody antigenicity and enabling good penetration and clearance of fixative and antibodies. Exposure to cold depolymerizes all but stable microtubules and this was a key factor when modifying the various protocols. We found that increasing the ethylenediaminetetraacetic acid (EDTA) concentration from 3 mM to 30 mM gave efficient detachment of villi and crypts in the small intestine while 3 mM EDTA was sufficient for colonic crypts. The developed formaldehyde/methanol fixation protocol gave very good structural preservation while also preserving antigenicity for effective labeling of microtubules, actin, and the end-binding (EB) proteins. It also worked for the centrosomal protein ninein although the methanol protocol worked more consistently. We further established that fixation and immuno-labeling of microtubules and associated proteins could be achieved with organoids isolated from or remaining within the basement matrix.

Immuno-fluorescent Labeling of Microtubules and Centrosomal Proteins in Ex Vivo Intestinal Tissue and 3D In Vitro Intestinal Organoids

We present protocols for isolation of intestinal 3D structures from in vivo tissue and in vitro basement matrix embedded organoids, and detail different fixation and staining protocols optimized for immuno-labeling of microtubule, centrosomal, and junctional proteins as well as cell markers including the stem cell protein Lgr5.

The advent of 3D in vitro organoids that mimic the in vivo tissue architecture and morphogenesis has greatly advanced the ability to study key biological ...

Developmental Biology

Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted in sophisticated in vitro models of the intestinal mucosa. Leveraging these emerging model systems will require adaptation of tools and techniques developed for 2D culture systems and animals. Here, we describe a technique for measuring epithelial barrier permeability in human intestinal organoids in real-time. This is accomplished by microinjection of fluorescently-labeled dextran and imaging on an inverted microscope fitted with epifluorescent filters. Real-time measurement of the barrier permeability in intestinal organoids facilitates the generation of high-resolution temporal data in human intestinal epithelial tissue, although this technique can also be applied to fixed timepoint imaging approaches. This protocol is readily adaptable for the measurement of epithelial barrier permeability following exposure to pharmacologic agents, bacterial products or toxins, or live microorganisms. With minor modifications, this protocol can also serve as a general primer on microinjection of intestinal organoids and users may choose to supplement this protocol with additional or alternative downstream applications following microinjection.

This protocol describes the measurement of epithelial barrier permeability in real-time following pharmacologic treatment in human intestinal organoids using fluorescent microscopy and live cell microscopy.

Advances in 3D culture of intestinal tissues obtained through biopsy or generated from pluripotent stem cells via directed differentiation, have resulted ...

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 ...

Medicine

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 ...

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 ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

A Fluorescence-Based Assay of Membrane Potential for High-Throughput Functional Study of Two Endogenous Ion Channels in Two Epithelial Cell Lines

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery campaigns targeting recombinant ion channel proteins overexpressed in cells such as HEK-293 cells. However, there is increasing emphasis on the use of tissue-relevant cell lines for studying the effects of small molecule interventions. The following protocol describes the adaptation of a fluorescence-based membrane potential assay for the study of ion channels endogenously expressed in epithelial cell lines. The membrane potential assay details a high-throughput assay for chloride channel activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) in two commonly studied epithelial cell lines, Caco-2 and Calu-3. In addition, this paper describes a novel application of this system to measure the activity of the Epithelial Sodium Channel (ENaC) in a high-throughput format in the same epithelial cell lines. Together, these fluorescence-based assays provide a robust and flexible platform for studying small molecule modulators, targeting two epithelial channels in a relevant cellular context.

This protocol describes a method for the study of electrogenic membrane proteins by measuring changes in membrane potential. This assay provides a platform for the functional readout of multiple ion channels endogenously expressed in epithelial cell lines.

Fluorescence-based studies are suitable for high-throughput plate reader assays of cells in culture. They have been commonly employed for drug discovery ...

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. Traditionally, research in this area has relied on animal experiments, but there's a growing demand for alternative methods that adhere to the 3R principles-replace, reduce, and refine. Porcine organoids have emerged as a promising tool, offering a more accurate in vitro replication of the in vivo conditions than traditional cell models. One major challenge with intestinal organoids is their inward-facing apical surface and outward-facing basolateral surface. This limitation can be overcome by creating two-dimensional (2D) organoid layers on transwell inserts (from here on referred to as insert(s)), providing access to both surfaces. In this study, we successfully developed two-dimensional cultures of porcine jejunum and colon organoids. The cultivation process involves two key phases: First, the formation of a cellular monolayer, followed by the differentiation of the cells using tailored media. Cellular growth is tracked by measuring transepithelial electrical resistance, which stabilizes by day 8 for colon organoids and day 16 for jejunum organoids. After a 2-day differentiation phase, the epithelium is ready for analysis. To quantify and track active electrogenic transport processes, such as chloride secretion, we employ the Ussing chamber technique. This method allows for real-time measurement and detailed characterization of epithelial transport processes. This innovative in vitro model, combined with established techniques like the Ussing chamber, provides a robust platform for physiologically characterizing the porcine GIT within the 3R framework. It also opens opportunities for investigating pathophysiological mechanisms and developing potential therapeutic strategies.

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. ...

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

Samenvatting

Meer video's verkennen