This work was supported by grants from the German Research Foundation (DFG) [KFO257, project 4 to M.S. and project 1 to C.B.; FOR2438, project 2 to M.S. and E.N. and project 5 to C.B.; SFB1181 project C05 to C.B.; TRR241, project A06 to N.B.L. and M.S., project A03 to C.B., BR5196/2-1 to N.B.L. and BE3686/2 to C.B.]; the Interdisciplinary Center for Clinical Research (IZKF) of the Clinical Center Erlangen (to M.S., E.N., and M.B.), the W. Lutz Stiftung (to M.S.) and the Forschungsstiftung Medizin of the Clinical Center Erlangen (to M.S.). The present work was performed in (partial) fulfillment of the requirements for obtaining the degree Dr. Med. of Marco Bardenbacher.

All steps were completed in accordance and compliance with all relevant regulatory and institutional animal care guidelines.
1. Plating of Organoids
<ol>
	<li>Isolate organoids as described previously3. The procedure is briefly described below.
	<ol>
		<li>Collect the small intestines from mice.</li>
		<li>Open the small intestines longitudinally and remove villi tips by scraping the inner intestinal tissue with a coverslip.</li>
		<li>Cut the intestinal tissue in small pieces using scissors.</li>
		<li>Wash pieces 5x in cold phosphate-buffered saline (PBS) by pipetting the pieces 10x up and down with a 25 mL pipette.</li>
		<li>Incubate the tissue pieces in cold 2 mM EDTA solution on ice for 30 min on a horizontal shaking platform. Allow the tissue pieces to sediment.</li>
		<li>Replace the EDTA solution with PBS buffer once the tissue pieces settle at the bottom. Discard the supernatant and add 20 mL of PBS.</li>
		<li>Release the intestinal crypts from the tissue by vigorously pipetting 10x up and down with a 10 mL pipette.</li>
		<li>Collect the supernatant in centrifugation tubes and inspect it by phase contrast microscopy. To do this, add a drop of the supernatant to a 96 well cell culture plate. Keep centrifugation tubes on ice.</li>
		<li>Repeat steps 1.1.6&#8211;1.1.8 until the number of intestinal crypts in the collected supernatant decreases.</li>
		<li>Pass the fractions containing the most crypts through a 70 &#181;m cell strainer.</li>
		<li>Centrifuge the crypt suspension at 300 x g, 4 &#176;C for 5 min.</li>
		<li>Discard the supernatant and resuspend the pellet in cold PBS in order to wash the crypts. Then repeat the centrifugation step as described in 1.1.11.</li>
		<li>Resuspend the pellet in a total of 25 &#181;L per well of a 1:1 mixture of cell matrix solution and murine organoid culture medium and plate the organoids in a 48 well cell culture plates.</li>
		<li>Incubate the organoids at 37 &#176;C, 5% CO2, for 20 min to allow the cell matrix solution to solidify.</li>
		<li>Cover the organoids with 300 &#181;L of murine organoid medium per well.</li>
		<li>Culture the organoids at 37 &#176;C, 5% CO2, changing the medium every 2&#8211;3 days.</li>
		<li>Use the organoids for experiments after 7 days of culture.</li>
	</ol>
	</li>
	<li>Prepare the organoids for the barrier integrity measurement.
	<ol>
		<li>Precoat all the centrifugation tubes that will be used for storing the organoids during the plating process with bovine serum albumin (BSA) by adding enough of a 0.1% BSA solution in PBS to cover all plastic surfaces. Then remove the BSA solution again and store the centrifugation tubes on ice.</li>
		<li>Thaw the cell matrix solution and organoid culture medium on ice.</li>
	</ol>
	</li>
	<li>To separate the organoids, carefully remove the culture medium and resuspend the organoids from one well of a 48 well plate in 1 mL of cold PBS. Dissolve the cell matrix by vigorous pipetting. Always keep the organoid suspension in centrifugation tubes precoated with BSA and always keep on ice. 
	NOTE: The density, size, and position of the organoids within the chambered coverslip slide are influenced by the split ratio, cell matrix solution concentration, and handling of the organoid-cell matrix suspension. It is recommended to practice the handling of the cell matrix solution in advance. Usually eight well chambered glass coverslips are suitable for the assay. Organoids derived from one well of a confluent 48 well plate can be split into two wells of an eight well chambered coverslip (40 &#956;L of the organoid-cell matrix pellet per well).</li>
	<li>Centrifuge the organoid suspension at 300 x g at 4 &#176;C for 5 min.</li>
	<li>Carefully discard the supernatant and resuspend the pellet with 1 mL of cold PBS.</li>
	<li>Centrifuge the organoid suspension at 300 x g, 4 &#176;C for 5 min.</li>
	<li>Discard the supernatant completely and resuspend the organoids derived from one well from a 48 well plate in 40 &#181;L of cold medium. Fragment large organoid structures by pipetting the organoid suspension 5x through a 10 &#181;L pipette tip to collect structures with a size of 40&#8211;60 &#181;m for seeding. 
	NOTE: Use the 10 &#181;L tip on a 100 &#181;L pipette tip for the fragmentation of the organoid structures, and practice step 1.7 in advance to ensure consistent results. Control the size of the organoids by phase contrast microscopy within the centrifugation tube. Ensure that there are no more multibranched organoids present and that the organoid fragments are roughly 40&#8211;60 &#181;m long.</li>
	<li>Once the organoids have obtained the desired size, mix them with 40 &#181;L of the cell matrix solution (medium:cell matrix solution = 1:1). 
	NOTE: The medium to cell matrix solution ratio must be kept constant to achieve consistent results. The dilution of the cell matrix solution reduces the stiffness of the organoid blob and impacts its diffusion properties. Use precooled pipet tips (-20 &#176;C) for all suspensions containing cell matrix solution.</li>
	<li>Place 40 &#181;L of the organoid-cell matrix solution suspension in the center of each well of an 8 well chambered coverslip.</li>
	<li>Keep the slide on an ice pack for 5 min. This preserves the cell matrix organoid suspension liquid and increases the organoid concentration at the coverslip surface by gravity.</li>
	<li>Incubate for 20 min at 37 &#176;C and 5% CO2 to enable polymerization of the organoid-cell matrix blob.</li>
	<li>Add 150 &#181;L of organoid culture medium per well and incubate for 24 h at 37 &#176;C and 5% CO2 prior to proceeding with the experimental treatment.
	<ol>
		<li>Use this period to treat the organoids and modulate their barrier integrity according to the corresponding scientific hypothesis. For the positive control, treat the organoids for 48 h with IFN-&#947; in order to investigate IFN-&#947; associated tight junction degradation and permeability increase. Stimulate the positive control with 10 U/mL (10 ng/mL) recombinant murine IFN-&#947;. Leave the organoids of one well untreated.</li>
	</ol>
	</li>
	<li>Culture organoids at 37 &#176;C and 5% CO2 for up to 48 h.</li>
</ol>
2. OrganoidPermeability Assay
<ol>
	<li>Bring the incubation chamber of the microscope to 37 &#176;C at least 2 h before starting the experiment to reduce thermal drift while imaging the organoids.</li>
	<li>Prepare a 100 mM solution of LY in PBS. Store on ice protected from light.</li>
	<li>Prepare a 200 mM solution of EGTA in PBS. Store it on ice.</li>
	<li>Transfer the chambered coverslip including the organoids into the incubation chamber of an inverted confocal microscope and turn on the CO2 incubation (5%). Make sure the chambered coverslip is tightly locked within the stage of the microscope.</li>
	<li>Using the organoids in one well as a reference, adjust the imaging settings of the microscope. Add LY (3 &#181;L of 100 mM LY in 150 &#181;L of medium) to obtain a final volume of 1 mM LY in 300 &#181;L of medium. Incubate on the microscope for 1 h and adjust the focus for the imaging of the organoids&#8217; lumen. Define the required laser energy for LY excitation (488 nm) and respective detection sensitivity of the instrument and try to image the LY fluorescence at 30&#8211;40% of the available dynamic range of the instrument being used. 
	NOTE: Adjust the laser excitation energy and detection efficiency on untreated organoids 70 min after the addition of LY. Ensure that the excitation energy is high enough to get a well exposed image. To avoid saturation of LY fluorescence within the microscopic images, it is recommended to adjust these settings after the LY diffusion reaches a steady state.</li>
	<li>Define the position of the organoids by differential interference contrast (DIC) live imaging. Try to image organoids with comparable diameters (80 &#177; 30 &#956;m) and focus on the central slice of the organoids to image their lumen. Define roughly 10 organoids per well and try to image only organoids close to the coverslip surface with a spherical structure. 
	NOTE: The number of organoids that can be imaged per run depends on the speed of the microscope. It is recommended to image the organoids within a 5 min interval. On a regular laser scanning microscope, 40 positions in total are a reasonable starting point.</li>
	<li>Record the DIC and the LY fluorescence of every position to document the organoid&#39;s shape and autofluorescence before adding the LY to the wells, used for the barrier integrity assay.</li>
	<li>Do not image organoids that display high autofluorescence. This is due to the accumulation of dead cells within the organoid&#39;s lumen, and the results of autofluorescent organoids are hard to analyze afterwards.</li>
	<li>Dilute 3 &#181;L of the prepared LY solution (100 mM LY in 150 &#181;L of medium) and add this carefully to each well without touching the chambered coverslip. The recommended concentration of LY per well is 1 mM. The final volume per well should be 300 &#181;L.</li>
	<li>Quickly check the focus of the defined positions and correct if needed. 
	NOTE: LY diffuses quickly through the cell matrix. Therefore, the confocal imaging must be started within 3 min after the addition of the fluorophore.</li>
	<li>Start a time-lapse imaging on the microscope. Take a fluorescence image of every position every 5 min for a total of 70 min. 
	NOTE: The organoids were imaged in 5 min intervals to visualize the LY uptake over time. To measure the intestinal barrier breakdown, it is sufficient to record the fluorescence before and 60 min after LY addition and once again 10 min after the addition of EGTA.</li>
	<li>Add 3 &#181;L of the prepared EGTA solution per well without touching the chambered coverslip. The recommended concentration within the chambered coverslip of EGTA is 2 mM. The total volume per well should be 300 &#181;L.</li>
	<li>Start a second time-lapse. Record the fluorescence of the defined organoids again with an interval of 5 min for a total of 30 min.</li>
	<li>Discard everything according to local safety regulations. 
	NOTE: The protocol can be paused here.</li>
</ol>
3. Data Analysis
<ol>
	<li>Only analyze the results of the organoids that took up LY after EGTA addition.</li>
	<li>Results can be quantified with Fiji ImageJ.</li>
	<li>Open dataset in ImageJ by clicking File |Open and select image data. In the following BIO-Formats Import options dialog select View stack with: Hyperstack.</li>
	<li>Open the region of interest (ROI) manager by clicking Analyze | Tools | ROI Manager.</li>
	<li>Draw an oval ROI by clicking on the Oval selection button in the ImageJ menu bar. Draw a selection containing the inner lumen of the organoid. Then press Add in the ROI Manager.</li>
	<li>Repeat the steps for three representative areas outside of the organoid.</li>
	<li>Click on Analyze in the menu bar and select Set Measurements. Enable only Mean Gray Value and disable any other measurement. Then click OK.</li>
	<li>Make sure that all ROIs are selected in the ROI Manager. In the ROI Manager, click More | Multi measure. In the option dialog select Measure all [&#8230;] slices and One row per slice. Then click OK.</li>
	<li>Select all the values in the Results window and copy them into a spreadsheet application for further analysis. 
	NOTE: If the position of the organoid moved during the time-lapse imaging, the ROI must be adjusted accordingly. To do so, select the correct ROI in the ROI manager and move it to the new position. Then click Update in the ROI manager. Perform the measurement for each timepoint individually by clicking Measure in the ROI manager, then switch to the next timepoint in the image window using the bar on the bottom. Collect all measurements in a spreadsheet. The individual shape and the movement of organoids during the imaging period requires the analysis of the data in a manual manner.</li>
	<li>Calculate the mean intensity value of the three ROIs outside of the organoid for each timepoint.</li>
	<li>Divide the intensity of the ROI inside the organoid&#8217;s lumen by the mean intensity of the ROI outside and the mean intensity inside the organoid.</li>
	<li>In order to calculate the relative increase of luminal organoid fluorescence, divide the relative fluorescence (see step 3.11) at each timepoint imaged by the minimal relative fluorescence. 
	NOTE: Use the minimal relative fluorescence, because sometimes the diffusion of the fluorophore can be slow at the beginning of the experiment.</li>
</ol>

<ol>
	<li>L&#243;pez-Posadas, R., St&#252;rzl, M., Atreya, I., Neurath, M. F., Britzen-Laurent, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+of+GTPases+and+Cytoskeleton+in+Cellular+Barrier+Defects+during+Gut+Inflammation.">Interplay of GTPases and Cytoskeleton in Cellular Barrier Defects during Gut Inflammation.</a> Frontiers in Immunology. 8, 1240 (2017).</li><li>Zhang, Y. Z., Li, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+bowel+disease:+pathogenesis.">Inflammatory bowel disease: pathogenesis.</a> World Journal of Gastroenterology. 20 (1), 91-99 (2014).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Leslie, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistence+and+toxin+production+by+Clostridium+difficile+within+human+intestinal+organoids+result+in+disruption+of+epithelial+paracellular+barrier+function.">Persistence and toxin production by Clostridium difficile within human intestinal organoids result in disruption of epithelial paracellular barrier function.</a> Infection and Immunity. 83 (1), 138-145 (2015).</li><li>Zietek, T., Rath, E., Haller, D., Daniel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+organoids+for+assessing+nutrient+transport,+sensing+and+incretin+secretion.">Intestinal organoids for assessing nutrient transport, sensing and incretin secretion.</a> Scientific Reports. 5 (1), 16831 (2015).</li><li>Pearce, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marked+differences+in+tight+junction+composition+and+macromolecular+permeability+among+different+intestinal+cell+types.">Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types.</a> BMC Biology. 16 (1), 19 (2018).</li><li>Hill, D. R., Huang, S., Tsai, Y. H., Spence, J. R., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+Measurement+of+Epithelial+Barrier+Permeability+in+Human+Intestinal+Organoids.">Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids.</a> Journal of Visualized Experiments. (130), e56960 (2017).</li><li>Bardenbacher, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+analyses+and+three+dimensional+imaging+of+interferon+gamma-induced+barrier+disintegration+in+intestinal+organoids.">Permeability analyses and three dimensional imaging of interferon gamma-induced barrier disintegration in intestinal organoids.</a> Stem Cell Research. 35, 101383 (2019).</li><li>Tomita, M., Hotta, Y., Ohkubo, R., Awazu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarized+transport+was+observed+not+in+hydrophilic+compounds+but+in+dextran+in+Caco-2+cell+monolayers.">Polarized transport was observed not in hydrophilic compounds but in dextran in Caco-2 cell monolayers.</a> Biological and Pharmaceutical Bulletin. 22 (3), 330-331 (1999).</li><li>Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+mucosal+barrier+function+in+health+and+disease.">Intestinal mucosal barrier function in health and disease.</a> Nature Reviews: Immunology. 9 (11), 799-809 (2009).</li></ol>

The authors have nothing to disclose.

This assay offers a technique to study the intestinal barrier integrity within living organoids. The whole assay is based on small intestinal mouse organoids and confocal live cell microscopy. Therefore, it is mandatory to practice the proper handling of organoids in advance. Upon isolation, organoids can be routinely split and stored by cryofreezing3,9. For this assay we recommend splitting the organoids 48 h before the treatment is started. This period gives the organoids the chance to totally close and form spherical structures. The seeding of the organoids for the experiment is a critical step within the assay. To reduce individual handling variations, we recommend a routine procedure for the seeding process. This step is crucial, and a routine handling protocol clearly reduces experimental variations.
During the seeding procedure (step 1.7) the organoids get fragmented by repetitive passaging through a standard 10 &#956;L pipette tip. The pore size of this product varies from company to company. This procedure should be practiced in advance, and the result should always be checked by phase contrast microscopy. Once the organoids obtained reach the desired size, do not change the procedure.
The seeding of the organoids must be optimized and adapted for the available microscopic setup. To be able to culture and image organoids for at least 48 h, an incubated microscope chamber is absolutely required. Choose a chambered coverslip that matches your requirements. When seeding the organoids, make sure to concentrate the organoids on the coverslip surface. This is possible by keeping the chambered coverslip on an ice pack for 5 min after placing the cell matrix-organoid suspension. This step is important to increase the quality of confocal live cell imaging. The axial resolution and working distance of confocal microscope lenses is especially limited. The closer you bring the sample to the lens, the better you can image it and the less laser energy is needed to excite the LY fluorescence.
Phototaxis is an important issue when it comes to live cell microscopy. Within this assay we exclude this option. A functional AJC is visible by exclusion of LY from the organoid&#8217;s lumen (Figure 1, PBS). The addition of EGTA at the end of the experiment causes sequestering of bivalent ions, which are cofactors for AJC proteins. LY is excluded from the organoid&#39;s lumen only in vital organoids with a functional AJC complex. In general, fluorescent molecules can be used to measure the integrity of the intestinal barrier. We chose LY instead of other commonly used fluorophores such as fluorescein labeled dextran because those are transported transcellularly in intestinal cells from the basal to the apical compartment9. We also chose LY because of its small size. LY has a molecular weight of 457 Da and therefore facilitates the investigation of the barrier permeability for small molecules. The fluorescent molecule has to be chosen depending on the scientific question investigated. Because phototoxic AJC defects are present, laser excitation energy has to be reduced or the imaging interval extended. The optimal confocal imaging technique for this assay is spinning disc microscopy. Respective instruments enable confocal imaging with short exposure time at low laser power.
Different models have already been developed to study intestinal barrier integrity in vitro. While the use of assays based on cell line monolayers or experiments in vivo are declining, organoid-based methods increasing. In contrast to methods previously described4,5,6,7, our method allows quantification of barrier function over time. This allows exposure of the organoids to additional stimuli over the course of the experiment. Here we apply EGTA as a second stimulus at the end of the experiment as a positive control.
In contrast to the situation in vivo, in our assay LY is added into the medium and penetrates the organoid from the outside basolateral epithelial side towards the inside apical lumen. The LY is small and is only used to visualize the tightness of the intestinal barrier. Molecules and stimuli that modulate the epithelial layer at the apical surface need to be injected into the organoid&#39;s lumen7. To reduce the experimental effort and to be able to measure the barrier integrity of many organoids at the same time, we chose to apply the fluorescent dye from the outside.
We used the assay to investigate the function of IFN-&#947; on the tight junction of small intestinal mouse organoids. The fact that we were able to analyze the barrier integrity in living organoids offers future possibilities to apply this technique to describe inhibitors for the inflammation-induced breakdown of the intestinal barrier. Substances that counteract the impaired barrier function caused by IFN-&#947; could be candidates for the treatment of inflammatory bowel diseases, in which impaired barrier function is one of the pathogenic factors10.

Integrity of the epithelial barrier is maintained by the apical junctional complex (AJC), which consist of tight junction (TJ) and adherence junction (AJ) proteins1. The polarized structure of the AJC is crucial for its function in vivo. Dysregulation of the AJC is present in various diseases and is suspected to be an important trigger of inflammatory bowel pathogenesis. Loss of intestinal barrier function represents the initiating event of the disease. The following translocation of commensal bacteria and inflammatory responses are the painful consequences2.
Various in vitro and in vivo models have been developed to investigate the regulation of the AJC. The Transwell assay is based on two-dimensional (2D) cell monolayers that were derived from tumor cell lines. These systems are good to assess by optical and biochemical methods and enable the analysis of many samples at the same time but lack many features of primary cells and the differentiation processes present in vivo. Investigating the barrier integrity is also possible in animal models. In terminal experiments, the effects of specific treatments in vivo on the permeability of the whole intestine can be quantified. However, these models require a large number of animals, and they do not allow detailed visualization of the underlying molecular processes. Nowadays improved 3D in vitro models are available that closely recapitulate cell differentiation processes, cell polarization, and represent the crypt-villus structure of the intestine3. The application of 3D intestinal organoids for functional analyses requires the adaptation of available methods from 2D models. Here we describe a model to investigate intestinal barrier integrity in living small intestinal mouse organoids. The assay was established to investigate the effect of IFN-&#947; on the barrier integrity and respective tight junction proteins8.
In contrast to the technique applied by Leslie4, Zietek5, or Pearce6, which measures fluorescence after removing lucifer yellow (LY) from the medium, our approach allows quantification of the luminal uptake of the fluorophore over time. Therefore, the result represents a dynamic uptake kinetic and our assay enables the application of additional stimuli or inhibitors during the course of the experiment. The fact that both assays measure the uptake from the outside basolateral side to the inside apical surface is in clear contrast to the situation in vivo. In a model described by Hill et al.7, this topic was explored. Upon microinjection of the fluorophore into the organoid&#8217;s lumen, the fluorescence was quantified. The direction of diffusion represents the direction present in vivo. The technical effort of microinjection clearly reduces the throughput of this method. In contrast to the model described here, the microinjection method enables the measurement of effects that require biological activation on the apical epithelial surface.
The organoid barrier integrity model presented here is based on live cell microscopy and enables the analysis of dynamic changes within the AJC regulation over time. The setup can be applied to test the pharmacological impact of substances inducing and inhibiting the integrity of the intestinal barrier. Furthermore, organoid-based models help reduce the number of animals used for pharmacological studies.

<table><tbody><tr><td>48-well culture plate</td><td>Thermo Fisher Scientific</td><td>#150687</td><td></td></tr><tr><td>8-well chamber slides</td><td>Ibidi</td><td>#80826</td><td></td></tr><tr><td>96-well culture plate</td><td>Greiner Bio-One</td><td>#655101</td><td></td></tr><tr><td>Axio Observer.Z1 - spinning disc</td><td>Zeiss</td><td></td><td>excitation laser 488 nm / emission filter 525/25</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A3059-100G</td><td></td></tr><tr><td>Cell strainer</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Centrifugation tube 15 mL</td><td>Thermo Fisher Scientific</td><td>11507411</td><td></td></tr><tr><td>Centrifugation tube 50 mL</td><td>Thermo Fisher Scientific</td><td>10788561</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>431788-25g</td><td></td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>431788</td><td></td></tr><tr><td>Lucifer Yellow CH dilithium salt</td><td>Sigma-Aldrich</td><td>L0259</td><td></td></tr><tr><td>Matrigel, growth factor reduced, phenol red free</td><td>Corning</td><td>356231</td><td>Cell matrix solution</td></tr><tr><td>Mice</td><td>The Jackson Laboratory</td><td>&lt;em&gt;M. musculus&lt;/em&gt; C57/Bl6</td><td></td></tr><tr><td>Microscope coverslip</td><td></td><td>24 mm x 60 mm</td><td></td></tr><tr><td>Organoid Growth Medium mouse</td><td>Stemcell Technologies</td><td>#06005</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Biochrom</td><td>L182-05</td><td></td></tr><tr><td>Recombinant murine IFN-&amp;gamma;</td><td>Biolegend</td><td>Cat#575304</td><td></td></tr></tbody></table>

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.

To validate the application of 3D small intestinal mouse organoids as a model to quantify the effect of compounds regulating the intestinal barrier integrity, we applied IFN-&#947;. To do so, we isolated and cultured organoids derived from IFN-&#947; responsive wild type and IFN-&#947;-receptor-2 knockout mice, which do not respond to IFN-&#947;8. Upon treatment for 48 h with IFN-&#947; or PBS (control), all organoids were exposed to LY and imaged by confocal spinning disc live cell microscopy in 5 min intervals for a period of 70 min. The functional integrity of the intestinal barrier in this model resulted in exclusion of LY from the organoid&#39;s lumen while intraluminal accumulation of LY signified destruction of the TJ. The representative fluorescence microscopic images after 70 min of incubation with LY clearly demonstrate that intraluminal LY fluorescence was only visible in organoids from wild type animals treated with IFN-&#947;. In unstimulated (PBS) controls nor in organoids derived from knock out animals (IFN-&#947;R2&#916;IEC, Figure 1), no intraluminal LY fluorescence was present after 70 min.
The addition of EGTA causes an unspecific breakdown of the intestinal barrier integrity by sequestering TJ cofactors. This control was always utilized at the end of the experiment to demonstrate the ability of the respective organoid to take up LY (Figure 1). If no intraluminal LY fluorescence was detected upon EGTA treatment, the organoid was excluded from the experiment.
For the quantitative evaluation of the microscopic results, LY fluorescence was measured within the organoid&#39;s lumen and outside of the organoid. Relative intensity values were calculated (fluorescence inside/ fluorescence outside + inside) and are shown for each time point imaged. It is recommended to avoid imaging of organoids of varying sizes. We chose to focus on organoids with a diameter of 80 &#177; 30 &#181;m (Figure 2). A schematic of the protocol with representative images is shown in Figure 3. Some major problems and troubleshooting techniques are shown and discussed in Figure 4.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60546/60546fig01.jpg" /> 
Figure 1: Intestinal barrier integrity can be analyzed in mouse organoids.&#160;Intestinal organoids from IFN-&#947;R2WT and IFN-&#947;R2&#916;IEC were cultured in the presence of IFN-&#947; for 48 h or left untreated. To investigate the integrity of the intestinal barrier, LY (457 Da) was added and confocal fluorescent images were captured in 5 min intervals for a total of 70 min. Representative images at time point 0 min, 70 min, and after addition of EGTA are shown (green = Lucifer yellow; Scale bar = 20 &#181;m). This figure has been modified from Bardenbacher et al.8. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60546/60546fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60546/60546fig02.jpg" /> 
Figure 2: Small intestinal organoid barrier integrity model provides quantitative results.&#160;(A) LY fluorescence was determined inside and outside the organoid. Relative intensity values were calculated (inside/fluorescence outside + inside) relative to the initial relative intensity + SEM and are shown for each time point. (B) Size distribution of analyzed organoids. To reduce the standard deviation and errors due to changes in the surface-to-volume ratio, we only analyzed organoids with a diameter of 80 &#177; 30 &#181;m. Mean values of the respective organoid diameters are shown + SD (IFN-&#947;R2WT, n = 20; IFN-&#947;R2&#916;IEC, n = 18). The mean diameter values did not vary significantly between the different groups (one-way ANOVA). (C) The permeability of the organoids was determined 70 min after the addition of LY. It was defined by dividing the intraluminal fluorescence intensities after 70 min by the minimal relative fluorescence intensities measured during the observation period. Each bar represents mean values + SD, measured in 10 organoids derived from two independent experiments (IFN-&#947;R2WT, n = 20; IFN-&#947;R2&#916;IEC, n = 18). IFN-&#947; significantly increased the LY uptake only in IFN-&#947;R2WT organoids. ***p-value &#60;0.001 in the Student&#8217;s t-test. This figure has been modified from Bardenbacher et al.8. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60546/60546fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60546/60546fig03.jpg" /> 
Figure 3: Schematic protocol with representative images.&#160;(A) Schematic description of the main steps of the protocol. (B) Representative pictures of the major steps of the protocol. (B1) DIC microscopy image of a central slice through a suitable organoid that was selected for permeability analysis. The dotted line represents a width of 89 &#181;m. (B2) Fluorescence microscopy picture of the same organoid in (B1) before adding LY. The image shows the autofluorescence of the organoid. (B3) An organoid 70 min after the addition of LY. The depicted organoid shows no uptake of LY and therefore an intact barrier function. Dotted lines show the ROIs for further analysis. The inner lumen of the organoid and three representative areas around the organoid are marked. (B4) An organoid after the addition of EGTA. The organoid is usable for further analysis because it shows LY uptake after the EGTA treatment. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60546/60546fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60546/60546fig04.jpg" /> 
Figure 4: Troubleshooting of common problems.&#160;(A) Table with common problems and solutions. (B) Exemplary images. (B1) DIC image of a large multibranched organoid that is not suitable for this assay. (B2). Confocal image of an organoid displaying high autofluorescence before LY was added to the medium. The organoid was excluded from quantification. (B3) Confocal image of an organoid displaying low autofluorescence before LY was added to the medium. The fluorescence was quantified in this case. (B4) Organoid showing no LY uptake from the medium 30 min after addition of EGTA and therefore excluded from quantification. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60546/60546fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Investigating Intestinal Barrier Breakdown in Living Organoids at JoVE.com

Investigating Intestinal Barrier Breakdown in Living Organoids

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 ...

investigating-intestinal-barrier-breakdown-in-living-organoids

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.9K Views.  Friedrich-Alexander-Universität (FAU) Erlangen-Nürnberg and Universitätsklinikum Erlangen. 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.

Video: Investigating Intestinal Barrier Breakdown in Living Organoids

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 ...

Developmental Biology

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

Bioengineering

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 (BSL-3) Conditions

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived from all sections of the GI tract (gastric, jejunum, duodenum, ileum, colon, rectum) and, upon differentiation, contain most of the cell types that are naturally found in each individual section. For example, intestinal organoids contain nutrient-absorbing enterocytes, secretory cells (Goblet, Paneth, and enteroendocrine), stem cells, as well as all lineage-specific differentiation intermediates (e.g.,&#160;early or immature cell types). The greatest advantage in using gastrointestinal tract-derived organoids to study infectious diseases is the possibility of precisely identifying which cell type is targeted by the enteric pathogen and to address whether the different sections of the gastrointestinal tract and their specific cell types similarly respond to pathogen challenges. Over the past years, gastrointestinal models, as well as organoids from other tissues, have been employed to study viral tropism and the mechanisms of pathogenesis. However, utilizing all the advantages of using organoids when employing highly pathogenic viruses represents a technical challenge and requires strict biosafety considerations. Additionally, as organoids are often grown in three dimensions, the basolateral side of the cells is facing the outside of the organoid while their apical side is facing the inside (lumen) of the organoids. This organization poses a challenge for enteric pathogens as many enteric infections initiate from the apical/luminal side of the cells following ingestion. The following manuscript will provide a comprehensive protocol to prepare human intestinal organoids for infection with enteric pathogens by considering the infection side (apical vs. basolateral) to perform single-cell RNA sequencing to characterize cell-type-specific host/pathogen interactions. This method details the preparation of the organoids as well as the considerations needed to perform this work under biosafety level 3 (BSL-3) containment conditions.

Adapting Gastrointestinal Organoids for Pathogen Infection and Single Cell Sequencing under Biosafety Level 3 BSL-3 Conditions

This protocol describes how to infect human intestinal organoids from either their apical or basolateral side to characterize host/pathogen interactions at the single-cell level using single-cell RNA sequencing (scRNAseq) technology.

Human intestinal organoids constitute the best cellular model to study pathogen infections of the gastrointestinal tract. These organoids can be derived ...

Immunology and Infection

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 ...

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 ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

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 ...

Biology

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 ...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...