The authors would like to thank Drs. Stephanie Spohn and Basel Abuaita for many useful discussions on organoid microinjection. JRS is supported by the Intestinal Stem Cell Consortium (U01DK103141), a collaborative research project funded by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and the National Institute of Allergy and Infectious Diseases (NIAID). JRS and VBY are supported by the NIAID Novel, Alternative Model Systems for Enteric Diseases (NAMSED) consortium (U19AI116482). DRH is supported the Mechanisms of Microbial Pathogenesis training grant from the National Institute of Allergy and Infectious Disease (NIAID, T32AI007528) and the Clinical and Translational Science award to the Michigan Institute for Clinical and Health Research (UL1TR000433).
The complete data files and data analysis code used in this manuscript are available at https://github.com/hilldr/HIO_microinjection.

<ol>
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The authors have no competing financial interests or other conflicts of interest to disclose.

This protocol establishes a general-purpose method for the microinjection of hPSC-derived HIOs and tissue-derived intestinal organoids and the measurement of epithelial barrier permeability in real time. We have also demonstrated our approach to analysis and interpretation of the data generated using these methods. Given the growing adoption of intestinal organoids model systems16,20,21,28 and the long-standing interest in intestinal barrier permeability as a physiologically relevant functional outcome3,4,5,6, we anticipate that others working in this field will be able to apply and build upon these methods.
There are several steps which are critical to the application of this technique. Access to high quality hPSC- or tissue derived HIO tissue should be established prior to extensive experimentation with microinjection. HIO macrostructure may be heterogenous, with variation in both size and shape, although tissue identity and cellular morphology is highly reproducible when utilizing established methodology to generate HIOs24. Spherical HIOs consisting of a single semi-transparent lumen and measuring approximately 1 mm in diameter are ideal for microinjection and measurement of luminal fluorescence in real-time. In some cases, microinjection will fail, resulting in collapse of the HIO or obvious leakage of injected material. Failed HIOs can be removed from the culture well at the user&#39;s discretion using a standard micropipette. Consider the objective lenses available on an imaging platform when selecting HIOs for microinjection and imaging. In general, 2 - 4X objective lenses are ideal for capturing the complete HIO fluorescent signal, although a 10X objective may be used if low power lenses are not available or if the available HIOs are &#60;1 mm in diameter. Imaging software must allow for the automated capture of fluorescent images at defined points over time.
Several modifications of this protocol are possible in order to suit the experimental requirements. For example, the results of barrier function tests may be dependent on the molecular size of the compounds in use43 and it may be appropriate to test dextran preparations of varying molecular weight. In addition, brightfield imaging may be performed in addition to fluorescence imaging as an indicator of the overall structural integrity of the tissue25. When performing microinjection of live bacteria25,28,31,32,33,44, it may be necessary to add penicillin and streptomycin or gentamicin to the HIO culture media prior to or after microinjection. The outside of the microcapillary will become contaminated during filling with the bacterial culture suspension and this may be transferred to the HIO media. Alternately, microinjection can be performed on HIOs suspended in extracellular matrix (e.g., Matrigel) without media, adding the media after the microinjection is completed. This may limit contamination to the extracellular matrix and external face of the HIO. When planning microbial growth assays, it may be necessary to remove antibiotics in the media after 1 - 2&#160;h to avoid slowing or preventing growth of microinjected organisms.
Finally, recognizing that not all researchers will have access to microscopy equipment suited to in vitro imaging, it is important to point out that the procedures outlined in this protocol for collecting fluorescence data can be applied to images taken at fixed timepoints using standard epifluorescent microscopy without automated image capturing or environmental controls. Examples of this approach can be found in the reports by Leslie and Huang et al.25, who examined C. difficile toxin activity in hPSC-derived intestinal organoids, and Karve and Pradan et al.44, who examined epithelial barrier permeability in similar hPSC-derived intestinal organoids microinjected with live E. coli. Manual operation of imaging equipment may result in greater variation and difficulty in normalizing the fluorescent signal. When performing manual imaging of FITC-dextran injected HIOs it is essential to maintain fixed magnification, fluorescent excitation intensity, and exposure times throughout the experiment to avoid distorting the fluorescent intensity measurements.

The intestinal epithelium forms a selective barrier that mediates the directional transport of nutrients, H2O, ions, and waste products while minimizing nonspecific diffusion-mediated exchange of other particles between the lumen and the mesenchymal tissue or blood supply1,2. The nonspecific permeability of the intestinal epithelial barrier has long been considered a key functional parameter in both health and disease3,4,5,6, that reflects the rate of diffusion of small molecules across the epithelium via the paracellular space. Measurement of epithelial barrier permeability can be conducted in animal models7 and in human patients8 through the ingestion of lactulose, which has no specific transporter in the gastrointestinal tract, and the subsequent collection and measurement of lactulose concentrations in peripheral blood. Alternate ingested markers of barrier function such as fluorescently labeled carbohydrates are also available9,10. This approach has been adapted for intestinal epithelial cell cultures grown on Transwell supports11, a simplified approach that allows for greater experimental control but has also been criticized as a poor predictor of in vivo permeability due to the absence of differentiated epithelial subtypes and tissue structure12. Using chambers represent yet another approach and allow for the measurement of epithelial barrier function in whole intestinal mucosa ex vivo13. Application of this technique is frequently limited by tissue availability and condition13,14. Thus new methods which balance reproducibility and throughput with physiologic relevance are necessary.
Recent developments in in vitro organogenesis have led to the adoption of 3D tissue culture model systems as a sophisticated platform for recapitulating the dynamics of complex tissues15,16,17,18,19,20,21,22,23. In particular, human pluripotent stem cell (hPSC) derived human intestinal organoids (HIOs)19,24 have emerged as a reproducible and experimentally tractable model system for the study of host-microbial interactions and epithelial barrier dynamics25,26,27,28. Similarly, human tissue-derived organoids (also known as enteroids) can be derived from a simple biopsy procedure and can be used as a tractable system to study human physiology and disease15,29,30. Microinjection of human intestinal organoids allows for the delivery of experimental compounds25 or live microbes25,31,32,33 to the apical epithelial surface of the organoid lumen. Leslie and Huang et al.25 recently adapted this technique to measure barrier permeability in HIOs microinjected with fluorescein isothiocyanate (FITC) labeled dextran following exposure to bacterial toxins.
This protocol is intended as a guide for the measurement of epithelial barrier permeability in hPSC-derived HIOs and tissue-derived HIOs using fluorescent microscopy. With minor modifications, it can also serve as a general primer on microinjection of HIOs with experimental compounds. Users may supplement this protocol with additional or alternative downstream applications after microinjection.

<table><tbody><tr><td>EGTA 0.5 M sterile (pH 8.0)</td><td>Bioworld</td><td>405200081</td><td></td></tr><tr><td>Cell matrix solution (Matrigel)</td><td>Corning</td><td>354230</td><td></td></tr><tr><td>Deltavision RT live cell imaging system</td><td>GE Life Sciences</td><td>29065728</td><td>http://www.gelifesciences.com/webapp/wcs/stores/servlet/catalog/en/GELifeSciences/brands/deltavision/</td></tr><tr><td>Camera</td><td>GE Life Sciences</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>softWoRx Imaging software</td><td>GE Life Sciences</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>Biosafety cabinet</td><td>Labconco</td><td>Cell Logic+</td><td>http://www.labconco.com/product/purifier-cell-logic-class-ii-type-a2-biosafety-cabinets-2/4262</td></tr><tr><td>1X PBS</td><td>Life Technologies</td><td>10010-023</td><td></td></tr><tr><td>Advanced DMEM-F12</td><td>Life Technologies</td><td>12634-010</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>B27 supplement (50X)</td><td>Life Technologies</td><td>17504044</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>L-glutamine (100X)</td><td>Life Technologies</td><td>25030-081</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>HEPES buffer</td><td>Life Technologies</td><td>15630080</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Manipulator</td><td>Narshge</td><td>UM-3C</td><td></td></tr><tr><td>Micromanipulator</td><td>Narshge</td><td>UM-1PF</td><td></td></tr><tr><td>Pipette Holder</td><td>Narshge</td><td>UP-1</td><td>Alternate to Xenoworks pipette holder</td></tr><tr><td>Magnetic stand</td><td>Narshge</td><td>GJ-1</td><td></td></tr><tr><td>Dissecting scope</td><td>Olympus</td><td>SX61</td><td>Recommended scope, although other models are likely compatible</td></tr><tr><td>Olympus IX71 Fluorescent microscope</td><td>Olympus</td><td>IX71</td><td>Included with Deltavision system</td></tr><tr><td>CoolSNAP HQ2</td><td>Photometrics</td><td>29065728</td><td>Included with Deltavision system</td></tr><tr><td>Recombinant &lt;em&gt;C. difficile&lt;/em&gt; Toxin A/TcdA Protein</td><td>R&amp;amp;D Systems</td><td>8619-GT-020</td><td></td></tr><tr><td>EGF</td><td>R&amp;amp;D Systems</td><td>236-EG</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>R-spondin 1</td><td>R&amp;amp;D Systems</td><td>4645-RS</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Noggin</td><td>R&amp;amp;D Systems</td><td>6057-NG</td><td>Component of ENR media; see McCraken et al. &lt;sup class=&quot;xref&quot;&gt;24&lt;/sup&gt;</td></tr><tr><td>Mineral oil</td><td>Sigma-Aldrich</td><td>M8410</td><td></td></tr><tr><td>FITC-dextran (4 kDa)</td><td>Sigma-Aldrich</td><td>46944</td><td></td></tr><tr><td>Micropipette puller</td><td>Sutter Instruments</td><td>P-30</td><td></td></tr><tr><td>Nunc Lab-Tek II Chamber Slides</td><td>ThermoFisher Scientific</td><td>154526PK</td><td></td></tr><tr><td>Glass filaments</td><td>WPI</td><td>TW100F-4</td><td></td></tr><tr><td>Micropipette holder</td><td>Xenoworks</td><td>BR-MH2</td><td>Preferred device</td></tr><tr><td>Analog Tubing kit</td><td>Xenoworks</td><td>BR-AT</td><td></td></tr><tr><td>1/16 in clear ferrule</td><td>Xenoworks</td><td>V001104</td><td></td></tr><tr><td>1-1.2 mm O-ring</td><td>Xenoworks</td><td>V300450</td><td></td></tr></tbody></table>

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.

HIOs were differentiated from human pluripotent stem cells and cultured in cell matrix solution as previously described19,24. After 4 weeks in culture, the HIOs had expanded sufficiently to allow for microinjection. HIOs were microinjected with 4 kDa FITC-conjugated dextran suspended in PBS or PBS containing Clostridium difficile toxin TcdA. C. difficile is an opportunistic gastrointestinal pathogen that exhibits toxin-mediated epithelial toxicity in HIOs25. As a positive control, EGTA was added to the HIO culture media in a subset of HIOs injected with PBS and FITC-dextran. EGTA is a calcium chelator that causes rapid de-polymerization of the actin cytoskeleton42. FITC fluorescence was monitored in real time on a live imaging microscope within a controlled environmental chamber and images were captured in 10-minute intervals.
Post-hoc analysis of imaging data revealed substantial differences in the retention of FITC fluorescence (Figure 5). HIOs injected with PBS retained nearly all of the fluorescent signal present at t = 0, however HIOs that were also injected with TcdA of treated with EGTA exhibited a substantial decrease in fluorescent intensity by 8 hours post-microinjection (Figure 5A). Imaging data were quantified for all HIOs at all time points to generate a high-resolution dataset representing the relative change in fluorescent intensity over time in each experimental condition (Figure 5B). Differences in epithelial permeability were evaluated by calculating the mean elimination time (t1/2 ) of FITC for each treatment group (Table 1) and comparing differences in t t1/2 between groups using the Student&#39;s t-test. Control-treated HIOs retained the majority of FITC fluorescent signal for more than 16 hours (t1/2 = 17 &#177; 0.3 h). Treatment with EGTA significantly reduced FITC-dextran elimination time relative to control HIOs (t1/2 = 2.6 &#177; 0.2 h; P = 1.3 x 10-8). Consistent with previously published results25, microinjection of TcdA significantly increased epithelial barrier permeability relative to control treatment (t1/2 = 7.6&#160;&#177; 0.6 h; P = 5.4 x 10-4). Thus, both external (EGTA) and microinjected (TcdA) compounds are capable of inducing significant alterations in epithelial barrier permeability in HIOs. These results suggest that the effects of a wide range of pharmacologic agents, metabolites, bacterial products, cytokines, growth factors, and other compounds on epithelial barrier function may be evaluated using this approach.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Treatment</td>
			<td>Half-life (h)</td>
			<td>SEM (h)</td>
			<td>Lower 95% CI (h)</td>
			<td>Upper 95% CI (h)</td>
			<td>n</td>
		</tr>
		<tr>
			<td>Control</td>
			<td>17.11</td>
			<td>0.3</td>
			<td>16.45</td>
			<td>17.78</td>
			<td>11</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>2.58</td>
			<td>0.19</td>
			<td>1.78</td>
			<td>3.38</td>
			<td>3</td>
		</tr>
		<tr>
			<td>TcdA</td>
			<td>7.56</td>
			<td>0.63</td>
			<td>5.93</td>
			<td>9.18</td>
			<td>6</td>
		</tr>
	</tbody>
</table>
Table 1: Mean elimination time (t1/2) for FITC-dextran in HIOs treated with EGTA or TcdA. Units are hours post-microinjection.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56960/56960fig1.jpg" /> 
Figure 1: Basic layout of a microinjector and micromanipulator for HIO microinjection. This image shows the complete complement of equipment used for performing microinjection of HIOs. Key components are labeled and ordering information can be found in the Materials table. <a href="https://cloudflare.jove.com/files/ftp_upload/56960/56960fig1large.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/56960/56960fig2.jpg" /> 
Figure 2: Micropipette puller. The copper heating coil hc, top clamp c1, bottom clamp (c2), puller arm (pa), heat selection toggle (ht) are identified by the arrows. The correct setting for HEAT 1 and PULL are indicated in red text. The inset shows a correctly mounted glass filament ready for heating. <a href="https://cloudflare.jove.com/files/ftp_upload/56960/56960fig2large.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/56960/56960fig3.jpg" /> 
Figure 3: Representative images of a tissue-derived HIO injected with FITC-dextran. (A) Image demonstrating the positioning of the glass microcapillary just prior to insertion into the HIO and microinjection. (B) Brightfield image of a tissue-derived human intestinal organoids after microinjection of FITC-dextran. Note that the fluorescence signal is apparent even without the use of a specific filter set. This coloration aids microinjection precision. 3X magnification <a href="https://cloudflare.jove.com/files/ftp_upload/56960/56960fig3large.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/56960/56960fig4.jpg" /> 
Figure 4: Live imaging workflow. (A) Screenshot illustrating the steps necessary to set the microscope parameters, find the HIOs on the slide, and save the position of the stage for each HIO to be imaged. (B) Pre-imaging settings for capturing the FITC channel at regular intervals at each of the positions specified in A. (C) Post-imaging export of DV format data files as TIFF images with one TIFF image per HIO per time point. <a href="https://cloudflare.jove.com/files/ftp_upload/56960/56960fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56960/56960fig5.jpg" /> 
Figure 5: Representative results. (A) Stem-cell derived human intestinal organoids (HIO) microinjected with 2 mg/mL FITC-dextran (4 kDa) imaged for 20 hours. HIOs were also microinjected with PBS (control) or the Clostridium difficile toxin TcdA (12.8 ng/&#181;L) or treated with 2 mM EGTA added to the external culture media. 4X Magnification. (B) Plot of mean normalized FITC intensity over time in HIOs treated with PBS (control), TcdA, or EGTA. Error bars represent S.E.M. and n = 11 HIOs (Control), 3 HIOs (EGTA), 6 HIOs (TcdA). <a href="https://cloudflare.jove.com/files/ftp_upload/56960/56960fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

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

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 ...

real-time-measurement-epithelial-barrier-permeability-human

Research

JoVE Journal

Developmental Biology

14.1K Views.  University of Michigan. Dit protocol beschrijft de meting van epitheliale barrière permeabiliteit in real-time volgende farmacologische behandeling in menselijke intestinale organoids met behulp van fluorescentie microscopie en levende cel microscopie.

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

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

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

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

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

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

Medicine

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, ...

Bioengineering

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

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

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

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

Immunology and Infection

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 ...

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 ...

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 ...

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 ...

Biology

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

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

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

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

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

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

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

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

Developing 2D Monolayer Cultures from Bovine Intestinal Organoids

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

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

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

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

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

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

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