Name: real-time measurement of epithelial barrier permeability in human intestinal organoids
Uploaded: 2017-12-18T15:00:00
Duration: 8 min 4 s
Description: Watch this Scientific Journal Video about Real-time Measurement of Epithelial Barrier Permeability in Human Intestinal Organoids at JoVE.com

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. 24</td>
		</tr>
		<tr>
			<td>B27 supplement (50X)</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td>Component of ENR media; see McCraken et al. 24</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. 24</td>
		</tr>
		<tr>
			<td>HEPES buffer</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td>Component of ENR media; see McCraken et al. 24</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 C. difficile Toxin A/TcdA Protein</td>
			<td>R&#38;D Systems</td>
			<td>8619-GT-020</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>R&#38;D Systems</td>
			<td>236-EG</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>R-spondin 1</td>
			<td>R&#38;D Systems</td>
			<td>4645-RS</td>
			<td>Component of ENR media; see McCraken et al. 24</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>R&#38;D Systems</td>
			<td>6057-NG</td>
			<td>Component of ENR media; see McCraken et al. 24</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 ...

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