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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JoVE Journal

Developmental Biology

14.0K Views.  University of Michigan. 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.

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

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease phenotypes. Recent advances have identified more efficient and safe methods for introduction of reprogramming factors. However, there are few tools to monitor and track the progression of reprogramming. Current methods for monitoring reprogramming rely on the qualitative inspection of morphology or staining with stem cell-specific dyes and antibodies. Tools to dissect the progression of iPSC generation can help better understand the process under different conditions from diverse cell sources. 
This study presents key approaches for kinetic measurement of reprogramming progression using flow cytometry as well as real-time monitoring via imaging. To measure the kinetics of reprogramming, flow analysis was performed at discrete time points using antibodies against positive and negative pluripotent stem cell markers. The combination of real-time visualization and flow analysis enables the quantitative study of reprogramming at different stages and provides a more accurate comparison of different systems and methods. Real-time, image-based analysis was used for the continuous monitoring of fibroblasts as they are reprogrammed in a feeder-free medium system. The kinetics of colony formation was measured based on confluence in the phase contrast or fluorescence channels after staining with live alkaline phosphatase dye or antibodies against SSEA4 or TRA-1-60. The results indicated that measurement of confluence provides semi-quantitative metrics to monitor the progression of reprogramming.

The protocol presented in this study describes methods for the real-time monitoring of reprogramming progression via the kinetic measurement of positive and negative pluripotent stem cell markers using flow cytometry analysis. The protocol also includes the imaging-based assessment of morphology, and marker or reporter expression during iPSC generation.

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

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

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

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

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

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

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Generation of Multicellular Human Primary Endometrial Organoids

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can also become diseased and cause morbidity and even death. Model systems to study human endometrial biology have been limited to in vitro culture systems of single cell types. In addition, the epithelial cells, one of the major cell types of the endometrium, do not propagate well or retain their physiological traits in culture, and thus our understanding of endometrial biology remains limited. We have generated, for the first time, endometrial organoids that consist of both epithelial and stromal cells of the human endometrium. These organoids do not require any exogenous scaffold materials and specifically organize so that epithelial cells encompass the spheroid-like structure and become polarized with stromal cells in the center that produce and secrete collagen. Estrogen, progesterone and androgen receptors are expressed in the epithelial and stromal cells and treatment with physiological levels of estrogen and testosterone promote the organization of the organoids. This new model system can be used to study normal endometrial biology and disease in ways that were not possible before.

A protocol to generate human primary endometrial organoids that consist of epithelial and stromal cells and retain characteristics of the native endometrial tissue is presented. This protocol describes methods from uterine tissue acquisition to the histologic processing of endometrial organoids.

The human endometrium is one of the most hormonally responsive tissues in the body and is essential for the establishment of pregnancy. This tissue can ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Recapitulating Suckling-to-Weaning Transition In Vitro using Fetal Intestinal Organoids

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to digest solid food. This process is termed suckling-to-weaning transition and results in the replacement of neonatal epithelium with adult epithelium which goes hand in hand with metabolic and morphological adjustments. These complex developmental changes are the result of a genetic program that is intrinsic to the intestinal epithelial cells but can, to some extent, be modulated by extrinsic factors. Prolonged culture of mouse primary intestinal epithelial cells from late fetal period, recapitulates suckling-to-weaning transition in vitro. Here, we describe a detailed protocol for mouse fetal intestinal organoid culture best suited to model this process in vitro. We describe several useful assays designed to monitor the change of intestinal functions associated with suckling-to-weaning transition over time. Additionally, we include an example of an extrinsic factor that is capable to affect suckling-to-weaning transition in vivo, as a representation of modulating the timing of suckling-to-weaning transition in vitro. This in vitro approach can be used to study molecular mechanisms of the suckling-to-weaning transition as well as modulators of this process. Importantly, with respect to animal ethics in research, replacing in vivo models by this in vitro model contributes to refinement of animal experiments and possibly to a reduction in the use of animals to study gut maturation processes.

This protocol describes how to mimic suckling-to-weaning transition in vitro using mouse late fetal intestinal organoids cultured for 30 days.

At the end of the suckling period, many mammalian species undergo major changes in the intestinal epithelium that are associated with the capability to ...

Generation of a Human iPSC-Based Blood-Brain Barrier Chip

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the blood that can disrupt delicate brain function. As such, the BBB is a major obstacle to the delivery of therapeutics to the CNS. Accumulating evidence suggests that the BBB plays a key role in the onset and progression of neurological diseases. Thus, there is a tremendous need for a BBB model that can predict penetration of CNS-targeted drugs as well as elucidate the BBB&#39;s role in health and disease.
We have recently combined organ-on-chip and induced pluripotent stem cell (iPSC) technologies to generate a BBB chip fully personalized to humans. This novel platform displays cellular, molecular, and physiological properties that are suitable for the prediction of drug and molecule transport across the human BBB. Furthermore, using patient-specific BBB chips, we have generated models of neurological disease and demonstrated the potential for personalized predictive medicine applications. Provided here is a detailed protocol demonstrating how to generate iPSC-derived BBB chips, beginning with differentiation of iPSC-derived brain microvascular endothelial cells (iBMECs) and resulting in mixed neural cultures containing neural progenitors, differentiated neurons, and astrocytes. Also described is a procedure for seeding cells into the organ chip and culturing of the BBB chips under controlled laminar flow. Lastly, detailed descriptions of BBB chip analyses are provided, including paracellular permeability assays for assessing drug and molecule permeability as well as immunocytochemical methods for determining the composition of cell types within the chip.

The blood-brain barrier (BBB) is a multicellular neurovascular unit tightly regulating brain homeostasis. By combining human iPSCs and organ-on-chip technologies, we have generated a personalized BBB chip, suitable for disease modeling and CNS drug penetrability predictions. A detailed protocol is described for the generation and operation of the BBB chip.

The blood brain barrier (BBB) is formed by neurovascular units (NVUs) that shield the central nervous system (CNS) from a range of factors found in the ...

Real-Time Imaging of CCL5-Induced Migration of Periosteal Skeletal Stem Cells in Mice

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of therapies to enhance fracture healing.&#160; Periosteal cells rapidly migrate to an injury to supply new chondrocytes and osteoblasts for fracture healing. Traditionally, the efficacy of a cytokine to induce cell migration has only been conducted in vitro by performing a transwell or scratch assay. With advancements in intravital microscopy using multiphoton excitation, it was recently discovered that 1) P-SSCs express the migratory gene CCR5 and 2) treatment with the CCR5 ligand known as CCL5 improves fracture healing and the migration of P-SSCs in response to CCL5. These results have been captured in real-time. Described here is a protocol to visualize P-SSC migration from the calvarial suture skeletal stem cell (SSC) niche towards an injury after treatment with CCL5. The protocol details the construction of a mouse restraint and imaging mount, surgical preparation of the mouse calvaria, induction of a calvaria defect, and acquisition of time-lapse imaging.

This protocol describes the detection of CCL5-mediated periosteal skeletal stem cell migration in real-time using live animal intravital microscopy.

Periosteal skeletal stem cells (P-SSCs) are essential for lifelong bone maintenance and repair, making them an ideal focus for the development of ...

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development and biology is scarce. In particular, not much is known about the tooth's epithelial stem cells and their function. We succeeded in developing a novel organoid model starting from human tooth tissue (i.e., dental follicle, isolated from extracted wisdom teeth). The organoids are robustly and long-term expandable and recapitulate the proposed human tooth epithelial stem cell compartment in terms of marker expression as well as functional activity. In particular, the organoids are capable to unfold an ameloblast differentiation process as occurring in vivo during amelogenesis. This unique organoid model will provide a powerful tool to study not only human tooth development but also dental pathology, and may pave the way toward tooth-regenerative therapy. Replacing lost teeth with a biological tooth based on this new organoid model could be an appealing alternative to the current standard implantation of synthetic materials.

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Teeth are of key importance in life not only for food mastication and speech but also for psychological well-being. Knowledge on human tooth development ...

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...