Name: three-dimensional culture of murine colonic crypts to study intestinal stem cell function ex vivo
Uploaded: 2022-10-11T14:00:00.000+00:00
Duration: 7 min 46 s
Description: Watch this Scientific Journal Video about Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo at JoVE.com

This study was funded by NIH DK103166.

All animal experiments and procedures were approved by the East Carolina University (ECU) Animal Care and Use Committee (IACUC) and conducted in compliance with guidelines from the National Institutes of Health and ECU on laboratory animal care and use. Inducible, intestinal-specific claudin-7 knockout mice were generated by crossing C57BL6 claudin-7-flox transgenic mice with Villin-CreERT2 mice19. Male and female mice aged 3 months were used in this study.
1. Reagent/equipment preparation
<ol>
	<li>Cool the following reagents/equipment before starting their associated experiments: phosphate buffered saline (PBS) for washing colon tissue during crypt isolation; a rocker/rotator (place in a 4 &#176;C refrigerator) for incubation with epithelial dissociation media.
	<ol>
		<li>Remove the gel matrix (see Table of Materials) from -20 &#176;C and thaw on ice before plating.</li>
		<li>Pre-cool the centrifuge to 4 &#176;C prior to spinning down crypts for plating.</li>
		<li>Cool 0.1% sodium citrate buffer (see Table of Materials) and keep on ice prior to in situ cell death detection.</li>
	</ol>
	</li>
	<li>Warm the following reagents before starting their associated experiments: a 96-well culture plate for 24 h before plating; L-WRN media (see Table of Materials) before adding to plated crypts and before each media change.
	<ol>
		<li>Heat the water bath to 94 &#176;C before staining.</li>
	</ol>
	</li>
</ol>
2. Murine colonic crypt isolation
<ol>
	<li>Prepare the necessary media following the steps below. 
	NOTE: The media volumes are calculated here for the colonic tissue of two mice.
	<ol>
		<li>Prepare epithelial dissociation media: 30 mL of 1x PBS + 400 &#181;L of 0.5 M EDTA + 50 &#181;L of 10 mM Y-27632 dihydrochloride (see Table of Materials). Keep on ice until use.</li>
		<li>Prepare crypt dissociation media: 10 mL of 1x PBS + 10 &#181;L of 10 mM Y-27632 dihydrochloride. Keep on ice until use.</li>
		<li>Supplement L-WRN media: 50 mL of L-WRN media + 47.5 mL of DMEM high glucose with L-Glutamine + 500 &#181;L of L-glutamine + 500 &#181;L of pennicilin/streptomycin + 1,000 &#181;L of B-27 supplement (50x) + 500 &#181;L of N2 supplement (100x) + 50 &#181;L of HEPES (1 M) buffer solution (see Table of Materials).</li>
		<li>Filter the complete (supplemented) L-WRN media and aliquot for storage at -20 &#176;C for up to 3 months. 
		NOTE: Thawed media is stable at 4 &#176;C for up to 2 weeks.</li>
	</ol>
	</li>
	<li>Perform the crypt isolation.
	<ol>
		<li>For general anesthesia, add 1 mL of isoflurane to cotton and place it inside a plastic cassette within a 0.09 cubic feet anesthesia chamber (see Table of Materials). Place the mouse in the anesthesia chamber until breathing halts (approximately 3-5 min) and then perform cervical dislocation20.</li>
		<li>Make an approximately 2 in incision down the midline of the mouse, pinning the back skin to expose the abdomen. Isolate the colon by cutting just below the cecum from the proximal side and above the rectum from the distal side21.</li>
		<li>Using forceps, remove the adipose tissue attached to the colon. Gently push feces out utilizing the flat end of the forceps and cut the tissue open longitudinally.</li>
		<li>Wash the tissue 10-15 times with cold 1x PBS using forceps to &#34;swirl&#34; the tissue around in PBS between washes.</li>
		<li>Using clean, sharp scissors, cut the tissue into small pieces, approximately 3-5 mm in size.</li>
		<li>Repeat the process for the second mouse and combine the tissue pieces in a 50 mL tube containing cold epithelial dissociation media (step 2.1.1).</li>
		<li>Incubate the colon tissue pieces in epithelial dissociation media for 90 min at 4 &#176;C with gentle rocking.</li>
		<li>Allow the tissue fragments to sink to the bottom of the tube, then carefully discard the epithelial dissociation media without disrupting the tissue. Repeat this process when washing the tissue 10-15 times with cold 1x PBS. Discard as much PBS as possible during the final wash.</li>
		<li>Add crypt dissociation media (step 2.1.2) to the 50 mL tube containing colon tissue pieces and continuously shake for 5-10 min by hand. 
		NOTE: The media must become cloudy from the dissociated crypts.</li>
		<li>Under the cell culture hood, filter the tissue and media with a 70 &#181;m nylon cell strainer (see Table of Materials) into a fresh 50 mL tube.</li>
		<li>Centrifuge at 200 x g for 10 min at room temperature and discard the supernatant without disturbing the crypt-containing pellet. 
		NOTE: Depending on the equipment, one may transfer the strained media to a 15 mL tube for centrifuging.</li>
		<li>Resuspend the pellet in ~3-4 mL of cold 1x PBS.</li>
		<li>Pipette 10 &#181;L of the isolated crypts in a line on a microscope slide. Under the microscope, count the number of full, long crypts to estimate crypt concentration per 10 &#181;L.</li>
		<li>Calculate the appropriate volume of crypts to spin down in order to plate 10 crypts/&#181;L in a 96-well plate.</li>
	</ol>
	</li>
</ol>
3. Crypt plating
<ol>
	<li>Centrifuge an appropriate volume of isolated crypts in a 1.5 mL microcentrifuge tube at 200 x g for 5 min at 4 &#176;C.</li>
	<li>Carefully remove the supernatant using a 1,000 &#181;L pipette without disrupting the pelleted crypts.</li>
	<li>Add 100 &#181;L of gel matrix (enough for nine wells) to the pelleted crypts and pipette carefully to avoid introducing air bubbles. 
	NOTE: See the Discussion section for a detailed description of how to mix the gel matrix and crypts. Typically, 100 &#181;L is sufficient for nine wells.</li>
	<li>Allow the gel matrix to partially solidify (~1-2 min).</li>
	<li>Plate 10 &#181;L of the gel matrix mixed with the crypts in each well of a pre-warmed 96-well culture plate to form a dome shape. 
	NOTE: Place the dome in the center of the well. Be careful not to allow the gel matrix to spread to the sides of the well. See the Discussion section for a detailed description on solidification and plating.</li>
	<li>Allow the gel matrix to be fully set for 10-20 min in an incubator at 37 &#176;C with 5% CO2.</li>
	<li>Prepare the final working solution of L-WRN media.
	<ol>
		<li>Add 100 &#181;L pennicilin/streptomycin to a 10 mL aliquot of L-WRN media (see step 2.1.3) (stable for 2 weeks at 4 &#176;C).</li>
		<li>Add supplements to 900 &#181;L of L-WRN media with pennicilin/streptomycin: 0.9 &#181;L of 1 mg/mL EGF + 0.9 &#181;L of 10 mM Y-27632 dihydrochloride. 
		NOTE: The volumes are based upon nine wells. Y-27632 dihydrochloride is only added on day 0 (at the time of plating).</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of media to each well. Be careful not to disrupt the dome.</li>
	<li>Incubate at 37 &#176;C with 5% CO2 for 24 h.</li>
</ol>
4. Creating claudin-7 knockout in culture
<ol>
	<li>Allow the crypts to grow normally in culture for 24 h after plating.</li>
	<li>In a 1.5 mL microcentrifuge tube, add 2.7 &#181;L of 1 mmol/L 4-hydroxytamoxifen (4OH-Tamoxifen) to 900 &#181;L of L-WRN media (final concentration of 3 &#181;mol/L) + 0.9 &#181;L of 1 mg/mL EGF. 
	NOTE: The stock solution of 4OH-Tamoxifen is prepared by mixing powdered 4OH-Tamoxifen (see Table of Materials) with 1x PBS to a concentration of 1 mmol/L.</li>
	<li>Remove old media from the wells via vacuum suction.</li>
	<li>Add 100 &#181;L of 4OH-Tamoxifen-containing media to each well and label them as claudin-7 cKO/4OH-TAM. 
	NOTE: DMSO needs to be added to the control wells. Fresh 4OH-Tamoxifen-containing media must be added every 2 days.</li>
	<li>Incubate at 37 &#176;C until the next media change (~2-3 days).</li>
</ol>
5. Colonic organoid maintenance
NOTE: Media must be changed every 2-3 days. Culture up to 12 days.
<ol>
	<li>Prepare a fresh final working solution of L-WRN media: 900 &#181;L of L-WRN media + 0.9 &#181;L of 1 mg/mL EGF.</li>
	<li>Remove old media from the wells via vacuum suction. Add fresh media to the wells. 
	NOTE: Be careful not to disrupt the dome.</li>
	<li>Incubate at 37 &#176;C until the next media change. 
	&#8203;NOTE: Cultures can be maintained and passaged for the duration desired for the experiment. The cultures for the present study were typically maintained for between 9-12 days, but one may wish to continue further, for 15 or 20 days.</li>
</ol>
6. Harvesting and embedding colonic organoids
<ol>
	<li>Remove old media from the wells via vacuum suction.</li>
	<li>Fix the organoids with 4% paraformaldehyde (PFA) for 1 h at room temperature. 
	NOTE: PFA is a toxic material. Take precautions when using this reagent.</li>
	<li>Remove 4% PFA from the wells via vacuum suction and add 30% sucrose for 24 h at 4 &#176;C.</li>
	<li>Label a plastic mold and fill it to 90% with optimum cutting temperature (OCT) compound (see Table of Materials).</li>
	<li>Remove 30% sucrose from the wells via vacuum suction. Add 10 &#181;L of 1x PBS to each well.</li>
	<li>With a pipette tip, gently scratch the bottom of the well to dissociate the dome containing organoids.</li>
	<li>With a pipette, remove the PBS containing the dissociated organoids and load the liquid into the mold containing OCT. 
	NOTE: Avoid introducing bubbles into the OCT compound.</li>
	<li>Continue this process until all organoids have been removed from all wells.</li>
	<li>In a stainless-steel Dewar flask, add dry ice pellets and 2-methylbutane (enough to cover the dry ice pellets) (see Table of Materials).</li>
	<li>Steadily hold the organoid-containing OCT block above 2-methylbutane to flash freeze.</li>
	<li>Store the organoid-containing OCT block at -80 &#176;C until ready to section (can be stored for up to 1 year).</li>
</ol>
7. Immunofluorescence
<ol>
	<li>Section the organoid-containing OCT block at a 5 &#181;m thickness using a cryostat (see Table of Materials) at -20 &#176;C.</li>
	<li>For each section, check under the microscope to ensure an organoid was captured. When sectioning is complete, store the slides at -80 &#176;C until ready to stain (can be stored for up to 6 months).</li>
	<li>Heat the slides in 10 mM sodium citrate buffer for 10 min at 94 &#176;C. 
	NOTE: The buffer is made by dissolving sodium citrate in deionized water. Adjust the pH to 6 with hydrochloric acid.</li>
	<li>Allow the slides to cool on the benchtop for 20 min. Rinse with distilled water for 5 min.</li>
	<li>Assemble the slides in a staining rack (see Table of Materials) with 0.2% Triton X-100 and incubate with 100 mM glycine for 15 min.</li>
	<li>Rinse three times with 1x PBS for 5 min each. Block with 5% bovine serum albumin (BSA) for 45 min at room temperature.</li>
	<li>Incubate with claudin-7 anti-murine rabbit primary antibody22 (diluted in 1% BSA) overnight at 4 &#176;C. Rinse three times with 1x PBS for 10 min each.</li>
	<li>Incubate with Cy3 anti-rabbit secondary antibody23 (diluted in 1% BSA) for 1 h at room temperature. Rinse three times with 1x PBS for 10 min each.</li>
	<li>Mount the slides with an appropriate mounting medium (see Table of Materials) with DAPI and add a coverslip.</li>
</ol>
8. Cell death detection
<ol>
	<li>Fix the organoids inside the wells with 4% paraformaldehyde for 1 h at room temperature. Rinse with 1x PBS for 5 min.</li>
	<li>Incubate the culture plate on ice with 0.1% Triton X-100 in cold 0.1% sodium citrate buffer for 2 min. Rinse twice with 1x PBS for 5 min each.</li>
	<li>Prepare TUNEL reaction24,25 using TMR Red, an in situcell death detection kit (see Table of Materials). Add 50 &#181;L of TUNEL reaction reagents to each well.</li>
	<li>Incubate in a humidified atmosphere for 1 h at 37 &#176;C in the dark. 
	NOTE: All the remaining steps must be completed in the dark.</li>
	<li>Rinse three times with 1x PBS for 5 min each. Incubate with 1:2,500 Hoechst (diluted in 1x PBS, see Table of Materials) for 3 min.</li>
	<li>Rinse three times with 1x PBS for 5 min each. Utilize the TRITC filter to visualize images on a fluorescent microscope (see Table of Materials).</li>
</ol>

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The authors declare no conflicts of interest.

Organoid culture is an excellent model for studying stem cell function, intestinal physiology, drug discovery, human intestinal diseases, and tissue regeneration and repair7,8,9,10,11,26. While it has many advantages, it can be challenging to establish. Care must be taken in all steps throughout the protocol, but most importantly during the plating stage. When mixing the isolated crypts with a gel matrix, ensure to thoroughly pipette up and down to break up the crypt pellet formed following centrifugation and distribute the crypts evenly throughout the matrix. Concurrently, avoid introducing air bubbles into the matrix while pipetting. In order to do this, pipetting must be done slowly, with the pipette tip toward the bottom of the 1.5 mL tube.
Additionally, the gel matrix must not be fully solidified throughout this process. To prevent premature solidification, mix by careful pipetting, then place the tube on ice, and repeat this process. Once the isolated crypts and gel matrix are sufficiently mixed, allow the gel matrix to solidify partially. This process may take 1-5 min, depending on the type/brand of gel matrix used. It must resemble a gel that will move slightly if the tube is tipped, but should not be too runny that it would spill out if inverted. At this point, one may begin plating 10 &#181;L into the center of each well. The gel matrix must form a 3D dome and should not touch the sides of the well. If the gel matrix spreads and hits the wall of the well, it is not solidified enough; wait until it is sufficiently solidified to form a dome, as the crypts will not survive and grow if the dome is not formed. Once plating is completed properly, and crypts are sufficiently supplemented, as explained above, the organoids are expected to grow without issue.
This protocol establishes a colon organoid system with or without claudin-7 to observe its effects on colonic stem cell survival. While colon organoid culture is an innovative and advantageous system, the model still has limitations. Depending on the type of study, the lack of immune cells and the microbiota in intestinal organoids may be an advantage or disadvantage26. For the present study, it is advantageous to investigate claudin-7&#39;s regulatory role on stem cell functions without the immune component. It was concluded that a certain effect is specifically due to claudin-7, rather than other potential variables such as the immune response that would be present in in vivo animal models. Conversely, this factor may be a limitation for other types of studies. Establishing colon organoid culture may also be more costly and time intensive than traditional 2D cell lines. However, they can mimic the cellular microenvironment of tissues providing in vivo relevance, are much more representative of tissue than 2D cell culture, and are still less costly than animal models4,7. Given intestinal organoid culture&#39;s vast application and enormous potential, this system is likely to become the ideal model in laboratory research worldwide.

Intestinal organoid culture is a three-dimensional (3D) ex vivo system in which stem cells are isolated from the intestinal crypts of primary tissue and plated into a gel matrix1,2. These stem cells are capable of self-renewal, self-organization, and organ functionality2. Organoids mimic the tissue microenvironment and are more similar to in vivo models than two-dimensional (2D) in vitro cell culture models, although less manipulatable than cells3,4. This model eliminates obstacles encountered in 2D models, such as a lack of proper cell-cell adhesions, cell-matrix interactions, and homogenous populations, and also reduces the limitations of animal models, including high costs and lengthy periods of time5. Intestinal organoids-also referred to as colonoids for those grown from colonic crypt-derived stem cells-are essentially mini-organs that contain an epithelium including all cell types that would be present in vivo, as well as a lumen. This model allows manipulation of the system to study many aspects of the intestine, such as the stem cell niche, intestinal physiology, pathophysiology, and gut morphogenesis3,5,6. It also provides a great model for drug discovery, studying human intestinal disorders such as inflammatory bowel disease (IBD) and colorectal cancer, patient-specific personalized treatment development, and studying tissue regeneration4,7,8,9. In addition, the organoid system can also be used to study cellular communication, drug metabolism, viability, proliferation, and response to stimuli7,8. While animal models may be used to test potential therapeutics for intestinal pathologic conditions, they are quite limited, as studying multiple drugs at once poses a challenge. There are more confounding variables in vivo, and associated cost and time are high and long, respectively. On the other hand, the organoid culture system allows for the screening of many therapeutics at once in a shorter time period and also allows for personalized treatment through the use of patient-derived organoid culture4,8. The ability of colonic organoids to mimic tissue organization, microenvironment, and functionality also makes them an excellent model for studying regeneration and tissue repair9. Our lab has established a small intestine organoid culture system to study the effect of claudin-7 on small intestine stem cell functions10. In this study, a large intestinal organoid culture system is established to study stem cells&#39; ability, or lack of ability, to self-renew, differentiate, and proliferate in a conditional claudin-7 knockout (cKO) model.
Claudin-7 is a very important tight junction (TJ) protein that is highly expressed in the intestine and is essential for maintaining TJ function and integrity11. cKO mice suffer from an IBD-like phenotype, exhibiting severe inflammation, ulcerations, epithelial cell sloughing, adenomas, and increased cytokine levels11,12. While it is widely accepted that claudins are vital for epithelial barrier function, new roles for claudins are emerging; they are involved in proliferation, migration, cancer progression, and stem cell function10,12,13,14,15,16,17. It is currently unknown how claudin-7 impacts the stem cell niche and function of colonic stem cells. As the intestine rapidly self-renews approximately every 5-7 days, maintenance of the stem cell niche and proper functioning of the active stem cells is vital18. Here, a system is established to examine the potential regulatory effects of claudin-7 on the colonic stem cell niche.

<table><tbody><tr><td>0.09 cubic feet space-saver vacuum desiccator&amp;nbsp;</td><td>United States Plastic Corp</td><td>78564</td><td>anesthesia chamber</td></tr><tr><td>0.5 M EDTA pH 8.0</td><td>Invitrogen</td><td>AM9261</td><td></td></tr><tr><td>1.5 mL microcentrifuge tubes</td><td>ThermoFisher</td><td>69715</td><td></td></tr><tr><td>15 mL conical centrifuge tubes</td><td>Fisher Scientific</td><td>14-959-53A</td><td></td></tr><tr><td>1x Dulbecco&amp;rsquo;s Phosphate buffered saline</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>2-methylbutane</td><td>Sigma</td><td>277258</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>ThermoFisher</td><td>J61899.AK</td><td></td></tr><tr><td>4-hydroxytamoxifen (4OH-TAM)</td><td>Sigma</td><td>579002</td><td></td></tr><tr><td>50 mL conical centrifuge tubes</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>70 &amp;micro;m nylon cell strainer</td><td>Corning</td><td>352350</td><td></td></tr><tr><td>96 well culture plate</td><td>Greiner Bio-One</td><td>655180</td><td></td></tr><tr><td>B-27 Supplement (50x)</td><td>Gibco</td><td>12587-010</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Fisher Scientific</td><td>BP1605-100</td><td></td></tr><tr><td>Claudin-7 anti-murine rabbit antibody</td><td>Immuno-Biological Laboratories&amp;nbsp;</td><td>18875</td><td></td></tr><tr><td>Cover glass (24 x 50-1.5)</td><td>Fisher Scientific</td><td>12544E</td><td></td></tr><tr><td>Cryomolds</td><td>vwr</td><td>25608-916</td><td></td></tr><tr><td>Cultrex RCF BME, Type 2</td><td>R&amp;amp;D Systems</td><td>3533-005-02</td><td>gel matrix</td></tr><tr><td>Cy3 anti-rabbit antibody</td><td>Jackson Immunoresearch</td><td>111-165-003</td><td></td></tr><tr><td>Dewar Flask</td><td>Thomas Scientific</td><td>1173F61</td><td></td></tr><tr><td>DMEM High Glucose with L-Glutamine</td><td>ATCC</td><td>30-2002</td><td></td></tr><tr><td>EVOS FLoid Imaging System</td><td>ThermoFisher</td><td>4477136</td><td></td></tr><tr><td>Fluoro-Gel II with DAPI</td><td>Electron Microscopy Sciences</td><td>17985-50</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Glycine</td><td>JT Baker</td><td>4059-02</td><td></td></tr><tr><td>HEPES (1 M) Buffer Solution</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Hoechst</td><td>ThermoFisher</td><td>62249</td><td></td></tr><tr><td>In situ cell death detection kit, TMR Red</td><td>Roche</td><td>12156792910</td><td></td></tr><tr><td>Isoflurane</td><td>Pivetal</td><td>07-893-8440</td><td></td></tr><tr><td>L-WRN Media</td><td>Harvard Medical School Gastrointestinal Organoid Derivation and Culture Core</td><td>N/A</td><td></td></tr><tr><td>Mouse surgical kit</td><td>Kent Scientific Corporation</td><td>INSMOUSEKIT</td><td></td></tr><tr><td>Murine EGF</td><td>PeproTech</td><td>315-09-500UG</td><td></td></tr><tr><td>N2 Supplement (100x)</td><td>Gibco</td><td>17502-048</td><td></td></tr><tr><td>Optimum cutting temperature (OCT) compound&amp;nbsp;</td><td>Agar Scientific</td><td>AGR1180</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Sequenza Rack</td><td>vwr</td><td>10129-584</td><td></td></tr><tr><td>Sodium Citrate</td><td>Fisher Scientific</td><td>S-279</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S9378</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>Vacuum filter (0.22 &amp;micro;m; cellulose acetate)</td><td>Corning</td><td>430769</td><td></td></tr><tr><td>Y-27632 dihydrochloride</td><td>Tocris Bioscience</td><td>1254</td><td></td></tr></tbody></table>

three-dimensional culture of murine colonic crypts to study intestinal stem cell function ex vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

In order to examine the regulatory effects of claudin-7 on colon stem cells, colonic crypts were isolated from murine colon tissue as described above and shown in Figure 1A. Once the crypts were isolated from the primary tissue, they were plated in a 3D matrix in a 96-well plate to grow for 11 days (Figure 1). Normal healthy crypts will close the lumen and become spheroids by day 2 and eventually begin budding and forming the various epithelial cell types at approximately day 5 (Figure 1B). Colonoids were allowed to grow until day 11, where they were then harvested for further experiments (Figure 1B). To knockout claudin-7 in culture, the crypts were allowed to grow normally for 24 h. After 24 h, the crypts were treated with 3 &#181;mol/L 4OH-Tamoxifen (TAM) and cultured for 10 additional days. The culture medium containing fresh 4OH-TAM was changed every 2 days. DMSO was used as a vehicle in control wells. Claudin-7 deficient crypts (claudin-7 KO) failed to form proper spheroids and began rapidly dying after 1 day of 4OH-TAM treatment (Figure 1B).
Figure 2A highlights the successful growth of colonoids from normal crypts containing claudin-7 (control) as they progress throughout the 9 days of culture. These crypts began to form spheroids by day 2, started budding on day 5, and continued growing and budding until they were harvested on day 9 (Figure 2A). In contrast, crypts lacking claudin-7 (claudin-7 KO) deteriorated very quickly (Figure 2B). Approximately 2-3 days after treatment with 4OH-TAM, claudin-7 KO crypts had not formed healthy-looking spheroids and appeared only as circular clumps of cells (Figure 2B). Crypts isolated from wild-type mice were treated with 4OH-TAM to confirm there is no toxic effect due to Tamoxifen treatment; these crypts were able to survive and grow normally. To examine claudin-7 deletion and the survival condition of claudin-7 KO colonoids, an immunostaining method and an in situ cell death detection kit were utilized (Figure 3). Immunofluorescent staining for claudin-7 in harvested control and cKO organoids confirmed successful knockout of claudin-7 in culture (Figure 3A). Day 9 control colonoids exhibited very little apoptotic signal (Figure 3B); however, claudin-7 KO colonoids displayed high apoptosis (Figure 3B). Without claudin-7, the stem cells could not survive, self-renew, or differentiate to form colonoids.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64534/64534fig01.jpg" /> 
Figure 1: Schematic representation showing crypt isolation and colonoid growth. (A) Graphical depiction of the crypt isolation process, plating in the 3D matrix, and growth until harvest. (B) Timeline of experiments and colonoid growth in control and claudin-7 KO-derived crypts. <a href="https://www.jove.com/files/ftp_upload/64534/64534fig01large.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/64534/64534fig02.jpg" /> 
Figure 2: Claudin-7 deficient colonoids are unable to survive and grow. (A) Representative images of control/DMSO organoids on day 3 and day 9. (B) Representative images of 4OH-TAM/cKO organoids on day 3 and day 9, n = 10. Scale bars = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64534/64534fig02large.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/64534/64534fig03.jpg" /> 
Figure 3: Claudin-7 deficient colonoids rapidly undergo apoptosis. (A) Claudin-7 staining in day 9 control/DMSO and claudin-7 cKO/4OH-TAM organoids, n = 3. Scale bars = 250 &#181;m. (B) Apoptotic staining in day 9 control and claudin-7 cKO/4OH-TAM colonoids, n = 3. Scale bars = 200 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64534/64534fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo at JoVE.com

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

three-dimensional-culture-murine-colonic-crypts-to-study-intestinal

Nanyang Technological University

Research

JoVE Journal

Cancer Research

3.2K Views.  East Carolina University. The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

Video: Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

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 ...

Developmental Biology

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Medicine

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Immunology and Infection

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Biology

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in the continuity of this barrier can result in inflammatory disorders such as inflammatory bowel disease. One of the limitations in the study of intestinal epithelial biology has been the lack of primary cell culture models, which has obliged researchers to use model cell lines derived from carcinomas. The advent of three dimensional (3D) enteroids has given epithelial biologists a powerful tool to generate primary cell cultures, nevertheless, these structures are embedded in extracellular matrix and lack the maturity characteristic of differentiated intestinal epithelial cells. Several techniques to generate intestinal epithelial monolayers have been published, but most are derived from established 3D enteroids making the process laborious and expensive. Here we describe a protocol to generate primary epithelial colon monolayers directly from murine intestinal crypts. We also detail experimental approaches that can be used with this model such as the generation of confluent cultures on permeable filters, confluent monolayer for scratch wound healing studies and sparse and confluent monolayers for immunofluorescence analysis.

In this protocol, we describe how to generate murine primary epithelial colon monolayers directly from intestinal crypts. We provide experimental approaches to generate confluent monolayers on permeable filters, confluent monolayers for scratch wound healing and biochemical studies, and sparse and confluent monolayers for immunofluorescence analysis.

The intestinal epithelium is comprised of a single layer of cells that act as a barrier between the gut lumen and the interior of the body. Disruption in ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Author Spotlight: The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

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

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

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

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

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

Construction of a 3D Intestinal Mucosa Model with Paraffin Embedding

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the pharmacological screening of potentially beneficial substances, and for toxicity studies on potentially harmful food components. Of relevance, there is a current demand for the development of cell-based in vitro models to substitute animal models. Here, a protocol for a basic, &#8220;healthy tissue&#8221; three-dimensional (3D) intestinal equivalent model using cell lines is presented with the dual benefit of providing both experimental simplicity (standardized and easily repeatable system) and physiological complexity (Caco-2 enterocytes with a supporting immune component of U937 monocytes and L929 fibroblasts). The protocol also includes paraffin embedding for light microscopic evaluation of fixed intestinal equivalents, thereby providing the advantage of analyzing multiple visual parameters from a single experiment. Hematoxylin and eosin (H&#38;E) stained sections showing the Caco-2 columnar cells forming a tight and regular monolayer in control treatments are used to verify the efficacy of the model as an experimental system. Using gluten as a pro-inflammatory food component, parameters analyzed from sections include reduced monolayer thickness, as well as disruption and detachment from the underlying matrix (H&#38;E), decreased tight junction protein expression as shown from occludin staining (quantifiable statistically), and immune-activation of migrating U937 cells as evidenced from the cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. As shown by using lipopolysaccharide to simulate intestinal inflammation, additional parameters that can be measured are increased mucus staining and cytokine expression (such as midkine) that can be extracted from the medium prior to fixation. The basic three-dimensional (3D) intestinal mucosa model and fixed sections can be recommended for inflammatory status and barrier integrity studies with the possibility of analyzing multiple visual quantifiable parameters.

Author Spotlight: Investigating the Effects of Compounds on Intestinal Tissue Using 3D Human Cell Line Models 

Here we describe constructing a basic three-dimensional (3D) intestinal cell line model system and a paraffin embedding protocol for light microscopic evaluation of fixed intestinal equivalents. Staining of selected proteins permits the analysis of multiple visual parameters from a single experiment for potential use in preclinical drug screening studies.

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

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3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

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The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

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Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component