Name: three-dimensional culture of murine colonic crypts to study intestinal stem cell function ex vivo
Uploaded: 2022-10-11T14:00:00
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. Rea...

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(80), e50611 (2013).</li><li>Sugimoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+adhesion+signals+regulate+the+nuclear+receptor+activity.">Cell adhesion signals regulate the nuclear receptor activity.</a> Proceedings of the National Academy of Sciences. 116 (49), 24600-24609 (2019).</li><li>Mansour, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+30+expression+and+frewuency+of+connexin+heterogeneity+in+astrocyte+gap+junction+plaques+increase+with+age+in+the+rat+retina.">Connexin 30 expression and frewuency of connexin heterogeneity in astrocyte gap junction plaques increase with age in the rat retina.</a> PLoS One. 8 (3), 57038 (2013).</li><li>Miranda, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antioxidants+rescue+photoreceptors+in+rd1+mice:+relationship+with+thiol+metabolism.">Antioxidants rescue photoreceptors in rd1 mice: relationship with thiol metabolism.</a> Free Radical Biology and Medicine. 48 (2), 216-222 (2010).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+ameliorate+oxidative+stress-induced+islet+endothelium+apoptosis+and+functional+impairment+via+Wnt4-&#946;-catenin+signaling.">Mesenchymal stromal cells ameliorate oxidative stress-induced islet endothelium apoptosis and functional impairment via Wnt4-&#946;-catenin signaling.</a> Stem Cell Research and Therapy. 8 (1), 188 (2017).</li><li>Almeqdadi, M., Mana, M., Roper, J., Yilmaz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gut+organoids:+mini-tissues+in+culture+to+study+intestinal+physiology+and+disease.">Gut organoids: mini-tissues in culture to study intestinal physiology and disease.</a> American Journal of Physiology-Cell Physiology. 317 (3), 405-419 (2019).</li></ol>

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 importa...

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.

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		<tr>
			<td>0.09 cubic feet space-saver vacuum desiccator&#160;</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&#8217;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 &#181;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&#160;</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&#38;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&#160;</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 &#181;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 app...

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 ...

This protocol provides a method to study the function of colon stem cells in a manipulatable environment with lower cost in time than animal models. Organoid culture allows the study of stem cell function in an immune-free environment. This allows for pinpointing the effects of a specific cause, such as the knockout of claudin-7.Understanding the mechanisms of stem cell function may shed light on new therapeutic targets for debilitating diseases such as inflammatory bowel disease and colorectal cancer. To begin the isolation of colonic crypts from the euthanized mouse, make an incision of approximately two inches down the midline of the mouse. Then, pin back the skin to expose the abdomen.Cut the colon just below the coecum from the proximal side and above the rectum from the distal side to isolate the colon. Next, remove the adipose tissue attached to the colon using forceps. After pushing out feces from the isolated colon with the flat end of the forceps, cut the tissue open longitudinally.Wash the tissue 10 to 15 times with cold PBS by swirling the tissue around in PBS between washes with forceps. Then using a pair of clean, sharp scissors, cut the tissue into small pieces ranging from approximately three to five millimeters. After isolating the colon from another euthanized mouse, combine all the tissue pieces in a 50-milliliter centrifuge tube containing cold epithelial dissociation media and incubate the colon tissue pieces in epithelial dissociation media for 90 minutes at four degrees Celsius with gentle rocking.After incubation, allow the tissue fragments to sink to the bottom of the tube. Once settled, discard the epithelial dissociation media without disrupting the tissue fragments. Repeat the process when washing the tissue 10 to 15 times with cold PBS.Discard as much PBS as possible during the final wash. Next, add cold crypt dissociation media to the washed colon tissue pieces in a 50-milliliter tube and shake for five to 10 minutes by hand. Under the cell culture hood, filter the media containing the tissue with a 70-micron nylon cell strainer into a fresh-50 milliliter centrifuge tube.After filtration, centrifuge the tube at 200 G for 10 minutes at room temperature, followed by discarding the supernatant without disturbing the pelleted crypts. Resuspend the pellet in three to four milliliters of cold PBS. Pipette 10 microliters of the crypt suspension in a line on a microscope slide.Using the microscope, count the number of full, long crypts to estimate crypt concentration and calculate the appropriate volume of crypts required to plate 10 crypts per microliter in a 96-well plate. Centrifuge an appropriate volume of isolated crypts in a 1.5-milliliter microcentrifuge tube at 200 G for five minutes at four degrees Celsius. Then, remove the supernatant using a 1, 000-microliter pipette without disrupting the pelleted crypts.Then, add 100 microliters of gel matrix to the pelleted crypts without introducing air bubbles. Wait for one to two minutes until the gel matrix is partially solidified. Then, plate 10 microliters of the gel matrix mixed with the crypts in each well of a pre-warmed 96-well culture plate to form a dome shape.Allow 10 to 20 minutes for the gel matrix to be fully set by placing the plate in an incubator at 37 degree Celsius under 5%carbon dioxide. Finally, add 100 microliters of the freshly prepared working solution of L-WRN media supplemented with antibiotics to each well and incubate the plate at 37 degrees Celsius under 5%carbon dioxide for 24 hours. To harvest the organoids, remove old media from the wells by applying vacuum suction and fix the organoids with 4%paraformaldehyde for one hour at room temperature.Then remove 4%paraformaldehyde from the wells by vacuum suction and treat the organoids with 100 microliters of 30%sucrose per well at four degrees Celsius. After 24 hours, remove 30%sucrose from the wells by vacuum suction before adding 10 microliters of PBS to each well. Use a pipette tip to gently scratch the bottom of the well to dissociate the dome-containing organoids.Next, remove the PBS containing the dissociated organoids from the well with a pipette and transfer it to a labeled plastic mold filled 90%with optimum cutting temperature, or OCT, compound. Continue the process until all organoids have been removed from all wells. Add 2-methylbutane to a stainless steel Dewar flask containing dry ice pellets, enough to cover the pellets.Flash-freeze the organoid containing OCT block by steadily holding it above the 2-methylbutane. Finally, store the organoid-containing OCT block at minus 80 degrees Celsius until ready to section. The representative image highlights the successful growth of colonoids from normal crypts containing claudin-7.The crypts containing claudin-7 began to form spheroids by day two, started budding on day five, and continued growing and budding until they were harvested on day nine. In contrast, crypts lacking claudin-7 failed to form proper colonoids. After treatment with 4-hydroxytamoxifen for two to three days, the claudin-7 knockout crypts appeared as circular clumps of cells.Unlike the control, the crypts did not grow into healthy spheroids. Immunofluorescence staining for claudin-7 in the harvested control and the conditional knockout organoids confirmed the successful knockout of claudin-7 in culture. The control colonoids exhibited very little apoptotic signal on day nine.However, claudin-7 knockout colonoids displayed high apoptosis at the same time. Ensure partial solidification prior to plating. Plating before partial solidification will cause the gel matrix to spread and the dome will not be formed, affecting the survival and growth of the crypts.This protocol may be utilized for drug discovery, studying cellular communication, drug metabolism, viability, proliferation, and developing patient-specific personalized treatment. The establishment of organoid culture has revolutionized the study of diseases and personalized medicine.

Research

JoVE Journal

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