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 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<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 ...

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

Research

JoVE Journal

Cancer Research

3.1K 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

Study of Viral Vectors in a Three-dimensional Liver Model Repopulated with the Human Hepatocellular Carcinoma Cell Line HepG2

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector systems. The model is obtained by repopulating the extracellular matrix of a decellularized rat liver with a human hepatocyte cell line. The model permits studies in a vascularized 3D cell system, replacing potentially harmful experiments with living animals. Another advantage is the humanized nature of the model, which is closer to human physiology than animal models.
In this study, we demonstrate the transduction of this liver model with a viral vector derived from adeno-associated viruses (AAV vector). The perfusion circuit that supplies the 3D liver model with media provides an easy means to apply the vector. The system permits monitoring of the major metabolic parameters of the liver. For final analysis, tissue samples can be taken to determine the extent of recellularization by histological techniques. Distribution of the virus vector and expression of the delivered transgene can be analyzed by quantitative PCR (qPCR), Western blotting and immunohistochemistry. Numerous applications of the vector model in basic research and in the development of gene therapeutic applications can be envisioned, including the development of novel antiviral therapeutics, cancer research, and the study of viral vectors and their potential side effects.

The recellularized extracellular matrix of a decellularized rat liver can be used as a humanized, three-dimensional ex vivo model to study the distribution and transgene expression of a virus or viral vector.

This protocol describes the generation of a three-dimensional (3D) ex vivo liver model and its application to the study and development of viral vector ...

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...