This work is supported by The National Natural Science Foundation of China (31971062, 31900326, and 31601022), The Natural Science Foundation of Jiangsu Province (BK20190043, BK20180838), Research Fund of State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University (KF-GN-202004). The Natural Science Foundation of the Jiangsu Higher Education Institutions of China (19KJB320003), The Livelihood and Technology Program of Suzhou City (SYS2019030), and The Research Innovation Program for College Graduates of Jiangsu Province (KYCX19-1981). This work is also supported by Tang Scholar of Soochow University.

This protocol was approved by the Animal Care and Use Committee of Cambridge-Suda Genomic Resource Center (CAM-SU) at Soochow University.
1. Intestinal Organoid Isolation and Culture
<ol>
	<li>Isolation of intestinal crypts and enteroid culture
	<ol>
		<li>Euthanize an 8-week-old wild-type mouse with CO2 inhalation. Use tissue forceps and fine iris scissors to dissect out approximately 8 cm of ileum.</li>
		<li>Flush out using a syringe with gavage feeding needle with about 40 mL of ice-cold Dulbecco&#39;s phosphate-buffered saline (DPBS), then cut lengthwise with scissors and open the ileum.</li>
		<li>Cut the ileum into small (0.5&#8722;1.0 cm) pieces. Place the pieces in 5 mL of sterile ice-cold DPBS in a 15 mL conical tube, then rock for 5 min on ice.</li>
		<li>Use a pipette controller to aspirate the DPBS and replace it with 10 mL of cold buffer 1 (2 mM EDTA in DPBS). Rock for 30 min on ice.</li>
		<li>Use the pipette controller to aspirate buffer 1 and replace with 10 mL of cold buffer 2 (54.9 mM D-sorbitol, 43.4 mM sucrose in DPBS). Shake for 2&#8722;3 min by hand (~80 shakes/min).</li>
		<li>Take a 20 &#181;L droplet of buffer 2 contents after shaking and inspect under a microscope.</li>
		<li>Filter buffer 2 contents with a 70 &#181;m sterile cell strainer, then collect the filtered buffer with a 50 mL conical tube.</li>
		<li>Pipette 20 &#181;L of filtrate onto the slide to count the crypts. Transfer a sufficient volume of buffer 2 from step 1.1.7 to ensure that there are ~500 crypts/well.</li>
		<li>Spin down at 150 x g for 10 min at 4 &#176;C. Once spinning is finished, carefully aspirate the supernatant.</li>
		<li>Suspend crypts in 50 &#181;L of basement membrane matrix (e.g., Matrigel) per 500 crypts, pipette up and down (being careful to avoid bubbles), then place a 50 &#181;L droplet of basement membrane matrix/crypt mix in the center of one well in a 24 well plate. Incubate for 30 min at 37 &#176;C to polymerize basement membrane matrix.</li>
		<li>After 30 min of polymerization, carefully add 600 &#181;L of minigut media (advanced Dulbecco&#39;s modified Eagle medium [DMEM]/F12, 2 mM L-alanine-L-glutamine, pen/strep [100 units/mL], 10 mM Hepes, N2 supplement [1:100], B27 supplement [1:50] with epidermal growth factor [EGF; 50 ng/mL], Noggin [100 ng/mL], R-spondin [500 ng/mL], and Y27632 [10 &#181;M]) to each well, then return the plate to the 37 &#176;C incubator. Observe under a microscope each day, and change the media every 2&#8722;3 days.</li>
	</ol>
	</li>
	<li>Enteroid passaging 
	NOTE: For long cultures, enteroids should be split approximately each week.
	<ol>
		<li>To passage the enteroids, place the tissue culture plate on ice. Aspirate the media and add 1 mL of cold DPBS to each well. Use a P1000 tip to pipette up and down until no solid basement membrane matrix chunks remain.</li>
		<li>Pass up and down once through a 1 mL insulin syringe (27 G) and into a 15 mL conical tube. Spin down for 5 min at 150 x g at 4 &#176;C.</li>
		<li>Use pipette to remove DPBS. Resuspend in 50 &#181;L of basement membrane matrix/well.</li>
		<li>Place the plate in a 37 &#176;C incubator for 30 min to allow basement membrane matrix to polymerize. Overlay each well with 600 &#181;L of ENR media (minigut media, 50 ng/mL EGF, 100 ng/mL Noggin, 500 ng/mL R-spondin), then return the plate to the incubator.</li>
	</ol>
	</li>
</ol>
2. Flow Cytometry Analysis of EdU-positive Cells in Enteroids
NOTE: Figure 1 shows the workflow for flow cytometry analysis of EdU-positive cells in enteroids.
<ol>
	<li>Incubation of enteroids with EdU
	<ol>
		<li>Grow enteroids from step 1.2.4 for 5&#8211;7 days. Passage enteroids into a new 24 well plate in a splitting ratio of 1:2. Add 600 &#181;L of ENR medium to each well. Incubate enteroids for 4&#8722;5 days in a 37 &#176;C incubator.</li>
		<li>Set the control group (untreated enteroids) and experimental group (enteroids treated with 5 ng/mL interleukin 22 [IL-22]). Prepare at least three replicates for each group.</li>
		<li>Add EdU to the ENR medium to prepare EdU medium with a concentration of 50 &#956;M. Add 600 &#956;L of EdU medium to each well and incubate for 2 h in a 37 &#176;C incubator. Set one well of enteroids without EdU treatment as a negative control for background subtraction.</li>
	</ol>
	</li>
	<li>Harvesting of enteroids from basement membrane matrix
	<ol>
		<li>Use a pipette controller to aspirate EdU medium and wash 1x with DPBS. Add 1 mL of DPBS.</li>
		<li>Use P1000 pipette tip and pipette up and down until no solid basement membrane matrix chunks remain. Transfer to a 15 mL tube and spin down at 300 x g for 5 min. Discard the supernatant.</li>
		<li>Add 500 &#956;L of cell-dissociation enzymes (Table of Materials) and incubate for 15 min at 37 &#176;C. Use P200 pipette tip and pipette up and down to break enteroids into single cells.</li>
		<li>Add 3 mL of DMEM medium containing 10% fetal bovine serum (FBS) and repeatedly pipette with a P1000 pipette tip.</li>
		<li>Spin down at 300 x g for 5 min and aspirate the supernatant medium. Resuspend the cells with 1 mL of DPBS.</li>
	</ol>
	</li>
	<li>Fixation and permeabilization of cells
	<ol>
		<li>Transfer the cell suspension to a 1.5 mL tube, spin down at 300 x g for 5 min, and discard the supernatant.</li>
		<li>Resuspend cells with 1 mL of 4% paraformaldehyde (PFA), fix for 15 min at room temperature (RT), and spin down at 300 x g for 5 min. Aspirate the supernatant with a pipette tip, wash 1x with DPBS, and spin down to remove the supernatant.</li>
		<li>Resuspend cells with 1 mL of 0.5% nonionic surfactant. Incubate for 10 min at RT.</li>
		<li>Spin down at 300 x g for 5 min and aspirate the supernatant. Wash 1x with DPBS and spin down to remove the supernatant.</li>
	</ol>
	</li>
	<li>Detection of EdU
	<ol>
		<li>Prepare stock solutions for each component in advance: 1) working solution of the Alexa Fluor azide, 2) working solution of 1x EdU reaction buffer, and 3) 10x stock solution of EdU buffer additive.</li>
		<li>Prepare 1x EdU buffer additive by diluting the 10x solution 1:10 in deionized water. Prepare this solution fresh.</li>
		<li>Prepare reaction cocktail according to Table 1. 
		NOTE: Add the ingredients in the order listed in the table. Use the reaction cocktail within 15 min of preparation.</li>
		<li>Add 100 &#956;L of reaction cocktails to each 1.5 mL tube. Resuspend the cells, protect from light, incubate for 30 min at RT.</li>
		<li>Centrifuge at 300 x g for 5 min and aspirate the reaction solution with pipette tip gently.</li>
		<li>Add 0.5% nonionic surfactant penetrant to each tube to wash 1x at RT. Centrifuge at 300 x g for 5 min, aspirate the supernatant with pipette tip gently, and resuspend the cells in 1 mL of DPBS.</li>
	</ol>
	</li>
	<li>Analysis of cells by flow cytometry
	<ol>
		<li>Filter the resuspended cells with a 40 &#956;m strainer, then collect the filtered cells with a 15 mL conical tube. Perform FACS on-machine detection as soon as possible. 
		NOTE: If conditions are limited, the cell stained samples should be stored in the dark at 4 &#176;C and flow-tested within 3 days.</li>
		<li>Select the appropriate channel (according to the type of Alexa Fluor azide in the EdU kit; here, red channel) and voltage (here, ~150&#8211;350 V) for flow cytometry analysis.</li>
		<li>Gating strategy
		<ol>
			<li>Draw an FSC-A (x-axis) vs. SSC-A (y-axis) pseudocolor plot and distribute most of the cells to the visible range of the dot map by adjusting the voltage. Select the cell population (R1) and exclude the cell debris in the bottom left corner.</li>
			<li>From the R1 cell population, establish an FSC-A (x-axis) vs. FSC-H (y-axis) pseudocolor plot, and set the gate to select single cells (R2) to exclude cell clumps.</li>
			<li>From the R2 cell population, establish the fluorescence intensity (x-axis) vs. cell number (y-axis) plot, and use the negative control to set the gate. The region of the fluorescent signal is a positive cell region (R3). Compare the ratio of EdU-positive cells (R3) between the experimental and control groups.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Flow Cytometry Analysis of PI-positive Cells in Enteroids
NOTE: Figure 2 shows the workflow for flow cytometry analysis of PI-positive cells in enteroids.
<ol>
	<li>Incubation of enteroid cells with PI
	<ol>
		<li>Grow enteroids from step 1.2.4 for 5&#8211;7 days. Passage enteroids into a new 24 well plate in a splitting ratio of 1:2. Add 600 &#181;L of ENR medium to each well. Incubate enteroids for 4&#8722;5 days in a 37 &#176;C incubator.</li>
		<li>Set the control group (untreated) and experimental group (IL-22 treated), using at least three replicates for each group.</li>
		<li>Add PI to the ENR medium to prepare PI medium with a concentration of 3 &#956;M.</li>
		<li>Add 600 &#956;L of PI medium to each well and incubate for 30 min in a 37 &#176;C incubator. Set one well of enteroids without PI treatment as a negative control for background subtraction.</li>
	</ol>
	</li>
	<li>Harvest enteroids from basement membrane matrix following steps 2.2.1&#8722;2.2.5.</li>
	<li>Analysis of cells by flow cytometry
	<ol>
		<li>Filter the resuspended cells with a 40 &#956;m strainer and collect the filtered cells with a 15 mL conical tube.</li>
		<li>Perform FACS on-machine detection as soon as possible. 
		NOTE: If conditions are limited, the cell stained samples should be stored in the dark at 4 &#176;C and flow-tested within 3 days.</li>
		<li>Select the appropriate channel (here, red channel) and voltage (here, ~150&#8211;350 V) for flow cytometry analysis.</li>
		<li>Use the same gating strategy as described in section 2.5.3.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Odenwald, M. A., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intestinal+epithelial+barrier:+a+therapeutic+target.">The intestinal epithelial barrier: a therapeutic target.</a> Nature reviews. Gastroenterology &amp; Hepatology. 14 (1), 9-21 (2017).</li><li>Buckley, A., Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Biology+of+Tight+Junction+Barrier+Regulation+and+Mucosal+Disease.">Cell Biology of Tight Junction Barrier Regulation and Mucosal Disease.</a> Cold Spring Harbor Perspectives in Biology. 10 (1), (2018).</li><li>Gehart, H., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tales+from+the+crypt:+new+insights+into+intestinal+stem+cells.">Tales from the crypt: new insights into intestinal stem cells.</a> Nature reviews. Gastroenterology &amp; Hepatology. 16 (1), 19-34 (2019).</li><li>Vancamelbeke, M., Vermeire, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intestinal+barrier:+a+fundamental+role+in+health+and+disease.">The intestinal barrier: a fundamental role in health and disease.</a> Expert Review of Gastroenterology &amp; Hepatology. 11 (9), 821-834 (2017).</li><li>Garcia-Carbonell, R., Yao, S. J., Das, S., Guma, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysregulation+of+Intestinal+Epithelial+Cell+RIPK+Pathways+Promotes+Chronic+Inflammation+in+the+IBD+Gut.">Dysregulation of Intestinal Epithelial Cell RIPK Pathways Promotes Chronic Inflammation in the IBD Gut.</a> Frontiers in Immunology. 10, 1094 (2019).</li><li>Li, B. R., 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=In+Vitro+and+In+Vivo+Approaches+to+Determine+Intestinal+Epithelial+Cell+Permeability.">In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability.</a> Journal of Visualized Experiments. (140), e57032 (2018).</li><li>Turner, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+mucosal+barrier+function+in+health+and+disease.">Intestinal mucosal barrier function in health and disease.</a> Nature Reviews Immunology. 9 (11), 799-809 (2009).</li><li>Graham, W. V., 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=Intracellular+MLCK1+diversion+reverses+barrier+loss+to+restore+mucosal+homeostasis.">Intracellular MLCK1 diversion reverses barrier loss to restore mucosal homeostasis.</a> Nature Medicine. 25 (4), 690-700 (2019).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Sato, T., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growing+self-organizing+mini-guts+from+a+single+intestinal+stem+cell:+mechanism+and+applications.">Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications.</a> Science. 340 (6137), 1190-1194 (2013).</li><li>Friedrich, M., Pohin, M., Powrie, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+Networks+in+the+Pathophysiology+of+Inflammatory+Bowel+Disease.">Cytokine Networks in the Pathophysiology of Inflammatory Bowel Disease.</a> Immunity. 50 (4), 992-1006 (2019).</li><li>Zha, J. 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=Interleukin+22+Expands+Transit-Amplifying+Cells+While+Depleting+Lgr5+Stem+Cells+via+Inhibition+of+Wnt+and+Notch+Signaling.">Interleukin 22 Expands Transit-Amplifying Cells While Depleting Lgr5 Stem Cells via Inhibition of Wnt and Notch Signaling.</a> Cellular and Molecular Gastroenterology and Hepatology. 7 (2), 255-274 (2019).</li></ol>

The authors have nothing to disclose.

This protocol details the steps necessary for the culture of enteroids in vitro and quantification of EdU- and PI-positive cells in the enteroids by flow cytometry. There are several advantages of this strategy. First, EdU labelling is used to detect proliferating cells in enteroids. Compared with traditional BrdU assay, EdU labelling method is faster, more sensitive, and more accurate. EdU is very similar to thymine (T), which replaces thymine in DNA synthesis during cell division. Compared to the BrdU antibody, EdU is easier to diffuse into the cell, and the detection of EdU does not require DNA denaturation and an antigen-antibody reaction. Secondly, flow cytometry analysis can quickly and accurately quantify the proliferating (EdU+) and dead (PI+) cells in enteroids.
To successfully perform the entire procedure, there are critical aspects to be considered. First, it is important to culture enteroids under sufficient conditions. Well-grown enteroids should have plenty of buds, which contain proliferative cells. Second, it is important to split enteroids in a timely manner. Debris accumulated in the lumen contains dead cells, which can be stained with PI. This is detrimental for the following flow cytometry analysis. Third, due to the multiple steps (i.e., cell staining, cell fixation, membrane rupture, and centrifugation), cells can be easily lost during the procedure. Thus, it is important to collect a sufficient number of enteroids. It is important to combine 2&#8722;3 wells (in a 24 well plate) of enteroids before fixation. Lastly, it is critical to add ingredients to the buffer in order to detect EdU, otherwise the reaction will not proceed optimally.
In summary, the protocol details steps for the culturing of mouse enteroids in vitro and the quantification of proliferating and dead cells by flow cytometry. Enteroids are useful tools for disease modelling and therapeutic drug discovery. This protocol helps exploration of the effects of inflammatory cytokines, pathogen, and drugs on cell proliferation and cell survival in enteroid culture models.

A fundamental function of intestinal epithelial cells is to protect the entry of luminal contents such as pathogenic bacteria and toxins1,2. To perform such a function, intestinal stem cells continuously proliferate and differentiate into a variety of epithelial cells, including enterocytes and secretory cells, which form a barrier by forming tight connections3. The rapid renewal of intestinal epithelial cells requires strict coordination of cell proliferation, cell differentiation, and cell death4,5. Reduced cell proliferation or excessive cell death leads to epithelial damage and compromised barrier function1,6. Dysfunction of intestinal barrier has been associated with inflammatory bowel diseases7,8.
A method to culture intestinal crypts has been previously developed. Using this technique, isolated mouse crypts grow into intestinal organoids (enteroids), which have crypt-villus like structures and contain all intestinal epithelial cell lineage9,10. 5-Ethynyl-2'-deoxyuridine (EdU) is a thymidine analog that is capable of replacing thymine (T) in DNA that is undergoing replication during cell proliferation. The proliferative cells can be quickly and accurately labeled by EdU staining. Propidium iodide (PI) is an analog of ethidium bromide that releases red fluorescence upon insertion into double-stranded DNA. PI specifically detects dead cells, since it only passes through the damaged cell membrane.
In this protocol, we first describe how to isolate crypts from the murine small intestine then culture them as enteroids in vitro. We then describe how to analyze the proliferative and dead cells in enteroids by EdU and PI incorporation and flow cytometry.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 ml centrifuge tube</td>
			<td>Corning</td>
			<td>430791</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G gavage needle</td>
			<td>VWR</td>
			<td>20068-608</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Nunc</td>
			<td>142475</td>
			<td></td>
		</tr>
		<tr>
			<td>40 mm sterile cell strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>70 mm sterile cell strainer</td>
			<td>BD</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>GIBCO</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Attune NxT Acoustic Focusing Cytometer</td>
			<td>Invitrogen</td>
			<td>A24863</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement</td>
			<td>GIBCO</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer 1</td>
			<td></td>
			<td></td>
			<td>2 mM EDTA in DPBS</td>
		</tr>
		<tr>
			<td>Buffer 2</td>
			<td></td>
			<td></td>
			<td>54.9 mM D-sorbitol, 43.4 mM sucrose in DPBS</td>
		</tr>
		<tr>
			<td>C57/B6 mice</td>
			<td>Nanjing Biomedical Research Institute of Nanjing University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-dissociation enzymes (TrypLE)</td>
			<td>Life technologies</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT Plus EdU Alexa Fluor 594 Imaging Kit</td>
			<td>Life technologies</td>
			<td>C10639</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Panasonic</td>
			<td>MCO-18AC</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>GIBCO</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>BBI</td>
			<td>SB0491</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>BBI</td>
			<td>EB0185</td>
			<td></td>
		</tr>
		<tr>
			<td>ENR media</td>
			<td></td>
			<td></td>
			<td>Minigut media, 50 ng/ml EGF, 100 ng/ml Noggin, 500 ng/ml R-spondin</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine Iris Scissors</td>
			<td>Tansoole</td>
			<td>2037454</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX Supplement</td>
			<td>GIBCO</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Serum</td>
			<td>Life technologies</td>
			<td>16210-064</td>
			<td></td>
		</tr>
		<tr>
			<td>HDMEM</td>
			<td>Hyclone</td>
			<td>SH30243.01B</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Minigut media</td>
			<td></td>
			<td></td>
			<td>Advanced DMEM/F12, 2 mM Glutamax, Penn/Strep (100 units/ml), 10 mM Hepes, N2 supplement (1:100), B27 supplement (1:50)</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>R&#38;D</td>
			<td>AR009</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonionic surfactant (Triton X)</td>
			<td>BBI</td>
			<td>TB0198-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating Scissor (12.5 cm)</td>
			<td>Tansoole</td>
			<td>2025785</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>sigma</td>
			<td>158127-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Penn/Strep</td>
			<td>Invitrogen</td>
			<td>15140-148</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>Nikon</td>
			<td>TS1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4170-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant EGF</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse Noggin</td>
			<td>PeproTech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse R-Spondin 1</td>
			<td>R&#38;D</td>
			<td>3474-RS-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine IL-22</td>
			<td>PeproTech</td>
			<td>210-22-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>BBI</td>
			<td>SB0498</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Forceps</td>
			<td>Tansoole</td>
			<td>2026704</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 2HC1</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantification of proliferative and dead cells in enteroids

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. Epithelial barrier function requires the integrity of intestinal epithelial cells. While epithelial cell proliferation maintains a continuous layer of cells that forms a barrier, epithelial damage leads to barrier dysfunction. As a result, luminal contents can across the intestinal barrier via an unrestricted pathway. Dysfunction of intestinal barrier has been associated with many intestinal diseases, such as inflammatory bowel disease. Isolated mouse intestinal crypts can be cultured and maintained as crypt-villus-like structures, which are termed intestinal organoids or &ldquo;enteroids&rdquo;. Enteroids are ideal to study the proliferation and cell death of intestinal epithelial cells in vitro. In this protocol, we describe a simple method to quantify the number of proliferative and dead cells in cultured enteroids. 5-ethynyl-2&rsquo;-deoxyuridine (EdU) and propidium iodide are used to label proliferating and dead cells in enteroids, and the proportion of proliferating and dead cells are then analyzed by flow cytometry. This is a useful tool to test the effects of drug treatment on intestinal epithelial cell proliferation and cell survival.

Small intestinal crypts were isolated and cultured as enteroids in basement membrane matrix. Enteroids started to form buds 2 days after isolation. On day 6, enteroids had many buds with lots of debris (dead cells) in the lumen. Enteroids were ready to be passaged at this stage (Figure 3).
Numerous studies have shown that inflammatory cytokines are essential for the maintenance of intestinal epithelial homeostasis. Abnormal expression of inflammatory cytokines is closely associated with the occurrence of inflammatory bowel diseases11. For instance, our previous study showed that IL-22 promotes proliferation of transit-amplifying cells but also depletes Lgr5+ stem cells12.
Enteroids were treated with IL-22 for 3 days, after which the synthetic DNA was labeled with EdU to indicate cell proliferation. IL-22-treated enteroids displayed an increased number of EdU+ cells (Figure 4A). IL-22 increased proliferating cells from 40.1% to 83.5% as analyzed by flow cytometry (Figure 4B). IL-22 treatment also increased the cell death in enteroids, indicated by PI staining (Figure 5A). IL-22 increased dead cells from 4.9% to 16.2% as analyzed by flow cytometry (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60501/60501fig01.jpg" /> 
Figure 1: Workflow diagram for flow cytometry analysis of EdU-positive cells in enteroids. <a href="https://www.jove.com/files/ftp_upload/60501/60501fig01large.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/60501/60501fig02.jpg" /> 
Figure 2: Workflow diagram for flow cytometry analysis of PI-positive cells in enteroids. <a href="https://www.jove.com/files/ftp_upload/60501/60501fig02large.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/60501/60501fig03.jpg" /> 
Figure 3: Brightfield images of enteroids at 2, 4, and 6 days post-crypt isolation.&#160;Scale bar = 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/60501/60501fig3alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60501/60501fig04.jpg" /> 
Figure 4: IL-22 increases enteroids proliferation.&#160;(A) Enteroids were cultured in medium without or with IL-22 (5 ng/mL) for 3 days and incubated with EdU (red) for 1 h. Scale bar = 100 &#956;m. (B) Flow cytometry data from enteroids cultured without (left) or with (right) IL-22. Data are representative of three separate experiments. <a href="https://www.jove.com/files/ftp_upload/60501/60501fig4alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60501/60501fig05.jpg" /> 
Figure 5: IL-22 promotes cell death in enteroids.&#160;(A) Enteroids were cultured in medium without or with IL-22 (5 ng/mL) for 3 days and stained with propidium iodide (red). Scale bar = 100 &#956;m. (B) Flow cytometry data from enteroids cultured without (left) or with (right) IL-22. Data are representative of three separate experiments. <a href="https://www.jove.com/files/ftp_upload/60501/60501fig5alarge.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td rowspan="2">Reaction components</td>
			<td colspan="6">Number of wells of 24-well plates</td>
		</tr>
		<tr>
			<td>1</td>
			<td>2</td>
			<td>5</td>
			<td>10</td>
			<td>20</td>
			<td>50</td>
		</tr>
		<tr>
			<td>1x Reaction buffer</td>
			<td>86 &#956;L</td>
			<td>172 &#956;L</td>
			<td>430 &#956;L</td>
			<td>860 &#956;L</td>
			<td>1.72 &#956;L</td>
			<td>4.3 &#956;L</td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>4 &#956;L</td>
			<td>8 &#956;L</td>
			<td>20 &#956;L</td>
			<td>40 &#956;L</td>
			<td>80 &#956;L</td>
			<td>200 &#956;L</td>
		</tr>
		<tr>
			<td>Alexa Fluor azide</td>
			<td>0.25 &#956;L</td>
			<td>0.5 &#956;L</td>
			<td>1.25 &#956;L</td>
			<td>2.5 &#956;L</td>
			<td>5 &#956;L</td>
			<td>12.5 &#956;L</td>
		</tr>
		<tr>
			<td>Reaction buffer additive</td>
			<td>10 &#956;L</td>
			<td>20 &#956;L</td>
			<td>50 &#956;L</td>
			<td>100 &#956;L</td>
			<td>200 &#956;L</td>
			<td>500 &#956;L</td>
		</tr>
		<tr>
			<td>Total volume</td>
			<td>100 &#956;L</td>
			<td>200 &#956;L</td>
			<td>500 &#956;L</td>
			<td>1 &#956;L</td>
			<td>2 &#956;L</td>
			<td>5 &#956;L</td>
		</tr>
		<tr>
			<td colspan="7">NOTE: Add the ingredients in the order listed in the table.</td>
		</tr>
	</tbody>
</table>
Table 1: EdU reaction cocktail.

Watch this Scientific Journal Video about Quantification of Proliferative and Dead Cells in Enteroids at JoVE.com

Quantification of Proliferative and Dead Cells in Enteroids

The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

The intestinal epithelium acts as a barrier that prevents luminal contents, such as pathogenic microbiota and toxins, from entering the rest of the body. ...

quantification-of-proliferative-and-dead-cells-in-enteroids

Nanyang Technological University

Research

JoVE Journal

Biology

8.3K Views.  The First Affiliated Hospital of Soochow University. The presented protocol uses flow cytometry to quantify the number of proliferating and dead cells in cultured mouse enteroids. This method is helpful to evaluate the effects of drug treatment on organoid proliferation and survival.

Video: Quantification of Proliferative and Dead Cells in Enteroids

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

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

Bioengineering

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...

Immunology and Infection

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to establish human enteroid ex vivo culture systems are important to model intestinal pathophysiology and to study the particular cellular responses involved. In recent years, enteroids from mice and humans are being cultured, passaged, and banked away for future use in several laboratories across the world. This enteroid platform can be used to test the effects of various treatments and drugs and what effects are exerted on different cell types in the intestine. Here, a protocol for establishing primary stem cell-derived small intestinal enteroids derived from neonatal mice and premature human intestine is provided. Moreover, this enteroid culture system was utilized to test the effects of species-specific breast milk. Mouse breast milk can be obtained efficiently using a modified human breast pump and expressed mouse milk can then be used for further research experiments. We now demonstrate the effects of expressed mouse, human, and donor breast milk on the growth and proliferation of enteroids derived from neonatal mice or premature human small intestine.

Breast Milk Enhances Growth of Enteroids: An Ex Vivo Model of Cell Proliferation

This protocol describes how to establish an enteroid culture system from neonatal mouse or premature human intestine as well as an efficient method to collect milk from mice.

Human small intestinal enteroids are derived from the crypts and when grown in a stem cell niche contain all of the epithelial cell types. The ability to ...

Establishment of a 2D Monolayer from 3D Bovine Ileal Enteroid Culture

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

Author Spotlight: Advanced Enteroid Model for Studying Host-Pathogen Interactions 

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have recently emerged as a promising system to model gastrointestinal pathologies. However, currently utilized methods for enteroid manipulation either lack access to the apical surface of the epithelium (three-dimensional [3D]) or are time-consuming and resource-intensive (two-dimensional [2D] monolayers). These methods often require additional steps, such as microinjection, for the model to become physiologically translatable. Here, we describe a physiologically relevant and inexpensive protocol for studying NEC in vitro by reversing enteroid polarity, resulting in the apical surface facing outward (apical-out). An immunofluorescent staining protocol to examine enteroid barrier integrity and junctional protein expression following exposure to tumor necrosis factor-alpha (TNF-&#945;) or lipopolysaccharide (LPS) under normoxic or hypoxic conditions is also provided. The viability of 3D apical-out enteroids exposed to normoxic or hypoxic LPS or TNF-&#945; for 24 h is also evaluated. Enteroids exposed to either LPS or TNF-&#945;, in combination with hypoxia, exhibited disruption of epithelial architecture, a loss of adherens junction protein expression, and a reduction in cell viability. This protocol describes a new apical-out NEC-in-a-dish model which presents a physiologically relevant and cost-effective platform to identify potential epithelial targets for NEC therapies and study the preterm intestinal response to therapeutics.

In Vitro Apical-Out Enteroid Model of Necrotizing Enterocolitis

This protocol describes an apical-out necrotizing enterocolitis (NEC)-in-a-dish model utilizing small intestinal enteroids with reversed polarity, allowing access to the apical surface. We provide an immunofluorescent staining protocol to detect NEC-related epithelial disruption and a method to determine the viability of apical-out enteroids subjected to the NEC-in-a-dish protocol.

Necrotizing enterocolitis (NEC) is a devastating disease affecting preterm infants, characterized by intestinal inflammation and necrosis. Enteroids have ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit ...

A Necrotizing Enterocolitis Model for Mechanistic Studies and Drug Discovery

Microfluidic Model of Necrotizing Enterocolitis Incorporating Human Neonatal Intestinal Enteroids and a Dysbiotic Microbiome

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, which remains incompletely understood. The pathophysiology of NEC includes disruption of intestinal tight junctions, increased gut barrier permeability, epithelial cell death, microbial dysbiosis, and dysregulated inflammation. Traditional tools to study NEC include animal models, cell lines, and human or mouse intestinal organoids. While studies using those model systems have improved the field&#39;s understanding of disease pathophysiology, their ability to recapitulate the complexity of human NEC is limited. An improved in vitro model of NEC using microfluidic technology, named NEC-on-a-chip, has now been developed. The NEC-on-a-chip model consists of a microfluidic device seeded with intestinal enteroids derived from a preterm neonate, co-cultured with human endothelial cells and the microbiome from an infant with severe NEC. This model is a valuable tool for mechanistic studies into the pathophysiology of NEC and a new resource for drug discovery testing for neonatal intestinal diseases. In this manuscript, a detailed description of the NEC-on-a-chip model will be provided.

Author Spotlight: Enhancing Understanding and Treatment Strategies with the NEC-on-a-Chip Model 

This protocol describes an in vitro model of necrotizing enterocolitis (NEC), which can be used for mechanistic studies into disease pathogenesis. It features a microfluidic chip seeded with intestinal enteroids derived from the human neonatal intestine, endothelial cells, and the intestinal microbiome of a neonate with severe NEC.

Necrotizing enterocolitis (NEC) is a severe and potentially fatal intestinal disease that has been difficult to study due to its complex pathogenesis, ...

Quantifying Cell Death in Human Colonoids in Response to Cytotoxic Perturbagens

Quantification of Cytokine-Induced Cell Death in Human Colonic Organoids Using Live Fluorescence Microscopy

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease (CD). This can contribute to defects in intestinal barrier function, exacerbation of inflammation, and disease immunopathogenesis. Cytokines and death receptor ligands are partially responsible for this increase in IEC death. IBD-relevant cytokines, such as TNF-&#945; and IFN-&#947;, are cytotoxic to IECs both independently and in combination. This protocol describes a simple and practical assay to quantify cytokine-induced cytotoxicity in CD patient-derived colonic organoids using a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software. We also demonstrate how to use the Bliss independence mathematical model to calculate a coefficient of perturbagen interaction (CPI) based on organoid cytotoxicity. The CPI can be used to determine if interactions between cytokine combinations or other types of perturbagens are antagonistic, additive, or synergistic. This protocol can be implemented to investigate the cytotoxic activity of cytokines and other perturbagens using patient-derived colonic organoids.

Author Spotlight: Understanding Cytokine-Induced Cell Death in Intestinal Epithelial Cells Using Human Organoids

This protocol describes a simple and cost-effective method to investigate and quantify cell death in human colonic organoids in response to cytotoxic perturbagens such as cytokines. The approach employs a fluorescent cell death dye (SYTOX Green Nucleic Acid Stain), live fluorescence microscopy, and open-source image analysis software to quantify single-organoid responses to cytotoxic stimuli.

Intestinal epithelial cell (IEC) death is increased in patients with inflammatory bowel diseases (IBD) such as ulcerative colitis (UC) and Crohn's disease ...

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. Traditionally, research in this area has relied on animal experiments, but there's a growing demand for alternative methods that adhere to the 3R principles-replace, reduce, and refine. Porcine organoids have emerged as a promising tool, offering a more accurate in vitro replication of the in vivo conditions than traditional cell models. One major challenge with intestinal organoids is their inward-facing apical surface and outward-facing basolateral surface. This limitation can be overcome by creating two-dimensional (2D) organoid layers on transwell inserts (from here on referred to as insert(s)), providing access to both surfaces. In this study, we successfully developed two-dimensional cultures of porcine jejunum and colon organoids. The cultivation process involves two key phases: First, the formation of a cellular monolayer, followed by the differentiation of the cells using tailored media. Cellular growth is tracked by measuring transepithelial electrical resistance, which stabilizes by day 8 for colon organoids and day 16 for jejunum organoids. After a 2-day differentiation phase, the epithelium is ready for analysis. To quantify and track active electrogenic transport processes, such as chloride secretion, we employ the Ussing chamber technique. This method allows for real-time measurement and detailed characterization of epithelial transport processes. This innovative in vitro model, combined with established techniques like the Ussing chamber, provides a robust platform for physiologically characterizing the porcine GIT within the 3R framework. It also opens opportunities for investigating pathophysiological mechanisms and developing potential therapeutic strategies.

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

The gastrointestinal tract (GIT) serves both in the digestion of food and the uptake of nutrients but also as a protective barrier against pathogens. ...