This work was supported by Grants-in-Aid for Scientific Research (C) to T.T. (grant numbers JP17K07495 and JP20K06751). We thank Prof. Mineko Kengaku for the use of equipment for the long-term time-lapse imaging (LCV100; Olympus).

All mouse experiments were approved by the Suntory Animal Ethics Committee (APRV000561), and all animals were maintained in accordance with the committee guidelines for the care and use of laboratory animals. A standard WT strain of Mus musculus (C57BL6/J) was used. Both male and female mice from 10 weeks to 20 weeks of age were used. The mice were euthanized with CO2 asphyxiation.
1. Isolation of the small intestine
<ol>
	<li>Excise the small intestines, including the duodenum and the proximal half of the jejunum, with laboratory scissors.</li>
	<li>Transfer the tissue into a Petri dish, and flush the small intestines with 5 mL of cold PBS-ABx (PBS + penicillin-streptomycin [1%] + gentamicin [0.5%]) in a 5 mL syringe to clear the luminal content.</li>
	<li>Cut the tissue open lengthwise with laboratory scissors, and manually wash with cold PBS-ABx while shaking. 
	NOTE: By scraping out the villi with a slide, villus contamination can be reduced12.</li>
	<li>Collect approximately 5 mm x 5 mm pieces of the intestinal segment using laboratory scissors. Transfer the fragments to a 50 mL tube with tweezers, and add 25 mL of cold PBS-ABx.</li>
	<li>Wash the fragments by agitating back and forth 10x with 25 mL of cold PBS-ABx to remove the intestinal contents in the 50 mL tube.</li>
</ol>
2. Crypt isolation
<ol>
	<li>Incubate the pieces in PBS-ABx containing 2 mM EDTA for 30 min on ice with no shaking.</li>
	<li>For easy solidification of the extracellular matrix (ECM), incubate a 24-well plate in a 37 &#176;C tissue culture incubator beforehand.</li>
	<li>Aspirate the EDTA solution from the cell culture system with a vacuum pump, add 25 mL of fresh, cold PBS-ABx, and then shake the pieces up and down vigorously by hand 30x-40x to release the crypt-villus complexes. 
	NOTE: The separated crypts and villi can be checked by the microscopic observation of a 25 &#181;L droplet from the suspension at 4x magnification.</li>
	<li>Next, filter the suspension through a 70 &#181;m strainer once.</li>
	<li>Centrifuge the suspension at 390 &#215; g for 3 min at 4 &#176;C.</li>
	<li>Resuspend the crypt pellet in 20 mL of sorbitol DMEM (advanced DMEM/F12 + penicillin-streptomycin [1%] + gentamicin [0.5%] + fetal bovine serum [1%] + sorbitol [2%]) with pipetting, and transfer the crypt suspension to two new 15 mL tubes for division into two 10 mL solutions to centrifuge at low speed. 
	NOTE: The large cell mass and cells/debris can be separated using low-speed centrifugation. The large cell mass is in the pellet, and cells/debris are in the supernatant.</li>
	<li>Centrifuge the two crypt suspensions at 80 &#215; g for 3 min at 4 &#176;C, and then aspirate the supernatant gently. 
	NOTE: As the pellet formation is weak, do not aspirate too much. Leave 2 mL of supernatant in each tube.</li>
	<li>Add 10 mL of sorbitol DMEM to each tube again. Centrifuge the suspension at 80 &#215; g for 3 min at 4 &#176;C.</li>
	<li>After aspirating the supernatant, leaving 2 mL of supernatant in each tube, add 10 mL of sorbitol DMEM for resuspension, and centrifuge the crypt suspension at 80 &#215; g for a final 3 min at 4 &#176;C.</li>
	<li>After aspirating the supernatant, leaving 2 mL of supernatant in each tube, add 10 mL of complete DMEM (advanced DMEM/F12 + penicillin-streptomycin [1%] + gentamicin [0.5%] + fetal bovine serum [1%]) for resuspension of the pellet by pipetting up and down, and leave it for 1 min. 
	NOTE: Wait for 1 min to obtain the floating crypts efficiently.</li>
	<li>After 1 min, collect each 10 mL suspension for a total of 20 mL, and filter once with a 70 &#181;m cell strainer to purify the crypts.</li>
	<li>Before seeding essentially pure crypts, count the number of crypts in the filtered complete DMEM, and then centrifuge at 290 &#215; g for 3 min at 4 &#176;C.
	<ol>
		<li>Drip 25 &#181;L droplets into a 6 cm dish at three points. Count the number of crypts under a microscope at 4x magnification, and calculate the concentration of crypts per 25 &#181;L droplet.</li>
	</ol>
	</li>
	<li>Suspend 100 crypts with 40 &#181;L of ECM per well. Pipet up and down 5x-10x to obtain a homogeneous suspension of crypts in the ECM, and then seed in a 37 &#176;C prewarmed 24-well plate. 
	NOTE: Always keep the ECM on ice to avoid polymerization. Pipette carefully to avoid making air bubbles in the ECM.</li>
	<li>Incubate the 24-well plate for 15 min in a 37 &#176;C, 5% CO2 incubator for the polymerization of the ECM.</li>
	<li>Finally, cover the ECM with 500 &#181;L of culture medium containing mouse epidermal growth factor (EGF), recombinant mouse R-spondin 1, and recombinant mouse Noggin at room temperature. The final concentration of materials per well is as follows: penicillin-streptomycin (1%), 50 U/mL each; gentamicin (0.5%), 25 &#181;g/mL; EGF, 20 ng/mL; Noggin, 100 ng/mL; R-spondin 1, 500 ng/mL; L-glutamine, 2 mM.</li>
	<li>Start the crypt culture at 37 &#176;C in a 5% CO2 incubator. 
	NOTE: For the culture medium for organoids in a 24-well plate, see Table 1.</li>
	<li>Carry out long-term live imaging to observe organoid morphogenesis with a recording time-lapse image microscope equipped with a 20x objective every 3 h for up to 7 days. Obtain serial z-stacked images at z-steps of 1 &#181;m (1 &#181;m x five steps).</li>
	<li>Change the medium every other day.</li>
</ol>
3. Fluorescence-activated cell sorting (FACS)
<ol>
	<li>Isolate crypts from the mice (see section 2).</li>
	<li>Treat the isolated crypts with 2 mL of trypsin for 30 min at 37 &#176;C.</li>
	<li>Stop the reaction with 10 mL of PBS, and then pass through a 20 &#181;m cell strainer.</li>
	<li>Centrifuge the solution at 390 &#215; g for 3 min at 4 &#176;C, and resuspend with 100 &#181;L of complete DMEM.</li>
	<li>Add anti-EphB2 APC-conjugated antibody (1/50), and incubate for 30 min on ice.</li>
	<li>Wash the cells 3x with PBS, and finally add 7-amino-actinomycin D (7-AAD) (1/100).</li>
	<li>Sort the stained cells via FACS.
	<ol>
		<li>Adjust the area scaling factor, and sort according to cell size (forward scatter, FSC-A) versus granularity (side scatter, SSC-A).</li>
		<li>Sort 7-AAD negative and positive cells for viability with the laser set at a wavelength of 488 nm and 50 mV power.</li>
		<li>Demarcate the gates to sort the EphB2-high (EphB2high), EphB2-medium (EphB2med), EphB2-low (EphB2low), and EphB2-negative (EphB2neg) cells with the laser set at a wavelength of 640 nm and 100 mV power.</li>
	</ol>
	</li>
	<li>Start the EphB2high cell culture at 37 &#176;C in a 5% CO2 incubator.</li>
</ol>
4. Single-cell cultured organoids
<ol>
	<li>Carry out the cell isolation method according to graded EphB2 surface levels11, and then obtain four distinct populations (high, medium, low, and negative).</li>
	<li>Collect, pellet with centrifugation at 390 &#215; g for 3 min at 4 &#176;C, and embed the single-sorted EphB2high cells in the ECM by pipetting, followed by seeding on a 24-well plate (100 singlets/40 &#181;L of ECM/well).</li>
	<li>As in step 2.14, allow the ECM to polymerize, and cover the ECM with a culture medium containing a Rho-associated kinase (ROCK) inhibitor (10 &#181;M) for the first 2 days to maintain the EphB2high cells. 
	NOTE: The ROCK inhibitor is effective against anoikis.</li>
	<li>Manually inspect the cells using an inverted microscope at 40x magnification, and observe viable organoids with spheroid formation and crypt protrusion.</li>
</ol>

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

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The authors have no conflicts of interest to declare.

This protocol describes a method for consistently isolating small intestinal crypts and the subsequent culture of 3D organoids. To improve the crypt-releasing rate, a mechanical isolation method involving vigorous shaking after treatment with EDTA was established. The medium composition is different from the original protocol of Sato et al.7. The original medium is relatively costly. Thus, a culture medium and customized media for murine small intestinal organoids containing pharmacological inhibitors, recombinant growth factors, and/or conditioned media are shown in Table 1. Wnt3A and N-acetylcysteine are not included in the culture medium in this protocol. As Paneth cells express Wnt3, the cells produce Wnt3 and support ISC maintenance. Additionally, during the course of crypt isolation, the conditioned medium is not used. The organoid model is dynamic and has cellular and structural heterogeneity (Paneth cells, enterocytes, goblet cells, enteroendocrine cells, tuft cells, and ISCs). Hence, these organoids can be used at scale to study fundamental issues of organoid biology.
The EphB2 gradient maintains ISC stemness and proliferation along the crypt-villus axis in the adult small intestine18. The advantage of making organoids from one single EphB2 cell compared to isolated crypts relates to understanding the biology of murine ISCs, as ISCs play key roles in various human intestinal disorders. Single EphB2high-expressing ISCs can be cultured to form organoids in a similar way to the development of organoids from single Lgr5-expressing ISCs. The most important step is to precisely divide the cells into four groups (EphB2high, EphB2med, EphB2low, and EphB2neg) according to the EphB2 expression in the crypts using FACS. Forward versus side scatter (FSC vs. SSC) plots are commonly used to identify cells of interest based on their size and granularity. FSC indicates the cell size, and SSC relates to the complexity or granularity of the cell in the P0 gate (Figure 2A). In this work, the cells that fell within the defined gate (P0) were subsequently analyzed for viability. Next, their viability was determined according to the negative and positive populations of 7-AAD fluorescence signals. The border between the 7-AAD-negative and -positive cells was strictly decided to gain the negative ones with minimal positive cell contamination. The EphB2 gates were roughly set based on the EphB2 graded expression. 
To confirm that the four groups were precisely divided, the mRNA expression of selected genes was analyzed. The mRNA levels of ISC markers are high in EphB2high cells20. Additionally, the mRNA levels of progenitor cell-specific markers are relatively high in EphB2med cells20. However, EphB2 exdpression in EphB2low and EphB2neg cells is low or negative compared with that of EphB2high and EphB2med cells20. The preceding measures should be taken to ensure the enrichment of the EphB2high cell population before plating. However, organoid growth of less than 6% from EphB2high cells may be due to the death of stem cells during the culture process, not the vigorous shaking during the crypt isolation. It has been shown that the application of a selective Rho-associated kinase (ROCK) inhibitor to human embryonic stem cells markedly diminishes dissociation-induced apoptosis22. Thus, as a technical change, it is worth trying to add the ROCK inhibitor at a higher concentration and with a longer incubation to improve the viability.
Wnt3A-secreting Paneth cells next to ISCs provide essential support to the ISCs8. Indeed, ISC-Paneth cell doublets display a strongly increased organoid-forming capacity compared to single ISCs8. Moreover, the addition of Wnt3A at the concentration of 100 ng/mL for the first 3 days of culture has been shown to increase the organoid-forming capacity8. Thus, as another technical change, adding exogenous Wnt3A could improve the organoid-forming capacity of single EphB2high-expressing ISCs.
Compared to in vivo approaches, organoids can be easily used for genetic manipulation, the analysis of malignancy phenotypes, and drug screening20,23. A combination of EDTA chelation and a mechanical isolation method is effective, reproducible, and time-efficient for creating small intestinal organoids from crypts and can be easily followed by laboratory staff without any advanced experience. Thus, the addition of the mechanical isolation with vigorous shaking after the treatment with EDTA can efficiently establish murine small intestinal organoids ex vivo and provide a potential tool for organoid cultivation and disease modeling of other adult epithelial tissues.
Intestinal epithelial cells are polarized and orientated with the apical side directed toward the lumen. However, the apical side facing the lumen of 3D organoids is in their interior. Thus, this organization prevents access to the apical side, which is an issue when studying the effects of luminal components, such as nutrients, microbes, and metabolites on epithelial cells. To circumvent this disadvantage, a culture of organoid cells as 2D monolayers has been developed24. In terms of future applications, the culture of organoid cell monolayers will be utilized, as this represents the most efficient and tractable system.

Intestinal organoids, which were first established in 2009, have emerged as a powerful in vitro tool for studying intestinal biology given their morphological and functional similarity to mature tissues. Recently, technological advances in cultured organoids derived from adult-tissue stem cells have allowed for the long-term culture of intestinal stem cells (ISCs) with self-renewal and differentiation potential. These organoids have been widely used for basic and translational research studies on gastrointestinal physiology and pathophysiology1,2,3,4,5,6. The 3D organoids developed by the Clevers group provide a powerful tool to study the intestinal epithelium with improved physiological relevance7. Since intestinal organoids are derived from tissue stem cells and are composed of multiple cell types, they recapitulate the functionality of the intestinal epithelium. Of note, a single-sorted leucine-rich repeats-containing G protein-coupled receptor 5-positive (Lgr5+) stem cell can also generate 3D organoids without any Paneth cells or an ISC niche such as the epithelial niche or stromal niche7. However, the organoid-forming capacity of single-sorted Lgr5+ cells is low compared to those of crypt and ISC-Paneth cell doublets8.
An increasing number of studies have shown that the methods of ethylenediaminetetraacetic acid (EDTA) incubation or collagenase dissociation cause loosening in the epithelium and the release of crypts. As enzymatic dissociation may have an effect on the cell state of crypts, a mechanical isolation method is usually used to dissociate the tissue. Though mechanical digestion is a rapid technique, this method can be associated with inconsistent crypt yields or poor cell viability9. Therefore, EDTA treatment and mechanical dissociation can be combined to generate better crypt yields. A feature of the methodology shown in this article is the use of vigorous shaking of the tissue fragments after EDTA chelation10. Vigorous shaking permits the efficient isolation of crypts from crypt-villus complexes in the small intestine. The degree of manual shaking determines the separation. Thus, obtaining crypts from complexes is important for experimenters in this field. Additionally, proper skill can reduce villus contamination to a minimum and increase the number of crypts.
Hence, this experimental protocol, which employs murine-derived small intestinal organoids, can better isolate crypts with physical force after treatment with EDTA for dissociation. It is known that the expression pattern of erythropoietin-producing hepatocellular receptor B2 (EphB2) in part reflects the crypt environment. For example, EphB2-positive cells are organized from bottom to top11. Fluorescence-activated cell sorting (FACS) was carried out based on the EphB2 expression, and the cells obtained were divided into four groups: EphB2high, EphB2med, EphB2low, and EphB2neg. Then, the organoid growth from single-sorted EphB2high cells in wild-type (WT) mice was demonstrated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL Eppendorf tube</td>
			<td>Eppendorf</td>
			<td>0030 125.215</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringe</td>
			<td>TERUMO</td>
			<td>SS-05SZ</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Falcon tube</td>
			<td>Iwaki</td>
			<td>2325-015</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;m cell strainer</td>
			<td>Sysmex</td>
			<td>04-004-2325</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Iwaki</td>
			<td>3820-024</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Falcon tube</td>
			<td>Iwaki</td>
			<td>2345-050</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm tissue culture dish</td>
			<td>FALCON</td>
			<td>353002</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m cell strainer</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Petri dish</td>
			<td>Iwaki</td>
			<td>3020-100</td>
			<td></td>
		</tr>
		<tr>
			<td>7-AAD</td>
			<td>BD Biosciences</td>
			<td>559925</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Goat Anti-Mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A-11004</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-EphB2 APC-conjugated antibody</td>
			<td>BD Biosciences</td>
			<td>564699</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL6/J mice</td>
			<td>Japan SLC, Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clean bench</td>
			<td>HITACHI</td>
			<td>CCV-1306E</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Olympus</td>
			<td>FV3000</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA (0.5 mol/L)</td>
			<td>Nacalai Tesque</td>
			<td>06894-14</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>FACSMelody</td>
			<td>BD Life Sciences-Biosciences</td>
			<td>661762</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Sigma</td>
			<td>173012</td>
			<td>1% (v/v)</td>
		</tr>
		<tr>
			<td>Fiji (software)</td>
			<td>https://fiji.sc/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin (10 mg/mL)</td>
			<td>Nacalai Tesque</td>
			<td>16672-04</td>
			<td>25 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Hammacher laboratory scissor</td>
			<td>SANSYO</td>
			<td>91-1538</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Panasonic</td>
			<td>MCO-170-PJ</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory tweezer</td>
			<td>AS-ONE</td>
			<td>7-164-04</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM</td>
			<td>Gibco</td>
			<td>25030081</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>BD Biosciences</td>
			<td>354230</td>
			<td>ECM for 3D organoids</td>
		</tr>
		<tr>
			<td>Mouse Anti-Human Lysozyme</td>
			<td>LSBio</td>
			<td>LS-B8704-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Murine EGF (20 &#956;g/mL stock solution)</td>
			<td>PeproTech</td>
			<td>315-09</td>
			<td>20 ng/mL</td>
		</tr>
		<tr>
			<td>PBS 1x</td>
			<td>Gibco</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>50 U/mL</td>
		</tr>
		<tr>
			<td>Pipetman (10 &#956;L, 20 &#956;L, 200 &#956;L, and 1,000 &#956;L)</td>
			<td>GILSON</td>
			<td>1-6855-12, -13, -15, and -16</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant murine Noggin (20 &#956;g/mL stock solution</td>
			<td>R&#38;D Systems</td>
			<td>1967-NG-025</td>
			<td>100 ng/mL</td>
		</tr>
		<tr>
			<td>Recombinant murine R-Spondin 1 (250 &#956;g/mL stock solution)</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>500 ng/mL</td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Nacalai Tesque</td>
			<td>32021-95</td>
			<td>2% (w/v)</td>
		</tr>
		<tr>
			<td>TE2000-S (inverted microscope)</td>
			<td>Nikon</td>
			<td>24131</td>
			<td></td>
		</tr>
		<tr>
			<td>Time-lapse image microscope</td>
			<td>Olympus</td>
			<td>LCV100</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express 1x</td>
			<td>Gibco</td>
			<td>12605-010</td>
			<td></td>
		</tr>
		<tr>
			<td>ULVAC</td>
			<td>ULVAC KIKO Inc.</td>
			<td>100073</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Fujifilm</td>
			<td>331752-47-7</td>
			<td>10 &#956;M</td>
		</tr>
	</tbody>
</table>

the 3d culturing of organoids from murine intestinal crypts and a single stem cell for organoid research

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

To generate mouse small intestinal organoids, a combination of EDTA treatment and a mechanical isolation method can be used to efficiently isolate crypts10,13. The results of this study showed that almost all the isolated crypts were immediately sealed and appeared cone-shaped after they were squeezed out of the epithelial niches (Figure 1A). To minimize villus contamination, the resulting suspension was passed through a 70 &#181;m cell strainer, and then the filtrate was centrifuged. As some crypts are disrupted during filtration and suspension, these steps should be carried out carefully. The results showed that almost all crypts in the final fraction were integrated and suitable for use in culture (Figure 1B). To visualize all the plated crypts individually, 100 crypts per well were plated (Figure 1C). After adding the specific crypt culture medium (Figure 1D), the development of organoids was monitored with a microscope daily. Furthermore, organoid growth from the crypts was observed by time-lapse images to monitor their development (Figure 1E and Supplementary Video S1). The cultured crypts behaved in a stereotypical fashion. The inner lumen of the organoid was filled with a mass of apoptotic cells. Active proliferation and differentiation of ISCs occurred in the crypt region with budding (Figure 1E and Supplementary Video S1). Budding was coupled with ISC migration and proliferation and Paneth cell differentiation. The differentiated Paneth cells were always located at the budding site (Supplementary Figure S1). As the organoids were confirmed to be stable in culture using an inverted microscope at 10x magnification, the technique could be used to examine crypt formation in the developing small intestine and to determine the capacity for tissue regeneration and ISC long-term survival for the production of new intestinal epithelial cells14,15,16.
Lgr5 is defined as an ISC marker, and murine Lgr5+ cells form 3D organoids7. However, as the cell surface abundance of LGR5 protein is low and there is a lack of high-affinity anti-LGR5 antibodies, it is challenging to efficiently isolate murine ISCs by FACS. EphB2 has been previously identified as a surface marker for the purification of murine and human ISCs from intestinal tissues17,18. The expression pattern of EphB2 increases the complexity involved in ISC markers. EphB2-positive cells are organized throughout the proliferative compartment, peaking at the bottom of the crypts, while they decrease in a gradient toward the top of the crypts11. Paneth cells and progenitor cells are also localized at the crypt. Paneth cells mainly express EphB3, which is required for their positioning, and the progenitor cells above them in the crypt express mainly EphB2. Thus, contamination of both cell types can occur during the course of ISC purification using the anti-EphB2 antibody. Accordingly, their marker gene expression and the organoid-forming capacity of cells isolated using EphB2 by FACS should be assessed.
Based on these facts, using FACS analysis, EphB2 surface-labeled cells can be isolated from WT crypts19. It has been investigated whether EphB2 expression can distinguish among four groups with the expression of specific markers, such as ISC-specific marker genes (Lgr5, Ascl2, and Olfm4) and progenitor cell-specific marker genes (Ki67, Myc, and FoxM1). This experiment demonstrated that EphB2high cells were predominantly ISCs, unlike EphB2med cells20,21. Finally, based on the cell isolation method, the cells obtained were divided into four groups (EphB2high, EphB2med, EphB2low, and EphB2neg cells) (Figure 2). Then, single cells expressing high levels of EphB2 sorted by FACS were cultured for organoid growth. A single EphB2high cell can independently be applied for localized treatment and recreate self-organizing crypt-villous structures reminiscent of the normal small intestine (Figure 3). However, the cells derived from other groups (EphB2med, EphB2low, and EphB2neg) do not generate organoids20.
In a previous study, ~6% of single-sorted Lgr5-GFPhi cells were able to initiate crypt-villous organoids7. However, the remaining cells were unable to generate organoids and died within the first 12 h7. The authors presumed that this was the result of physical and/or biological stress inherent in the isolation procedure7. Less than 6% organoid growth was also obtained from single-sorted EphB2high cells in WT mice. By day 5 of culture, spheroid-like structures formed (Figure 3). From day 7 to day 9, evagination of the spots to form crypts occurred (Figure 3). Importantly, the application of a selected ROCK inhibitor to the single-sorted EphB2high cells diminished dissociation-induced apoptosis and increased the efficiency of organoid growth.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65219/65219fig01.jpg" /> 
Figure 1: Generation of mouse small intestinal organoids. (A) Crypts prepared by a combination of EDTA chelation and mechanical dissociation. (B) Resultant purified crypts. (C) Crypts embedded in the extracellular matrix. (A-C) The black arrows indicate crypts. (D) Three-dimensional culture of crypts and organoids. (E) Representative images of a growing organoid derived from a crypt. The white arrows indicate crypt budding. Scale bars = (A-C) 100 &#181;m and (E) 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65219/65219fig01large.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/65219/65219fig02.jpg" /> 
Figure 2: Flow cytometry gating strategy to obtain a population of EphB2-positive (EphB2+) cells in wild-type mice. (A) Forward and side scatter plots are used to separate the cells according to their size and granularity, respectively. (B) Fluorescence scatter is used to separate viable cells according to the 7-AAD (PerCP) fluorescence intensity of the cells. The gate for the 7-AAD-negative cell population was chosen. (C) The gates for the EphB2-high (EphB2high), EphB2-medium (EphB2med), EphB2-low (EphB2low), and EphB2-negative (EphB2neg) cell populations were chosen. Abbreviations: FSC-A = forward scatter-peak area; SSC-A = side scatter-peak area; 7-AAD = 7-amino-actinomycin D. <a href="https://www.jove.com/files/ftp_upload/65219/65219fig02large.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/65219/65219fig03.jpg" /> 
Figure 3: Time course of single-sorted EphB2high cell organoid growth in wild-type mice. <a href="https://www.jove.com/files/ftp_upload/65219/65219fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Culture medium for a 24-well plate. <a href="https://www.jove.com/files/ftp_upload/65219/65219_JoVE_Table1.xlsx" target="_blank">Please click here to download this Table.</a>
Supplementary Video S1: Time-lapse images of a growing organoid. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65219/JoVE_Supplymental Movie1.avi" target="_blank">Please click here to download this File.</a>
Supplementary Figure S1: Representative image of anti-lysozyme antibody staining in an organoid. The white arrows indicate Paneth cells. Abbreviation: DIC = differential interference contrast microscope. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65219/65219_ supplementary Figure S1 (1).pdf" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research at JoVE.com

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

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

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

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

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7.6K Views.  Bioorganic Research Institute. We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

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

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia (SCG)

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide sympathetic innervations for the head and neck regions and they constitute a well-characterized and relatively homogeneous population (4).  Sympathetic neurons are dependent on nerve growth factor (NGF) for survival, differentiation and axonal growth and the wide-spread availability of NGF facilitates their culture and experimental manipulation (2, 3, 6).  For these reasons, cultured sympathetic neurons have been used in a wide variety of studies including neuronal development and differentiation, mechanisms of programmed and pathological cell death, and signal transduction (1, 2, 5, and 6).  Dissecting out the SCG from newborn rats and culturing sympathetic neurons is not very complicated and can be mastered fairly quickly.  In this article, we will describe in detail how to dissect out the SCG from newborn rat pups and to use them to establish cultures of sympathetic neurons.  The article will also describe the preparatory steps and the various reagents and equipment that are needed to achieve this.

Protocol for Culturing Sympathetic Neurons from Rat Superior Cervical Ganglia SCG

This is a protocol describing how to isolate and culture primary sympathetic neurons from superior cervical ganglia (SCG) of newborn rat pups.

The superior cervical ganglia (SCG) in rats are small, glossy, almond-shaped structures that contain sympathetic neurons.  These neurons provide ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Single-Cell Resolution Three-Dimensional Imaging of Intact Organoids

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development and function as well as disease. Fluorescence microscopy has played a major role in characterizing their cellular composition in detail and demonstrating their similarity to the tissue they originate from. In this article, we present a comprehensive protocol for high-resolution 3D imaging of whole organoids upon immunofluorescent labeling. This method is widely applicable for imaging of organoids differing in origin, size and shape. Thus far we have applied the method to airway, colon, kidney, and liver organoids derived from healthy human tissue, as well as human breast tumor organoids and mouse mammary gland organoids. We use an optical clearing agent, FUnGI, which enables the acquisition of whole 3D organoids with the opportunity for single-cell quantification of markers. This three-day protocol from organoid harvesting to image analysis is optimized for 3D imaging using confocal microscopy.

The entire 3D structure and cellular content of organoids, as well as their phenotypic resemblance to the original tissue can be captured using the single-cell resolution 3D imaging protocol described here. This protocol can be applied to a wide range of organoids varying in origin, size and shape.

Organoid technology, in vitro 3D culturing of miniature tissue, has opened a new experimental window for cellular processes that govern organ development ...

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

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

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

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

Generation of Murine Primary Colon Epithelial Monolayers from Intestinal Crypts

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

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

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

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.