This work was supported by grants from the Israel Science Foundation (MSC personal grant) and the joint program between the Israel Science Foundation and the National Natural Science Foundation of China.

All procedures described below were approved by the Institutional Animal Care and Use Committee (IACUC) at the Hebrew University of Jerusalem.
1. Preparation of reagents and buffers
<ol>
	<li>Preheat a water bath to 37 &#176;C.</li>
	<li>Prepare all the solutions in Table 1.</li>
</ol>
2. Intestinal mesenchyme isolation
<ol>
	<li>Euthanize the mouse by CO2 inhalation, immediately followed by cervical dislocation.</li>
	<li>Place the mouse in a supine position and spray the abdomen with 70% EtOH. Lift the abdominal skin and cut longitudinally along the midline to expose the peritoneal cavity (see Figure 1 I,II).</li>
	<li>Locate the stomach, cut from the esophagus, and slowly pull the intestine out of the peritoneal cavity. Clean excess fat and connective tissue using forceps. Excise the small intestine from the duodenum to approximately 0.5 cm from the cecum (Figure 1 III,IV).</li>
	<li>Wash the intestine in a Petri dish containing cold sterile PBS. Using ball-tip scissors, open the intestinal tube longitudinally and wash out the feces (Figure 1 V). Transfer the intestine into a new dish containing fresh cold PBS and wash again.</li>
	<li>Cut the small intestine into 1 cm long segments and transfer into a 15 mL conical tube filled with 8 mL of PBS. Shake the tube manually at one or two cycles/s for 1 min.</li>
	<li>Transfer the segments using forceps into a 50 mL conical tube filled with 20 mL of freshly made solution A (see Table 1). Place the tubes in an orbital shaker incubator at 37 &#176;C for 20 min. After incubation, shake the tube vigorously by hand at four or five cycles/s for 1 min to dissociate the epithelium.</li>
	<li>Repeat step 2.6 once.</li>
	<li>Transfer the segments into a new 50 mL tube filled with 10 mL of sterile PBS, and invert the tube at one or two cycles/s for 1 min.</li>
	<li>Transfer the segments into a new 15 mL tube filled with 10 mL of sterile PBS, and tilt up and down gently at one or two cycles/s for 2 min.</li>
	<li>Under a biosafety cabinet, use forceps to place the segments on a sterile laboratory wipe to dry them out. Once dried, cut the segments further into 0.5 cm pieces.</li>
	<li>Transfer the small segments using forceps into a 6-well plate filled with 4 mL of prewarmed digestion solution per well. Incubate at 37 &#176;C for 50 min. Gently shake the plate by hand every 20 min.</li>
	<li>Transfer the segments using a Pasteur pipette into a 15 mL conical tube filled with 4 mL of DMEM. Shake the tube manually at four or five cycles/s for 1 min to get a single-cell suspension.</li>
	<li>Filter the suspension through a 100 &#181;m strainer into a 50 mL conical tube. Centrifuge the filtrate at 700 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant by aspiration and resuspend the cell pellet in 5 mL of 2% FBS/PBS. Centrifuge the suspension at 700 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Discard the supernatant by aspiration, resuspend the cell pellet with 12 mL of culture medium, and seed 1 mL per well onto two 6-well plates. The following day, wash and aspirate any dying cells and replace the spent medium with fresh medium. 
	NOTE: For optimal maintenance of the culture, it is recommended to change the medium every 2 days. Depending on the mouse strain, genetic background, and age, 4 days to 2 weeks are needed for the mesenchyme to be ready for co-culture experiments. For further co-culture experiments, mesenchyme should reach confluency, displaying flat and fully stretched cellular morphology as shown in Figure 2. In general, cells with round morphology are not viable or functional.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64169/64169fig01.jpg" /> 
Figure 1: Mouse dissection. (I) Place the mouse in a supine position and spray the abdomen with 70% EtOH. Lift the abdominal skin. (II) Open the peritoneal cavity longitudinally along the midline. (III) While gently pulling the stomach, sever the esophagus. (IV) Pinch the stomach and slowly pull out the intestine. (V) Insert the tip of the ball-tip scissors in the lumen and open the intestinal tube longitudinally. <a href="https://www.jove.com/files/ftp_upload/64169/64169fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Mesenchyme resuspension for passaging or co-culture
<ol>
	<li>Resuspend the mesenchyme with 2 mL of 0.25% trypsin-0.5 mM EDTA/well in a 6-well plate. Incubate for 5 min at 37 &#176;C. Following incubation, if several cells have not already started detaching from the dish, incubate for an extra 2 min.</li>
	<li>Using a cell scraper, gently scrape the surface of the well. Transfer the cell suspension into a 15 mL conical tube. Add 2 mL of DMEM-F12/well and gently pipette the suspension up and down. 
	NOTE: It is important not to overfill the tube with more than 50% of the tube volume. It is therefore recommended to use a 15 mL conical tube filled with 8 mL of suspension for every two wells.</li>
	<li>Count the cells. 
	NOTE: A fully confluent well yields between 2 &#215; 106 cells and 2.5 &#215; 106 cells.</li>
	<li>Dilute the cell suspension to achieve a seeding density of 3-5 &#215; 105 cells/mL. Centrifuge for 5 min at 500 &#215; g at 4 &#176;C and discard the supernatant by careful aspiration. 
	NOTE: It is important to remove as much liquid as possible.</li>
	<li>Resuspend the cell pellet in prewarmed culture medium and plate.</li>
</ol>
4. Flow cytometry analysis for telocyte purification
<ol>
	<li>Obtain the mesenchymal cell pellet (step 2.14). Resuspend the cell pellet in 1 mL of FACS buffer and filter through a 40 &#181;m strainer.</li>
	<li>Incubate the cell suspension with allophycocyanin (APC)-conjugated CD326 (1:100), CD45 (1:400), and CD31 (1:250) antibodies in 400 &#181;L of FACS buffer for 15 min at room temperature to exclude epithelial, immune, and endothelial cells, respectively, from the sort.</li>
	<li>Wash the cells by adding 1 mL of FACS buffer and spin down at 700 &#215; g for 5 min at 4 &#176;C. Resuspend in 400 &#181;L of FACS buffer and add 4&#39;,6-diamidino-2-phenylindole (DAPI, 5 mg/mL, 1:1,000) for flow cytometry analyses. Gate the single cells according to the SSC-height by SSC-area. Gate the DAPI- live cells and gate out CD45+/CD31+/CD326+ to sort the GFP+ telocytes, as shown in Figure 3.</li>
</ol>

<ol><li>Carroll, T. D., Newton, I. P., Chen, Y., Blow, J. J., Näthke, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lgr5%2B+intestinal+stem+cells+reside+in+an+unlicensed+G1+phase%5BTitle%5D)+AND+%22The+Journal+of+Cell+Biology%22%5BJournal%5D)">Lgr5+ intestinal stem cells reside in an unlicensed G1 phase.</a> The Journal of Cell Biology. 217 (5), 1667-1685 (2018).</li>
<li>Kinchen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+remodeling+of+the+human+colonic+mesenchyme+in+inflammatory+bowel+disease%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Structural remodeling of the human colonic mesenchyme in inflammatory bowel disease.</a> Cell. 175 (2), 372-386 (2018).</li>
<li>Popescu, L. M., Faussone-Pellegrini, M. -S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TELOCYTES+-+a+case+of+serendipity%3A+The+winding+way+from+Interstitial+Cells+of+Cajal+%28ICC%29%2C+via+Interstitial+Cajal-Like+Cells+%28ICLC%29+to+TELOCYTES%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">TELOCYTES - a case of serendipity: The winding way from Interstitial Cells of Cajal (ICC), via Interstitial Cajal-Like Cells (ICLC) to TELOCYTES.</a> Journal of Cellular and Molecular Medicine. 14 (4), 729-740 (2010).</li>
<li>Gherghiceanu, M., Manole, C. G., Popescu, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telocytes+in+endocardium%3A+electron+microscope+evidence%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">Telocytes in endocardium: electron microscope evidence.</a> Journal of Cellular and Molecular Medicine. 14 (9), 2330-2334 (2010).</li>
<li>Ceafalan, L., Gherghiceanu, M., Popescu, L. M., Simionescu, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telocytes+in+human+skin%E2%80%94are+they+involved+in+skin+regeneration%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">Telocytes in human skin—are they involved in skin regeneration.</a> Journal of Cellular and Molecular Medicine. 16 (7), 1405-1420 (2012).</li>
<li>Hinescu, M. E., Gherghiceanu, M., Suciu, L., Popescu, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telocytes+in+pleura%3A+two-+and+three-dimensional+imaging+by+transmission+electron+microscopy%5BTitle%5D)+AND+%22Cell+and+Tissue+Research%22%5BJournal%5D)">Telocytes in pleura: two- and three-dimensional imaging by transmission electron microscopy.</a> Cell and Tissue Research. 343 (2), 389-397 (2011).</li>
<li>Popescu, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Telocytes+in+human+epicardium%5BTitle%5D)+AND+%22Journal+of+Cellular+and+Molecular+Medicine%22%5BJournal%5D)">Telocytes in human epicardium.</a> Journal of Cellular and Molecular Medicine. 14 (8), 2085-2093 (2010).</li>
<li>Shoshkes-Carmel, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Subepithelial+telocytes+are+an+important+source+of+Wnts+that+supports+intestinal+crypts%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Subepithelial telocytes are an important source of Wnts that supports intestinal crypts.</a> Nature. 557 (7704), 242-246 (2018).</li>
<li>Dekkers, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+functional+CFTR+assay+using+primary+cystic+fibrosis+intestinal+organoids%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">A functional CFTR assay using primary cystic fibrosis intestinal organoids.</a> Nature Medicine. 19 (7), 939-945 (2013).</li>
<li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Long-term+expansion+of+epithelial+organoids+from+human+colon%2C+adenoma%2C+adenocarcinoma%2C+and+Barrett%E2%80%99s+epithelium%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li>
<li>Vonk, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protocol+for+application%2C+standardization+and+validation+of+the+forskolin-induced+swelling+assay+in+cystic+fibrosis+human+colon+organoids%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">Protocol for application, standardization and validation of the forskolin-induced swelling assay in cystic fibrosis human colon organoids.</a> STAR Protocols. 1 (1), 100019(2020).</li>
<li>Wu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MAP3K2-regulated+intestinal+stromal+cells+define+a+distinct+stem+cell+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">MAP3K2-regulated intestinal stromal cells define a distinct stem cell niche.</a> Nature. 592 (7855), 606-610 (2021).</li>
<li>Bahar Halpern, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lgr5%2B+are+a+signaling+source+at+the+intestinal+villus+tip%5BTitle%5D)+AND+%22Nature+Communication%22%5BJournal%5D)">Lgr5+ are a signaling source at the intestinal villus tip.</a> Nature Communication. 11 (1), 1936(2020).</li>
<li>Koliaraki, V., Pallangyo, C. K., Greten, F. R., Kollias, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+cells+in+colon+cancer%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Mesenchymal cells in colon cancer.</a> Gastroenterology. 152 (5), 964-979 (2017).</li>
</ol>

The authors have no conflicts of interests to declare.

Here, we developed a protocol to isolate mesenchyme from mouse small intestine using the FOXL1-Cre: Rosa26-mTmG mouse model, which enables researchers to distinguish telocytes from other mesenchymal cells. There are some critical steps to follow in this protocol. First, it is important to shake the tube vigorously at four or five cycles/s, to discard the majority of epithelial cells during mesenchyme isolation. The incubation time for enzymatic digestion must be optimized based on digestion efficiency. During incubation, the plates are to be gently shaken horizontally for a few seconds every 20 min. Once the tissue becomes filament-like, the incubation must be stopped by proceeding to protocol step 2.12. Exposing the tissue for long digestion times may result in low cell viability rate and yield. Following enzymatic digestion, the tube must be mechanically shaken to release more single cells into suspension; ideally, the solution should look cloudy, and no tissue fragments should be visible. If this is not the case, prolong the enzymatic digestion to 60 min.
Keeping sterile conditions and avoiding potential bacterial contamination is one of the critical steps when working with primary tissue culture. Sterile dissecting tools, reagents, and buffers must be used; gloves must be changed, and the working area must be cleaned when working with animals is done. Once the cell suspension is obtained, work should be conducted under a laminar biological hood. Following plating, cells should be incubated overnight with no disturbances, since this can affect adherence. In addition, it is important to replace the culture medium one day post seeding, since non-adherent cells may affect culture viability.
Surface markers used in this protocol strongly reacted with their epitopes; however, it is possible that enzymatic digestion may affect binding reactivity, and therefore, FACS analysis results. Another limitation of this protocol is the underrepresentation of the muscularis layer. To improve the efficiency of muscle layer cell isolation, we recommend mechanical separation of the muscle from the mucosa layers, and separate enzymatic digestion for each of the layers. For dissociating the epithelium from the stroma, either mechanical separation or chelating agents (EDTA or DTT) can be used; however, enzymatic digestion for obtaining single cells has been optimized in this protocol.
Isolation of intestinal mesenchyme has been previously described8; scraping off the villi with a coverslip would cause the loss of some mesenchyme alongside the villus, especially villus tip mesenchymal cells such as Lgr5+ villus tip telocytes13. In this protocol, we utilize collagenase type VIII instead of dispase II and trypsin in combination with DNase I, as collagenase more efficiently releases mesenchymal cells from the matrix. Although it prolongs the processing time (&#62;90 min vs. 35 min), the two protocols yielded similar cell viability rates; the current protocol improved the yield of the mesenchymal cells in general, and more specifically of the telocyte fraction. The current protocol yielded approximately 10% GFP+ telocytes, confirmed both by visualization and by FACS analysis, while the earlier protocol yielded 2% GFP+ telocytes. The major differences between the current protocol and the two reference protocols are listed in Supplemental Table S1.
The identification of FOXL1+GFP+ cells as subepithelial telocytes is based on in vivo studies. The need to develop and modify available mesenchyme isolation protocols to produce higher yields of telocytes, and the knowledge of how to achieve this was based on our understanding of the structure and function of FOXL1+ telocytes in vivo, as large cells with long cellular projections closely attached to epithelial cells.
Interestingly, ex vivo GFP+ telocytes exhibit cellular characteristics similar to their features in vivo in the intestine and are, therefore, suggested to serve as an ideal support for organoid growth. Although this protocol mainly discusses telocyte isolation from the small intestine, a similar protocol with minor modification can be used and easily applied for colon mesenchymal cells, such as the recently described MAP3K2-regulated intestinal stromal cell (MRISC)12.
Once the mesenchymal cells have stretched and reached confluency, they can be used for several additional applications, such as 3D co-culture with mouse- or human-derived organoids, using growth factor-free Matrigel. The mesenchyme typically forms a network fully supporting organoid formation and growth with no exogenous growth factor supplementation. The intestinal stroma has intrinsic 3D features that can provide the epithelium with mechanical support14. Therefore, this protocol can also be used to isolate mesenchyme to be integrated into a 3D bio-printed scaffold and utilized for further xenograft experiments.

Both the small intestine and colon are highly regenerative tissues due to the presence of stem cells, which proliferate and fuel regeneration1. The mesenchyme surrounding the epithelium provides structural and functional support by secreting extracellular matrix proteins and signaling molecules2, which modulate the response of epithelial cells. Telocytes are large mesenchymal cells, mainly described so far by electron microscopy as cells with long cytoplasmic processes called telopodes, which overlap to create a labyrinthic network3,4,5,6,7. Recently, intestinal telocytes expressing the transcription factor FOXL1 have emerged as an important stem cell niche component providing Wnt proteins, which are crucial for stem and progenitor cell function. Intestinal telocytes express high levels of key signaling pathway proteins such as Wnt, Bmp, Tgfb, and Shh, as well as many growth factors8.
Given that telocytes are a critical stem cell niche component in vivo, developing protocols to isolate and culture them ex vivo will allow their use as a source for signaling molecules and growth factors, to support growth and differentiation ex vivo. Using well-established protocols, colon or intestinal epithelial crypts can be isolated and form 3D structures known as organoids9,10,11. Three-dimensional organoids represent a powerful tool to investigate both the physiology and pathology of the intestinal epithelium ex vivo. In an ex vivo system, organoids rely on exogenous supplementation of factors for survival and growth10. Isolated mesenchyme can be cultured with both mouse and human-derived organoids and used as a source of growth factors, instead of exogenous supplementation, to better reflect the situation in the original tissue. Studying telocytes ex vivo has numerous benefits in investigating normal or pathological cellular behaviors, mechanisms of tissue homeostasis, and cell-cell interactions in greater detail.
Although protocols describing how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures result in the efficient isolation of telocytes. Successful isolation of telocytes requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between the telocytes and neighboring cells, without affecting their viability. To overcome these limitations, this paper presents a modified protocol that consistently yields a highly viable, single-cell suspension containing a relatively high quantity of telocytes with sufficiently conserved antigenic and functional properties. These telocytes can be used for several applications, including co-culture with mouse- or human-derived organoids, to support growth with no growth factor supplementation. This in turn better reflects the situation in the original tissue.
We used the FOXL1-Cre: Rosa-mTmG mouse model8, in which telocytes are labeled with a membrane-bound version of green fluorescent protein (GFP) in green, which allows the investigator to follow telocytes in their entirety; all the other mesenchymal cells are labeled with a membrane-bound tdTomato in red. The current protocol was modified from a protocol isolating intestinal mesenchyme12 to improve telocyte yield and viability.

<table><tbody><tr><td>15 mL Centrifuge Tubes</td><td>Corning</td><td>430052</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>5 mL Polystyrene Round-Bottom Tube with Cell-strainer cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>6 Well Cell Culture Plate</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>APC Anti-Mouse CD31</td><td>Biolegend</td><td>102509</td><td></td></tr><tr><td>APC Anti-Mouse CD326</td><td>Biolegend</td><td>118213</td><td></td></tr><tr><td>APC Anti-Mouse CD45</td><td>Biolegend</td><td>103111</td><td></td></tr><tr><td>Cell Lifter</td><td>Corning</td><td>3008</td><td></td></tr><tr><td>Cell Strainer 100&amp;mu;m Nylon Yellow</td><td>Corning</td><td>CLS431752</td><td></td></tr><tr><td>Collagenase type VIII</td><td>Sigma</td><td>C2139-500MG</td><td></td></tr><tr><td>DL-Dithiothreitol (DTT)</td><td>Sigma</td><td>43815-1G</td><td></td></tr><tr><td>DMEM/F-12 (HAM) 1:1</td><td>Biological Industries</td><td>01-170-1A</td><td></td></tr><tr><td>DNase I</td><td>Sigma</td><td>DN25-1G</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Biological Industries</td><td>01-055-1A</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma</td><td>D1283-500ML</td><td>10x</td></tr><tr><td>EDTA 0.5 M, pH 8.0</td><td>Biological Industries</td><td>01-862-1B</td><td></td></tr><tr><td>FBS</td><td>Biological Industries</td><td>04-007-1A</td><td></td></tr><tr><td>Gentamicin</td><td>Sigma</td><td>G1914-250MG</td><td>100x</td></tr><tr><td>Gluta Max-I</td><td>Gibco</td><td>35050-038</td><td>100x</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution (HBSS)</td><td>Biological Industries</td><td>02-017-5A</td><td>10x</td></tr><tr><td>HEPES</td><td>Gibco</td><td>15630-080</td><td>100x</td></tr><tr><td>Penicillin-Streptomycin (Pen/Strep)</td><td>Biological Industries</td><td>03-033-1B</td><td>100x</td></tr><tr><td>RPMI 1640 medium</td><td>Gibco</td><td>21875-034</td><td></td></tr><tr><td>Trypsin EDTA Solution B</td><td>Sartorius</td><td>03-052-1A</td><td></td></tr></tbody></table>

isolation of murine intestinal mesenchyme resulting in a high yield of telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

The above intestinal mesenchyme isolation protocol was modified from protocols described in both Wu et al.12 and Shoshkes-Carmel et al.8. The protocol described in Wu et al. is for the colon, and the one by Shoshkes-Carmel et al. is for the small intestine, thus the digestion condition is different in enzyme combination, work concentration, and incubation time between these two protocols. Here, the described protocol has been successfully used to isolate and culture intestinal mesenchymal cells, including telocytes. Briefly, we dissected the small intestine from the duodenum to the ileum using a FOXL1-Cre: Rosa-mTmG mouse model8, in which the telocytes were labeled with membrane-GFP (green), while other mesenchymal cells were labeled with membrane-tdTomato (red). We dissociated the tissue using digesting enzymes and seeded the mesenchyme in a 6-well plate. Following dissociation, telocytes lose their cellular characteristics, showing round cellular morphology (Figure 2A) which is reflected in the under-quantification of GFP+ cells at day 1 compared with following days (Figure 2F). After a few days, telocytes exhibit a small stretched cell morphology with short cellular processes (Figure 2B,C). However, 7-10 days following seeding, telocytes regain their cellular characteristics, showing large stretched cell morphology with long cytoplasmic processes (Figure 2D,E), and are ready to be used in co-culture with organoids and support their growth.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64169/64169fig02.jpg" /> 
Figure 2: Cultured mesenchyme isolated from FOXL1Cre: Rosa-mTmG mouse intestine. FOXL1+ telocytes are labeled with GFP, while other mesenchymal cells are tdTomato+. (A-E) Representative images of cultured mesenchyme isolated using the current protocol, plated in a 6-well plate, and imaged following 1 (A), 4 (B,C), and 7 (D,E) days of culture. Scale bars = 100 &#181;m. (F) Quantification of GFP/tdTomato cell ratio per field of view (percentages) at days 1, 4, and 7 of culture. Abbreviations: FOXL1 = Forkhead box L1 protein; GFP = green fluorescent protein. <a href="https://www.jove.com/files/ftp_upload/64169/64169fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
To evaluate this isolation protocol and reveal the cell composition, we analyzed the obtained cell suspension by flow cytometry (Figure 3). Overall, 69% of the isolated cells were viable based on DAPI staining (Figure 3 III); out of the live cells, 60.9% represented epithelial contamination and immune and endothelial cells (CD326+, CD45+, and CD31+; Figure 3 IV). The telocyte fraction (GFP+) scattered above 100k and 70k FSC and SSC, respectively (Figure 3 VI), and represented almost 10% from the gated mesenchyme (live CD45-, CD326-, CD31-) (Figure 3 V).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64169/64169fig03.jpg" /> 
Figure 3: Flow cytometry gating strategy for sorting telocytes from isolated adult mouse intestinal mesenchyme. (I) Low-level side scatter events were excluded. (II) Single cells were gated according to SSC-height by SSC-area. (III) DAPI+ events were gated out to exclude dead cells from the sort. (IV) DAPI- CD45+/CD326+/CD31+ events were gated out to exclude immune, epithelial, and endothelial cells, respectively. (V) GFP+ telocytes accounted for 10.3% of DAPI- CD45-/CD326-/CD31- cells. (VI) Back-gating analysis revealed the coordinates of GFP+ telocytes in an FSC-A/SSC-A plot. Abbreviations: SSC-A = side scatter-peak area; FSC-A = forward scatter-peak area; SSC-H = side scatter-peak height; DAPI = 4&#39;,6-diamidino-2-phenylindole; GFP = green fluorescent protein; APC = allophycocyanin. <a href="https://www.jove.com/files/ftp_upload/64169/64169fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In parallel, we also isolated mesenchyme from a wild-type (WT) C57Bl6 mouse intestine, in which the telocyte fraction was evaluated by using a combination of surface markers previously analyzed on mesenchyme isolated from a FOXL1Cre: Rosa-mTmG mouse model, in which the telocyte fraction was GFP-labeled. We found that a subset of the telocytes can be defined by a positive staining to CD201 and podoplanin (GP38) (Figure 4). Moreover, using these markers in immunostaining 1 day following isolation and culture confirmed that, although the cells did not yet exhibit their cellular characteristics, they obtained the expression of these molecular markers, showing staining in 70%-80% of the GFP+ telocytes (Figure 5).
The telocyte fraction defined by surface markers is not identical to the one obtained by using the FOXL1-driven reporter mouse; the telocyte is highly heterogenous and contains several subsets. It is necessary to combine the surface marker with FOXL1 labeling for telocyte definition. In the small intestine of theFOXL1Cre: Rosa-mTmG mouse, 60%-70% of the GFP+ cells are CD201 positive, and 65%-80% are positive for GP38. When using surface markers, it is important to note that inappropriate storage and repetitive freeze-thawing cycles of antibodies decreases the binding efficiency. In addition, enzymatic digestion may disrupt surface marker expression. We observed that the expression of CD138, a transmembrane proteoglycan expressed on mesenchymal cells, was disrupted and greatly decreased with dissociation.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64169/64169fig04.jpg" /> 
Figure 4: FACS analysis of single-cell mesenchyme suspension isolated from FOXL1Cre: Rosa-mTmG mouse small intestine using the current protocol. FACS analysis on (I-II) single cells, (III) DAPI-, (IV-VI) Lin-(CD45-, CD326-, CD31-) GFP+ cells, showing that (VII) 65.3% of the GFP+ and 31.2% of the Tomato+ are positive for GP38, whereas (VIII) 60.5% of the GFP+ and 22.6% of the Tomato+ are positive for CD201. Abbreviations: SSC-A = side scatter-peak area; FSC-A = forward scatter-peak area; SSC-H = side scatter-peak height; DAPI = 4&#39;,6-diamidino-2-phenylindole; GFP = green fluorescent protein; APC = allophycocyanin; PE = phycoerythrin. <a href="https://www.jove.com/files/ftp_upload/64169/64169fig04large.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/64169/64169fig05.jpg" /> 
Figure 5: Day one cultured mesenchyme, fixed and stained for mesenchymal markers. (A-D) Representative images of cultured mesenchyme stained for GP38. (F-I) Representative images of cultured mesenchyme stained for CD201. Scale bars = 100 &#181;m. (E) Quantification of Tomato+ GP38+ and Tomato+ CD201+ double-positive from total Tomato+. (J) Quantification of GFP+ GP38+ and GFP+ CD201+ double-positive from total GFP+. <a href="https://www.jove.com/files/ftp_upload/64169/64169fig05large.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>Solution A:&#160;</td>
			<td colspan="4">HBSS supplemented with 2% FBS, 1 mM DL-dithiothreitol (DTT), 2 mM EDTA (pH 8.0).</td>
		</tr>
		<tr>
			<td>Complemented medium 1640 (CM1640):&#160;</td>
			<td colspan="4">RPMI 1640 medium supplemented with 10% FBS, Pen/Strep (100 units penicillin/mL, 100 &#181;g streptomycin/mL).&#160;</td>
		</tr>
		<tr>
			<td>Digestion solution:&#160;</td>
			<td colspan="4">100 U/mL collagenase type VIII, 75 mg/mL DNase I in 4 mL of prewarmed CM1640. Note: Add collagenase and DNase I just before digestion procedure starts.</td>
		</tr>
		<tr>
			<td>Culture media:&#160;</td>
			<td colspan="4">DMEM-F12 media supplemented with 10 &#181;g/mL gentamicin, 10 mM HEPES, glutamine, Pen/Strep (100 units penicillin/mL, 100 &#181;g streptomycin/mL).</td>
		</tr>
		<tr>
			<td>FACS buffer:&#160;</td>
			<td colspan="4">PBS supplemented with 5% FBS and 1 mM EDTA.</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of all the solutions used in the protocol. 
Supplemental Table S1: The major differences between the current protocol and the two reference protocols are listed here. <a href="https://www.jove.com/files/ftp_upload/64169/64169_Supp Table S1.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes at JoVE.com

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

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Research

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Medicine

1.9K Views.  The Hebrew University - Hadassah Medical School. Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue. 

Video: Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Immunology and Infection

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Isolation of Lamina Propria Mononuclear Cells from Murine Colon Using Collagenase E

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous antigens and modulate responses to potent microbially derived immune stimuli. For this reason, the intestine is a major target site of immune dysregulation and inflammation in many diseases including but, not limited to inflammatory bowel diseases such as Crohn&#8217;s disease and ulcerative colitis, graft-versus-host disease (GVHD) after bone marrow transplantation (BMT), and many allergic and infectious conditions. Murine models of gastrointestinal inflammation and colitis are heavily used to study GI complications and to pre-clinically optimize strategies for prevention and treatment. Data gleaned from these models via isolation and phenotypic analysis of immune cells from the intestine is critical to further immune understanding that can be applied to ameliorate gastrointestinal and systemic inflammatory disorders. This report describes a highly effective protocol for the isolation of mononuclear cells (MNC) from the colon using a mixed silica-based density gradient interface. This method reproducibly isolates a significant number of viable leukocytes while minimizing contaminating debris, allowing subsequent immune phenotyping by flow cytometry or other methods.

The goal of this protocol is to isolate mononuclear cells that reside in the lamina propria of the colon by enzymatic digestion of the tissue using collagenase. This protocol allows for the efficient isolation of mononuclear cells resulting in a single cell suspension which in turn can be used for robust immunophenotyping.

The intestine is the home to the largest number of immune cells in the body. The small and large intestinal immune systems police exposure to exogenous ...

Isolation of Primary Murine Skeletal Muscle Microvascular Endothelial Cells

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal muscles regulating the exchange of fluids and nutrients as well as the immune response against infectious agents by controlling immune cell migration. For these functions, MMEC form a functional &#34;myovascular unit&#34; (MVU), with further cell types, such as fibroblasts, pericytes and skeletal muscle cells. Consequently, a dysfunction of MMEC and therefore the MVU contributes to a vast variety of myopathies. However, regulatory mechanisms of MMEC in health and disease remain insufficiently understood and their elucidation precedes more specific treatments for myopathies. The isolation and in-depth investigation of primary MMEC functions in the context of the MVU might facilitate a better understanding of these processes.
This article provides a protocol to isolate primary murine MMEC of the skeletal muscle by mechanical and enzymatic dissociation including purification and culture maintenance steps.

Microvascular endothelial cells of skeletal muscles (MMEC) shape the inner wall of muscle capillaries and regulate both, exchange of fluids/molecules and migration of (immune) cells between muscle tissue and blood. Isolation of primary murine MMEC, as described here, enables comprehensive in vitro investigations of the &#34;myovascular unit&#34;.

The endothelial cells of skeletal muscle capillaries (muscle microvascular endothelial cells, MMEC) build up the barrier between blood stream and skeletal ...

Neuroscience

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, ...

Biology

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

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

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

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

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

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 ...