The authors wish to acknowledge support from R37 DK045729 (to JLM), R01 AR060837 (to HX) and the University of Michigan Gastrointestinal Research Center Molecular Core P30 DK034933.

All animal experiments described were approved by the University of Michigan&#39;s Committee on the Use and Care of Animals.
1. Preparation of Sterile Poly-D-lysine (PDL) and Laminin Solutions
<ol>
	<li>At least one day prior to cell isolation, prepare poly-D-lysine (PDL) and laminin coated plates. 
	NOTE: Both 6-well and 12-well plates were prepared depending on the experimental goals. Typically, 12-well plates were used for the quantitative analysis using western blots; whereas, 6-wel...

<ol>
	<li>Grubisic, V., Gulbransen, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia:+the+most+alimentary+of+all+glia.">Enteric glia: the most alimentary of all glia.</a> Journal of Physiology. 595 (2), 557-570 (2017).</li><li>Gulbransen, B. D., Sharkey, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+functional+roles+for+enteric+glia+in+the+gastrointestinal+tract.">Novel functional roles for enteric glia in the gastrointestinal tract.</a> Nature Reviews Gastroenterology &amp; Hepatology. 9 (11), 625-632 (2012).</li><li>Rao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+Glia+Regulate+Gastrointestinal+Motility+but+Are+Not+Required+for+Maintenance+of+the+Epithelium+in+Mice.">Enteric Glia Regulate Gastrointestinal Motility but Are Not Required for Maintenance of the Epithelium in Mice.</a> Gastroenterology. 153 (4), 1068-1081 (2017).</li><li>Rao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glia+express+proteolipid+protein+1+and+are+a+transcriptionally+unique+population+of+glia+in+the+mammalian+nervous+system.">Enteric glia express proteolipid protein 1 and are a transcriptionally unique population of glia in the mammalian nervous system.</a> Glia. , (2015).</li><li>McClain, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++responses+in+enteric+glia+are+mediated+by+connexin-43+hemichannels+and+modulate+colonic+transit+in+mice.">Ca2+ responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice.</a> Gastroenterology. 146 (2), 497-507 (2014).</li><li>Cunha Franceschi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enteric+glial+reactivity+to+systemic+LPS+administration+Changes+in+GFAP+and+S100B+protein.">Enteric glial reactivity to systemic LPS administration Changes in GFAP and S100B protein.</a> Neuroscience Research. 119, 15-23 (2017).</li><li>Kabouridis, P. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+enteric+glia+and+GDNF+during+gut+inflammation.">Distribution of enteric glia and GDNF during gut inflammation.</a> BMC Gastroenterology. 11, 3 (2011).</li><li>Rosenbaum, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+Myenteric+Glia+during+Acute+Inflammation+In+Vitro+and+In+Vivo.">Activation of Myenteric Glia during Acute Inflammation In Vitro and In Vivo.</a> Public Library of Science One. 11 (3), e0151335 (2016).</li><li>Zhuo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hGFAP-cre+transgenic+mice+for+manipulation+of+glial+and+neuronal+function+in+vivo.">hGFAP-cre transgenic mice for manipulation of glial and neuronal function in vivo.</a> Genesis. 31 (2), 85-94 (2001).</li><li>Boesmans, W., Lasrado, R., Vanden Berghe, P., Pachnis, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+and+phenotypic+plasticity+of+glial+cells+in+the+mammalian+enteric+nervous+system.">Heterogeneity and phenotypic plasticity of glial cells in the mammalian enteric nervous system.</a> Glia. 63 (2), 229-241 (2015).</li><li>Shaner, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+monomeric+red,+orange+and+yellow+fluorescent+proteins+derived+from+Discosoma+sp.+red+fluorescent+protein.">Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.</a> Nature Biotechnology. 22 (12), 1567-1572 (2004).</li><li>Madisen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robust+and+high-throughput+Cre+reporting+and+characterization+system+for+the+whole+mouse+brain.">A robust and high-throughput Cre reporting and characterization system for the whole mouse brain.</a> Nature Neuroscience. 13 (1), 133-140 (2010).</li><li>Kabouridis, P. 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The authors have nothing to disclose.

EGCs play important roles in gut homeostasis, and it is essential to isolate and study them in vitro. In this protocol, a simple method for isolating EGCs from the lamina propria of the adult mouse intestine was introduced to study enteric glial function.
Removing the adherent mesentery and LMMP with a cotton swab removes some of the inter-myenteric glia residing between the longitudinal and circular muscle, increases the accessibility of buffers to the submucosal surface and removes ...

Interest in the function of enteric glial cells (EGCs) has steadily increased due to their recognized roles in the gut integrity and homeostasis1,2. In addition, EGCs vary according to their location along the length of the GI tract3,4. EGCs release various trophic factors including glial cell-derived neurotrophic factor (GDNF), contribute to gut motility1,5 and respond to microbial byproducts6,7. Studies have indicated that the EGC population is heterogeneous and that their function varies depending on whether they are submucosal or reside within the myenteric plexus1,7. For example, EGCs within the submucosa contribute to tight junctions8. Differential GFAP expression and phosphorylation in EGCs have been linked to Parkinson&#39;s Disease, suggesting their possible link to the gut phenotype of this disorder9. Recently, it was observed that the loss of the nuclear protein menin in isolated cultures of EGCs from the proximal intestinal submucosa was sufficient to induce expression of the hormone gastrin10. As a result, it was proposed that EGCs might be the origin of duodenal gastrinomas, a type of neuroendocrine tumor10. Collectively, these examples underscore the relevance of studying the behavior and function of isolated EGCs in neuropathic disorders and cancer11.
The challenge in the field remains how to isolate and study either or both EGC populations in vitro. Lineage trace experiments demonstrated that EGCs in the submucosa and lamina propria originate from progenitor cells in the myenteric plexus7. Although there are several published isolation protocols available to generate cultures of myenteric EGCs12,13,14,15,16,17,18,19, none specifically targets isolation of the submucosal/lamina propria EGC population. Existing protocols for EGC isolation specifically use a combination of the mechanical separation or the microdissection of the smooth muscle combined with enzymatic dissociation, eventually discarding the mucosal cell layer.
The goal of this manuscript is to demonstrate the steps to non-enzymatically isolate primary EGCs from the lamina propria for in vitro studies. Since there are no markers that specifically distinguish myenteric EGCs from those in the submucosa, the spatial separation of the epithelial mucosa from the smooth muscle was exploited to isolate submucosal EGCs. In addition, by combining EDTA chelation with non-enzymatic dissociation, EGCs were isolated from the submucosa in contrast to the smooth muscle, which was discarded along with the associated inter-myenteric EGCs. Further separation of the submucosa and lamina propria EGCs occurred by culturing the cells on glial cell-friendly substrates, e.g., poly-D-lysine and laminin.

<table>
	<tbody>
		<tr>
			<td>Poly-D lysine (1 mg/ml stock)</td>
			<td>Sigma</td>
			<td>A-003-E</td>
			<td>Dilute 1:10</td>
		</tr>
		<tr>
			<td>Laminin (0.5 mg/ml stock)</td>
			<td>Sigma</td>
			<td>L4544</td>
			<td>Dilute to 10 &#181;g/mL on ICE</td>
		</tr>
		<tr>
			<td>EDTA (0.5M)</td>
			<td>Lonza</td>
			<td>51201</td>
			<td>Dilute 1:100 in DPBS</td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Corning</td>
			<td>36216004</td>
			<td>Dilute 1:100 in DPBS</td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td>HyClone</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (100X)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin (50mg/mL stock)</td>
			<td>Life Technologies</td>
			<td>15750060</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF (10 &#181;g stock)</td>
			<td>Sigma</td>
			<td>SRP3200</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine (200 mM stock)</td>
			<td>Life Technologies</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-GFAP</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA1-10004</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-a-Smooth Muscle Actin&#160;</td>
			<td>Abcam</td>
			<td>ab112022</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-Pgp9.5&#160;</td>
			<td>Novus Biologicals</td>
			<td>NB600-1160</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-E-cadherin&#160;</td>
			<td>R&#38;D Systems</td>
			<td>AF748</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit S100&#160;</td>
			<td>Abcam</td>
			<td>ab34686</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse p75 NTR&#160;</td>
			<td>Millipore</td>
			<td>MAB5592</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Chicken IgY</td>
			<td>Invitrogen</td>
			<td>A-11039</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey Anti-Goat IgG</td>
			<td>Invitrogen</td>
			<td>A-11055</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11004</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>R-37119</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold antifade Reagent with DAPI</td>
			<td>Thermo Fisher Scientific</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungizone (Amphotericin B) 250 &#181;g/ml</td>
			<td>Life Technologies</td>
			<td>15290-018</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Fura-2-AM</td>
			<td>Invitrogen</td>
			<td>F-14201</td>
			<td></td>
		</tr>
		<tr>
			<td>CCK peptide</td>
			<td>Anaspec, Fremont, CA</td>
			<td>AS-20741</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin peptide (Gastrin-17)</td>
			<td>Abbiotec, Bloomington, IN</td>
			<td>350188</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Celll Strainer (100 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>22363549</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon Mesh Celll Strainer (40 &#181;m)</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Serologic Pipet 5 ml</td>
			<td>Fisher Scientific</td>
			<td>13-678-11D</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1X)</td>
			<td>Life Technologies</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Preps were considered unsuccessful if GFAP+ cells did not adhere and spread within 24 h (Figure 4A). The number of glial cells could not be determined until after 24 h when the cells adhered and showed evidence of spreading into flat aggregates (Figure 4B). Cells at the edge of the clusters tended to extend long processes and expressed classic glial markers, e.g., GFAP, S100b and p75NTR (F...

Watch this Scientific Journal Video about Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse at JoVE.com

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

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 ...

This method can help answer key questions in the neuroscience field, specifically the function of the enteric nervous system. The main advantage of the technique is that it is a rapid, non-enzymatic method to isolate enteric cells from the lamina propria and submucosa rather than the smooth muscle. Begin by identifying the distal stomach and pylorus.Hold the pylorus with forceps while snipping away the mesentery. Then use blunt dissection with the back of the scissors to scrape away adherent pancreas. Then use scissors to remove seven centimeters of the proximal intestine without tearing.Place the intestinal segment into ice cold DPBS without calcium or magnesium. Next, use a five or 10 milliliter syringe attached to a blunt end 18 to 20 gauge needle to flush the fecal contents with ice cold DPBS. Divide the intestine into three centimeter segments to remove the longitudinal muscle.Rapid removal of the mesentery and longitudinal muscle is essential to isolating healthy enteric glia from the lamina propria and submucosa. Next, take a cotton swab and break off about four centimeters to separate the wooden stick from the cotton tip. Soak the wooden stick in DPBS.Slip one of the intestinal segments smoothly onto the wetted stick and then use needle nose forceps to remove any remaining adherent mesentery. Then use a clean razor blade or scalpel to make two longitudinal nicks along the intestine where the mesentery was attached. Hold the tip of the stick between the thumb and forefinger while stabilizing the segment with the middle and fourth finger.Wet the cotton tip with DPBS. Rub the wetted cotton tip longitudinally along the muscle to loosen the Longitudinal Muscle Mesenteric Plexus or LMMP. Then move the cotton tip horizontally to tease away the LMMP from the circular muscle.When complete, the LMMP will easily peel off the intestinal segment. Discard the LMMP unless harvesting EGCs from the mesenteric plexus is desired. Then use a razor blade to flay open the intestinal segment longitudinally to remove the wooden stick.Keep the stripped intestinal tissue consisting predominantly of mucosa and submucosa plus circular muscle in DPBS on ice. Repeat the dissection for other samples until all intestinal segments are prepared. To remove the epithelial mucosa, first use fine scissors to cut the intestines into approximately 0.5 centimeter pieces and collect the pieces in 30 milliliters of ice cold EDTA HEPES DPBS solution.Rock the tissue containing solution at four degrees Celsius for 10 minutes at approximately 60 tilts per minute. Pre-moisten a five milliliter plastic pipette by pipetting the EDTA HEPES DPBS buffer up and down one time and then use the pre-moistened pipette to triturate the tissue and buffer suspension 20 times to dislodge the epithelial mucosa. Collect the tissue by pouring the mixture through a 100 micron nylon cell strainer.Then use needle nose forceps to place the tissue retained in the strainer into a new 50 milliliter conical centrifuge tube containing 30 milliliters of ice cold EDTA HEPES DPBS. Repeat the EDTA HEPES DPBS incubation a second time by rocking the tissue at four degrees Celsius for 10 minutes at approximately 60 tilts per minute to strip off additional epithelial mucosa. Perform a gentle trituration using a pre-moistened five milliliter pipette.The tissue will tend to stick to the inside of the pipette as more of the epithelium is removed. Gentle trituration is required at this point since the enteric glia cells are more fragile. Collect the tissue after the second incubation by pouring the mixture through a 100 micron nylon cell strainer and discard the flow through.The second flow through should be noticeably less turbid than the first. Repeat the 10-minute EDTA incubation until the solution is nearly clear. Transfer the tissue from the nylon strainer to a 15 milliliter conical centrifuge tube containing five milliliters of the commercially available cell recovery solution.Then rock for 25 to 30 minutes at four degrees Celsius. Gently triturate 10 times to dissociate the enteric glial cells from the lamina propria. Then filter through a 40 micron filter and collect the filtrate into a clean 50 milliliter tube.Rinse the tissue on the nylon filter with one milliliter of DPBS. Discard the tissue and transfer the five to six milliliters of filtrate to a clean 15 milliliter conical centrifuge tube. Spin the filtrate at 2, 000 times g in a swinging bucket centrifuge for five minutes at four degrees Celsius.Resuspend the glial cell containing pellet in at least one milliliter of resuspension buffer by gently pipetting the pellet up and down with the 500 microliter pipette tip without introducing bubbles. Finally, plate the enteric glial cells by adding 200 microliters of the cell suspension into each well of a laminin-coated six-well plate or 100 microliters to each well of a 12-well plate containing glial growth media. Prep should be considered unsuccessful if enteric glial cells do not adhere and spread within 24 hours as seen here.Here is a representative example of an excellent prep 24 hours after cell isolation and resuspension where enteric glial cells have adhered in patches to the PDL laminin-coated plate. The number of glial cells can be determined after 24 hours when the cells adhere and display evidence of spreading into flat aggregates. Cells at the edge of the clusters tend to extend long processes and expressed classic glial markers such as GFAP, S100B, and p75NTR.Following this procedure, other methods like immunohistochemistry, western blots, flow cytometry, and imaging calcium flux can be performed in order to answer additional questions on enteric glial cell heterogeneity. After its development, this technique has paved the way for researchers studying the enteric nervous system to explore glial cell biology and its impact on the adjacent mucosa and microbiota in mice and by extension in humans.

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Research

JoVE Journal

Biology

12.7K ビュー.  University of Michigan. This method can help answer key questions in the neuroscience field, specifically the function of the enteric nervous system. The main advantage of the technique is that it is a rapid, non-enzymatic method to isolate enteric cells from the lamina propria and submucosa rather than the smooth muscle. Begin by identifying the distal stomach and pylorus.Hold the pylorus with forceps while snipping away the mesentery. Then use blunt dissection with the back of the scissors to scrape away ad...