Authors have nothing to disclose.

The protocol to perform animal experiments, the rationale and objectives of the research, were submitted and approved by the University of Southern California Institutional Animal Care and Use Committee.
The protocol for establishment of primary cultures from de-identified human esophagectomy specimens was approved by the University of Southern California Institutional Review Board.
1. Obtain Mouse or Human Esophagus
<ol>
	<li>Obtain Mouse Esophagus:
	<ol>
		<li>Euthanize the 8-12 day old mouse with 2% isoflurane inhalant overdose followed by cervical dislocation.</li>
		<li>Pin the animal down with belly facing up and wet the ventral surface with 70% ethanol. With forceps, grab and lift up the skin anterior to the urethral opening with forceps. Use standard straight surgical scissors to cut along the ventral midline from the urethral opening to the chin. Be careful to cut only the skin.</li>
		<li>Make an incision from the urethral opening downward to the knee on both sides of the animal, forming an upside down &#8220;Y&#8221;. Make another incision on both sides of the animal along the rib cage.</li>
		<li>Carefully peel the skin on both sides, off of the underlying peritoneum and the rib cage, and lay to the sides. Examine the rib cage overlying the thoracic cavity and the peritoneum overlying the abdominal cavity.
		<ol>
			<li>Lift up the peritoneum with forceps and cut through it up to the diaphragm and rib cage, pointing scissors upward to prevent damage to abdominal contents. Gently lift up the left lobe of the liver, expose the underlying stomach, the esophageal-gastric junction and the abdominal portion of the esophagus.</li>
		</ol>
		</li>
		<li>Use autoclaved surgical scissors to cut through the sternum and ribcage along the midline up to the cervical girdle. Point scissors upward to prevent damage to the thoracic contents. Fully expose the contents of the thoracic cavity by pulling the rib cage to the sides.</li>
		<li>Gently remove the heart and both lobes of the lungs. Absorb excess blood with Q-tips. The thoracic esophagus is a narrow, flexible tube lying posterior to the trachea and anterior to the thoracic vertebra. 
		NOTE: The esophagus can be difficult to identify in murine neonates. Localization of the esophageal-gastric junction makes esophageal identification straightforward.</li>
		<li>Carefully follow the esophagus up from the stomach, dissecting the surrounding vasculature, fat and mesentery all the way up to the esophageal origin in the cervical cavity. Surgical scissors with blunt tip work best for this purpose.</li>
		<li>Resect the entire length of the esophagus and place in Hanks&#8217; balanced salt solution (HBSS) for further processing described below. Remove a portion of the stomach for orientation purposes if desired. 
		NOTE: Due to small tissue size, separation of muscularis propria from the muscularis mucosa is not achievable in murine neonate esophagus and the entire esophagus is subjected to processing described below.</li>
	</ol>
	</li>
	<li>Obtain Human Esophagus:
	<ol>
		<li>Wash esophageal resection specimens (typically 5 cm or less) with HBSS and remove any attached connective tissue, fat, or vasculature.</li>
		<li>Resect a portion of the specimen and place in formalin for future histologic examination if desired.</li>
		<li>Separate the remaining mucosa from underlying muscularis propria by sharp dissection.</li>
		<li>Cut mucosa into sub centimeter fragments and subject to the protocol described below. 
		NOTE: Human esophagus resections can be stored in PBS for up to 6 hr prior to processing described below. Source of esophagectomy specimens will dictate specimen size and will be laboratory dependent.</li>
	</ol>
	</li>
</ol>
2. Isolate Murine and Human Esophageal Stromal Cells
<ol>
	<li>Isolate esophageal stromal cells from murine and human tissue by a combination of mechanical and enzymatic digestion.
	<ol>
		<li>Mechanically digest by mincing the tissue with scissors and multiple washes with HBSS. Enzymatically digest by incubating the tissue with 300 U/ml collagenase XI and 0.1 mg/ml dispase for 25 min (murine tissue) or for up to 6 hr (human tissue). Ensure that instruments used for mincing have been autoclaved and sterilized. 
		NOTE: Collagenases are enzymes that break down collagen, an extracellular matrix protein responsible for holding animal tissue together. Collagenase XI has high collagenase activity and has seen success in this isolation protocol. Dispase is a bacterial enzyme with mild proteolytic dissociation activity that preserves cell membrane activity and is used in combination with collagenase as a secondary enzyme.</li>
		<li>Place mucosal fragments obtained from mouse or human tissue in 1.7 ml micro-centrifuge or 5 ml tubes respectively, containing HBSS. Mince tissue further into 2-3 mm pieces using scissors with an open tip width that will fit into respective tubes. Allow enough time to allow for esophageal mucosal fragments to sediment to the bottom of the tube.</li>
		<li>Slowly decant HBSS, being careful not to inadvertently discard the tissue. Replace with fresh HBSS following by gentle shaking. Alternatively, remove HBSS with a 1 ml pipette.</li>
		<li>Wash tissue in this manner a total of 8 times with gentle shaking in between washes allowing time for esophageal fragments to sediment between each wash. 
		NOTE: Human tissue will require more mincing that neonate mouse tissue.</li>
	</ol>
	</li>
	<li>Incubate murine tissue with 300 U/ml collagenase XI and 0.1 mg/ml dispase for 25 min on a rocking shaker set at a slow speed at room temperature.
	<ol>
		<li>Incubate human tissue with 300 U/ml XI collagenase and 0.1 mg/ml dispase for up to 6 hr, on a rocking shaker set at a slow speed at room temperature. 
		NOTE: Availability of human esophagus is variable. Human esophageal mucosa incubated with enzymes for up to 6 hr results in successful generation of primary cultures.</li>
	</ol>
	</li>
	<li>After enzymatic digestion, mince tissue further with scissors and centrifuge at 200 x g for 10 min.</li>
	<li>Discard the supernatant. Transfer the pellet, consisting of a mixed cell suspension to a 5 ml tube and wash 5 times in Dulbecco&#8217;s Modified Eagle Medium (DMEM) supplemented with 2% sorbitol to eliminate nonviable cells and debris.</li>
	<li>Seed cells in 6-well plates and culture in DMEM with 10% fetal bovine serum (FBS), 10 mg/ml insulin, 10 &#181;g/ml transferrin,10 &#181;g/ml gentamicin, and 2 ng/ml epidermal growth factor (EGF). Vacuum filter (0.22 &#181;m) all components except EGF which can be added after filtration. Distribute cells obtained from human resection specimens among multiple 6-well plates, depending on the resection size.</li>
	<li>Once wells are 80% confluent, passage adherent cells to T25 flasks using 0.05% trypsin/ethylenediaminetetraacetic acid (EDTA). Neutralize with media and spin at 400 x g.
	<ol>
		<li>For a 6 well passage to a T25 flask, passage cells at a 1:1 ratio. Confluent T25 flasks can be passaged at a 1:2 or 1:3 ratio. To passage from a T25 flask to a T75 flask, use a 1:1 ratio. Change media every 2-3 days regardless of the vessel.</li>
		<li>Grow cells at 37 &#186;C in a humidified 5% CO2 incubator and culture in the myofibroblast media described above. 
		NOTE: Epithelial cells do not survive under these culture or passage conditions. Cells used for characterization and in the studies described below are between passages 5 and 15.</li>
		<li>Cryo-preserve cells in liquid N2 by adding 10% dimethyl sulfoxide to the myofibroblast media.</li>
	</ol>
	</li>
</ol>
3. Characterize Murine and Human Esophageal Stromal Cells
<ol>
	<li>Examine the morphology of cells with an inverted microscope. Observe the spindle-shaped morphology of adherent cells (Figure 1).</li>
	<li>Characterize cultured cells by evaluation of the following cytoskeletal markers: &#945;-SMA, vimentin, desmin, and cytokeratin. Stromal cells with a myofibroblast-like phenotype express cytoskeletal myofibroblast markers &#945;-SMA and vimentin (Figure 2).
	<ol>
		<li>To further distinguish cultured stromal cells from muscle cells, immunostain for desmin. To perform immunostaining on cultured cells, plate 1.5 x 104 cells in 4-well chamber slides and grow in myofibroblast media for 24 hr. Stromal cells with a myofibroblast-like phenotype will have weak or absent desmin expression. Stromal cells lack expression of the epithelial marker pan-cytokeratin (data not shown). 
		NOTE: Assessment of cultures includes evaluation of cytoskeletal markers by immunocytochemistry and cell surface markers by flow cytometry. Immunocytochemistry is used as the primary method of characterization as the expression profile of cytoskeletal proteins is unique for the fibroblast, myofibroblast or muscle cell. Flow cytometry establishes culture purity by evaluating cell surface expression of epithelial, endothelial, and hematopoietic cells markers. CD90 is helpful as an adjunctive method of identifying human stromal cells as it is expressed on both human fibroblasts and myofibroblasts. CD90 is not helpful in characterization of murine stromal cells as it also expressed by murine T cells.</li>
	</ol>
	</li>
	<li>After cells reach 60% confluence, rinse slides with phosphate-buffered saline (PBS) and fix with methanol. Incubate cells in methanol for 10 min at -20 &#176;C, then store in PBS at 4 &#176;C until use.</li>
	<li>Follow standard immunostaining protocol.</li>
	<li>Briefly, prior to applying the primary antibody, block the fixed cells with 5% goatserum in PBS. Dilute primary and secondary antibodies in 5% goat serum as well.
	<ol>
		<li>For detection of &#945;-SMA and vimentin, use the following antibodies and concentrations: Mouse mAB to &#945;-SMA at 1:250 dilution and Rabbit pAB to vimentin at 1:500 dilution.</li>
		<li>Incubate with primary antibody at room temperature overnight at 4 &#176;C, rinse 3 times with PBS and add secondary antibodies Rhodamine conjugated Goat anti mouse at a dilution of 1:200 and Cy2 conjugated Goat anti Rabbit at a dilution of 1:1,000 for 1 hr at room temperature.</li>
		<li>Counterstain nuclei with 4&#8217;, 6-Diamidino-2Phenylindole (DAPI) at a concentration of 1 &#181;g/ml for 1 min and then wash with PBS. Analyze slides with an inverted microscope.</li>
	</ol>
	</li>
	<li>Establish purity of cultures by examination of murine and human esophageal cells for hematopoietic (CD45) and endothelial (CD31) cell surface markers by flow cytometry (Figure 3). Characterize human cells further by expression of the stromal cell surface marker CD90. 
	NOTE: CD90 cannot be used to identify murine stromal cells as murine T cells also express CD90.
	<ol>
		<li>Detach cultured mouse esophageal stromal cells from the culture flasks by treatment with non-enzymatic Cell Dissociation Solution at 37 &#176;C for 10 min, washed with FACS buffer twice, counted and stained with FITC-CD45 and APC-CD31 with relevant isotype controls, according to standard staining procedures.</li>
		<li>Collect cells using FACS CAlibur and analyze data with FlowJo software. Prior to staining cells, optimize antibodies and titrate concentrations using isotype controls, normal mouse spleen, and bone marrow cells.</li>
	</ol>
	</li>
</ol>

<ol><li>Stappenbeck, T. S., Miyoshi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+stromal+stem+cells+in+tissue+regeneration+and+wound+repair%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The role of stromal stem cells in tissue regeneration and wound repair.</a> Science. 324 (5935), 1666-1669 (2009).</li>
<li>Powell, D. W., Pinchuk, I. V., Saada, J. I., Chen, X., Mifflin, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mesenchymal+cells+of+the+intestinal+lamina+propria%5BTitle%5D)+AND+%22Annu+Rev+Physiol%22%5BJournal%5D)">Mesenchymal cells of the intestinal lamina propria.</a> Annu Rev Physiol. 73, 213-237 (2011).</li>
<li>Rieder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((T-Helper+2+Cytokines%2C+Transforming+Growth+Factor+beta1%2C+and+Eosinophil+Products+Induce+Fibrogenesis+and+Alter+Muscle+Motility+in+Patients+With+Eosinophilic+Esophagitis%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">T-Helper 2 Cytokines, Transforming Growth Factor beta1, and Eosinophil Products Induce Fibrogenesis and Alter Muscle Motility in Patients With Eosinophilic Esophagitis.</a> Gastroenterology. 146 (5), 1266-1277 (2014).</li>
<li>Powell, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Paracrine+cells+important+in+health+and+disease%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">Paracrine cells important in health and disease.</a> The American Journal of Physiology. 277 (1 Pt. 1), C1-9 (1999).</li>
<li>Powell, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+subepithelial+myofibroblasts%5BTitle%5D)+AND+%22The+American+Journal+of+Physiology%22%5BJournal%5D)">Intestinal subepithelial myofibroblasts.</a> The American Journal of Physiology. 277 (2 Pt. 1), C183-201 (1999).</li>
<li>Shaker, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stromal+cells+participate+in+the+murine+esophageal+mucosal+injury+response%5BTitle%5D)+AND+%22American+Journal+of+Physiology+Gastrointestinal+and+Liver+Physiology%22%5BJournal%5D)">Stromal cells participate in the murine esophageal mucosal injury response.</a> American Journal of Physiology Gastrointestinal and Liver Physiology. 304 (7), 662-672 (2013).</li>
<li>Shaker, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epimorphin+deletion+protects+mice+from+inflammation-induced+colon+carcinogenesis+and+alters+stem+cell+niche+myofibroblast+secretion%5BTitle%5D)+AND+%22The+Journal+of+Clinical+Investigation%22%5BJournal%5D)">Epimorphin deletion protects mice from inflammation-induced colon carcinogenesis and alters stem cell niche myofibroblast secretion.</a> The Journal of Clinical Investigation. 120 (6), 2081-2093 (2010).</li>
<li>Kalabis, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+characterization+of+mouse+and+human+esophageal+epithelial+cells+in+3D+organotypic+culture%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Isolation and characterization of mouse and human esophageal epithelial cells in 3D organotypic culture.</a> Nature Protocols. 7 (2), 235-246 (2012).</li>
<li>Khalil, H., Nie, W., Edwards, R. A., Yoo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+primary+myofibroblasts+from+mouse+and+human+colon+tissue%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Isolation of primary myofibroblasts from mouse and human colon tissue.</a> Journal of Visualized Experiments. (80), (2013).</li>
<li>Rieder, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gastroesophageal+reflux+disease-associated+esophagitis+induces+endogenous+cytokine+production+leading+to+motor+abnormalities%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Gastroesophageal reflux disease-associated esophagitis induces endogenous cytokine production leading to motor abnormalities.</a> Gastroenterology. 132 (1), 154-165 (2007).</li>
</ol>

This work is supported by NIH/NCI K08CA153036-01A1 (AS), AGA-General Mills Bell Institute of Health and Nutrition Research Scholar Award in Gut Physiology and Health (AS) and a Wright Foundation Pilot Award (AS).

The techniques for primary culture generation are generally similar when using murine or human tissue with mincing and digestion times adapted for tissue size. Our experience with murine tissue suggests that using the methods described above consistently result in successful establishment of primary cultures. Critical steps within the protocol include sterilization of surgical equipment and following standard sterile techniques of tissue culture. The age of the mice is also a critical step. 8 - 12 day old neonates consistently yield successful primary cultures. Stromal cells isolated from younger neonates have not been not been successfully generated. Attempts to establish myofibroblast-like stromal cells from older mice are ongoing. Others have reported successful generation of esophageal fibroblasts from four to eight week old mice8.
Unlike establishment of colonic myofibroblasts, contamination is an infrequent issue in establishment of cultures of murine myofibroblasts when antibiotics are included in the culture medium. Because cultures are established from neonate mice, a limiting factor is small tissue size and stripping of muscularis propria is not readily feasible. On the other hand, in the human esophagus, muscularis propria is easily distinguishable and separated from the muscularis mucosa. The enzymatic digestion and culture conditions are not conducive to growth of immune or epithelial cells in cultures established from mouse or human esophagus. Human resection specimens remain vulnerable to bacterial contamination despite the use antibiotics in the culture medium, perhaps because of the larger quantify of tissue undergoing processing. Following standard tissue culture techniques and rigorously sterilizing surgical instruments mitigates risk of contamination.
The advantage of primary culture is that these cells are relatively unmanipulated and may better reflect the in vivo environment compared to immortalized cell lines9.
The described technique is straight-forward and is within the means of most laboratories. The disadvantage of primary cultures versus cell lines is that cells beyond passage 15 are susceptible to genetic mutation and eventual senescence. In addition,primary cultures obtained from mice or humans have inherent variability not observed in cell lines. Alternative methods for esophageal fibroblast isolation have been described10. Characterization of these cells in culture however has been limited to vimentin expression or have demonstrated cells that express vimentin and are only weakly &#945;-SMA positive10.
Once this technique has been mastered, primary cultures can be established from resection specimens of diseased human esophagus. These methods provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Cultured myofibroblast-like stromal cells can also be potentially adapted for use in organotypic or co-culture models with other cells such as eosinophils3 or in organotypic culture8.

Epithelial-stromal interactions are involved in the regulation of a variety of gastrointestinal tract functions including mucosal regeneration, repair, fibrosis, and carcinogenesis1,2. These interactions have been best studied in the small intestine and colon and may similarly play a role in esophageal mucosal disorders3. A subpopulation of intestinal and colonic stromal cells termed myofibroblasts has been demonstrated to participate in mediating tissue injury, inflammation and repair4,5. In the distal GI tract, these spindle shaped cells are located adjacent to the basement membrane at the interface between the epithelium and lamina propria and are defined as &#945;-SMA and vimentin positive, pan-cytokeratin negative, and weakly positive or desmin negative5.
The esophageal stroma has not been rigorously characterized at a cellular or molecular level. Our work in the murine esophagus has demonstrated &#945;-SMA and vimentin cells in the esophageal stroma, occasionally subjacent to the squamous epithelium6. Epithelial-stromal interactions have been implicated in esophageal mucosal disorders such as gastro-esophageal mediated injury6 and eosinophilic esophagitis3. Fibrotic strictures are also a known complication of esophageal injury and stromal cells have been implicated in the pathogenesis of gastrointestinal fibrosis. Isolation of these cells will help accomplish the necessary studies to investigate deranged signaling pathways.
This submission provides the techniques necessary to establish primary cultures of &#945;-SMA positive, vimentin positive myofibroblasts such that existing gaps in knowledge regarding signaling pathways mediating these interactions can be addressed. The technique described has been successfully used by the authors to establish primary murine colonic myofibroblasts7 and further adapted for establishment of murine6 and human myofibroblast-like esophageal stromal cells.
Herein we describe conditions needed to establish and characterize these cultures established from mouse or human esophagus prior to use in future functional studies. Cultures can be grown and utilized for up at 15 passages. Isolation and establishment of primary cultures via the methods outlined below generates stromal cells with a myofibroblast phenotype; &#945;-SMA, vimentin positive, and weakly positive or negative for desmin, and cytokeratin negative. This phenotype is distinct from the phenotype of the esophageal fibroblast which is predominantly vimentin positive, &#945;-SMA negative3 or the &#945;-SMA positive, vimentin negative phenotype of the muscularis mucosae6.

<table><tbody><tr><td colspan="4">Tissue culture reagents</td></tr><tr><td>HBSS</td><td>Sigma Aldrich</td><td>H6648</td><td></td></tr><tr><td>Dispase</td><td>GIBCO, Invitrogen</td><td>17105-041</td><td></td></tr><tr><td>Collagenase XI</td><td>Sigma-Aldrich</td><td>C9407</td><td></td></tr><tr><td>DMEM</td><td>GIBCO, Invitrogen</td><td>11965-092</td><td></td></tr><tr><td>Sorbitol</td><td>Sigma-Aldrich</td><td>S1876</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F42442</td><td></td></tr><tr><td>Transferrin</td><td>Roche</td><td>10-652-202-001</td><td></td></tr><tr><td>trypsin/EDTA</td><td>Corning</td><td>25-052-Cl</td><td></td></tr><tr><td>Epidermal growth factor</td><td>Sigma-Aldrich</td><td>E9644</td><td></td></tr><tr><td colspan="4">Reagents for immunostaining</td></tr><tr><td>Goat serum</td><td>Sigma Aldrich</td><td>G9023</td><td></td></tr><tr><td>Mouse mAB to &amp;alpha;-SMA&amp;nbsp;</td><td>abcam</td><td>ab7817</td><td></td></tr><tr><td>Rabbit pAB to vimentin</td><td>abcam</td><td>ab45939</td><td></td></tr><tr><td>Cy2 conjugated Goat anti Rabbit</td><td>Jackson ImmunoResearch</td><td>111-225-144</td><td></td></tr><tr><td>DAPI</td><td>Sigma-Aldrich</td><td>D8417</td><td></td></tr><tr><td>CD31 conjugated to eFluor 450</td><td>eBioscience</td><td>48-0319-41</td><td></td></tr><tr><td>CD90 conjugated to APC</td><td>eBioscience</td><td>17-0909-41</td><td></td></tr><tr><td>Annexin V and 7AAD</td><td>BD Pharmigen</td><td>559763</td><td></td></tr><tr><td>Mouse Fc block for CD16/CD32</td><td>BD Pharmigen</td><td>2136662</td><td></td></tr><tr><td colspan="4">Equipment</td></tr><tr><td>5 ml tube</td><td>Eppendorf</td><td>30108310</td><td></td></tr><tr><td>Inverted microscope</td><td>Motic</td><td>AE31</td><td></td></tr><tr><td>Biosafety cabinet and incubators</td><td>Nuaire</td><td></td><td>&lt;a href=&quot;http://www.nuaire.com/products/&quot; target=&quot;_blank&quot;&gt;http://www.nuaire.com/products/&lt;/a&gt;</td></tr><tr><td>4-well chamber slides</td><td>Thermo Scientific</td><td>177437</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>Olympus Vacuum-Driven Filter System</td><td>Genesee Scientific</td><td>25-227</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Nikon</td><td>Eclipse TE300</td><td></td></tr><tr><td>6-well plates</td><td>Corning</td><td>3516</td><td></td></tr><tr><td>T25 Flasks</td><td>TRP</td><td>90026</td><td></td></tr><tr><td>T75 Flasks</td><td>Corning</td><td>43064</td><td></td></tr><tr><td>Dissection scissors</td><td></td><td></td><td></td></tr><tr><td>Dissection forceps</td><td></td><td></td><td></td></tr><tr><td>Single tipped Q-Tips</td><td>Kendall</td><td>540500</td><td></td></tr><tr><td colspan="4">Software</td></tr><tr><td>Metamorph software (Molecular Devices)</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>FACSCAlibur</td><td>BD Bioscience</td><td></td><td></td></tr><tr><td>FACSVerse</td><td>BD Bioscience</td><td></td><td></td></tr></tbody></table>

isolation of myofibroblasts from mouse and human esophagus

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, and subjected to enzymatic digestion with collagenase and dispase for 25 min. Human esophageal resection specimens are stripped of muscularis propria and adventitia and the remaining mucosa is minced, and subjected to enzymatic digestion with collagenase and dispase for up to 6 hr. Cultured cells express &#945;-SMA and vimentin and express desmin weakly or not at all. Culture conditions are not conducive to growth of epithelial, hematopoietic, or endothelial cells. Culture purity is further confirmed by flow cytometric evaluation of cell surface marker expression of potential contaminating hematopoietic and endothelial cells. The described technique is straightforward and results in consistent generation of non-hematopoieitc, non-endothelial stromal cells. Limitations of this technique are inherent to the use of primary cultures in molecular biology studies, i.e., the unavoidable variability encountered among cultures established across different mice or humans. Primary cultures however are a more representative reflection of the in vivo state compared to cell lines. These methods also provide investigators the ability to isolate and culture stromal cells from different clinical and experimental conditions, allowing comparisons between groups. Characterized esophageal stromal cells can also be used in functional studies investigating epithelial-stromal interactions in esophageal disorders.

Esophageal stromal cells isolated using mechanical and enzymatic digestion are initially plated and cultured in 6 well plates. Examination of cells with an inverted microscope within hours of plating demonstrates a mixed cell suspension loosely adherent to the plate bottom (Figure 1A). Over the next 24 hr, spindle-shaped cells firmly adherent to the plate bottom are observed shooting from the mixed cell suspension (Figure 1B).These sprouting cells cover the entire area of a well within 5 days and can be passaged successfully, retaining morphology for at least 15 passages. Spindle-shaped adherent cells are observed at low density (Figure 1C) and when near-confluent (Figure 1D).
Immunostaining of primary cultures of esophageal stromal cells grown in chamber slides demonstrates abundant expression of myofibroblast cytoskeletal markers &#945;-SMA and vimentin (Figure 2), weak expression of desmin, and absent cytokeratin.
Primary cultures of esophageal myofibroblasts can be further examined for cell purity by examination of hematopoietic and endothelial cell surface markers. Forward and side scatter are established for primary murine esophageal stromal cells followed by gating and analysis of live cells for cell surface proteins (Figure 3A). Primary murine esophageal stromal cells lack expression of hematopoietic CD45 (Figure 3B) and endothelial cell CD31 (Figure 3C) cell surface markers.
<img alt="Figure 1" src="/files/ftp_upload/52215/52215fig1.jpg" /> 
Figure 1: Primary cultures of murine esophageal stromal cells at different passages. Examination of murine stromal cells with an inverted microscope within hours of isolation and plating demonstrates a cluster of mixed cells loosely adherent cells to the plate bottom (A). After 24-48 hours in culture, spindle-shaped cells sprout from the now adherent cluster (B). Spindle-shaped morphology of adherent cells persists at low (C) and high (D) density. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52215/52215fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a> (Figures used with permission from Shaker et al.6).
<img alt="Figure 2" src="/files/ftp_upload/52215/52215fig2.jpg" /> 
Figure 2: Murine esophageal stromal cells grown in primary culture express myofibroblast markers &#945;-SMA and vimentin. Primary murine esophageal stromal cells were grown on chamber slides and immunostained for &#945;-SMA and vimentin at moderate (A-C) and low density (D-F). Esophageal stromal cells abundantly express &#945;-SMA (A, D) and vimentin (B, E). Murine esophageal stromal cells co-express these myofibroblast markers (C, F). (Figures used with permission from Shaker et al.6). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52215/52215fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52215/52215fig3.jpg" /> 
Figure 3: Primary cultures of murine esophageal myofibroblasts cells lack hematopoietic and endothelial cell surface markers. The dot plot of murine esophageal stromal cells, demonstrates the FSC and SSC characteristics of this population of cells. A total of 32.4% of events were gated (A), Expression of hematopoietic (CD45) and endothelial (CD31) cell surface markers were evaluated by a single immunostain according to standard staining protocols. Nonspecific staining was determined for each antibody using isotype controls. Primary cultures of murine esophageal stromal cells do not express CD45 (B) or CD31 (C). FSC: forward scatter; SSC: side scatter. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52215/52215fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Myofibroblasts from Mouse and Human Esophagus at JoVE.com

Isolation of Myofibroblasts from Mouse and Human Esophagus

We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express &#945;-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions.

Murine and human esophageal myofibroblasts are generated via enzymatic digestion. Neonate (8-12 day old) murine esophagus is harvested, minced, washed, ...

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Nanyang Technological University

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JoVE Journal

Biology

10.9K Views.  Keck School of Medicine, University of Southern California. We present a protocol to generate primary cultures of murine and human esophageal stromal cells with a myofibroblast phenotype. Cultured cells have spindle shaped morphology, express α-SMA and vimentin, and lack epithelial, hematopoietic and endothelial cell surface markers. Characterized stromal cells can be used in functional studies of epithelial-stromal interactions. 

Video: Isolation of Myofibroblasts from Mouse and Human Esophagus

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Developmental Biology

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Bioengineering

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Ultrasonic-augmented Primary Adult Fibroblast Isolation

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary fibroblasts cannot divide indefinitely due to myofibroblast differentiation or senescence induction, new cultures must be established regularly. However, there are several obstacles to overcome during the processes of developing a reliable isolation protocol and primary fibroblast isolation itself: the method&#8217;s degree of difficulty (especially for beginners), the risk of bacterial contamination, the required time until primary fibroblasts can be used for experiments, and subsequent cell quality and viability. In this study, a fast, reliable and easy-to-learn protocol to isolate and culture primary adult fibroblasts from mouse heart, lung, liver and kidney combining enzymatic digestion and ultrasonic agitation is provided.

We present a protocol to isolate primary adult fibroblasts in an easy, fast and reliable way, performable by beginners (e.g., students). The procedure combines enzymatic tissue digestion and mechanical agitation with ultrasonic waves to obtain primary fibroblasts. The protocol can easily be adapted to specific experimental requirements (e.g., human tissue).

Primary adult fibroblasts have become an important tool to study fibrosis, fibroblast interactions and inflammation in all body tissues. Since primary ...

Medicine

Human Esophageal Organoids and Air-Liquid Interface Culture Protocols

Three-Dimensional Cell Culture Models to Investigate the Epithelial Barrier in Eosinophilic Esophagitis

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and microbes. Maintaining the integrity of the epithelial barrier is critical for preventing infections and avoiding inflammation caused by harmless food-derived antigens. This article provides simplified protocols for generating human esophageal organoids and air-liquid interface cultures from patient biopsies to study the epithelial compartment of the esophagus in the context of tissue homeostasis and disease. These protocols have been significant scientific milestones in the last decade, describing three-dimensional organ-like structures from patient-derived primary cells, organoids, and air-liquid interface cultures. They offer the possibility to investigate the function of specific cytokines, growth factors, and signaling pathways in the esophageal epithelium within a three-dimensional framework while maintaining the phenotypic and genetic properties of the donor. Organoids provide information on tissue microarchitecture by assessing the transcriptome and proteome after cytokine stimulation. In contrast, air-liquid interface cultures allow the assessment of the epithelial barrier integrity through transepithelial resistance (TEER) or macromolecule flux measurements. Combining these organoids and air-liquid interface cultures is a powerful tool to advance research in impaired esophageal epithelial barrier conditions.

Author Spotlight: Investigating the Pathophysiology of Eosinophilic Esophagitis

Here, a protocol for the culture of human esophageal organoids and air-liquid interface culture is provided. Esophageal organoids' air-liquid interface culture can be used to study the impact of cytokines on the esophageal epithelial barrier.

The squamous epithelium of the esophagus is directly exposed to the environment, continuously facing foreign antigens, including food antigens and ...

Immunology and Infection

Single Myofiber Isolation and Culture from a Murine Model of Emery-Dreifuss Muscular Dystrophy in Early Post-Natal Development

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate filament proteins that sustain the nuclear envelope and the components of the nucleoplasm. We recently reported that muscle wasting in EDMD can be ascribed to intrinsic epigenetic dysfunctions affecting muscle (satellite) stem cells regenerative capacity. Isolation and culture of single myofibers is one of the most physiological ex-vivo approaches to monitor satellite cells behavior within their niche, as they remain between the basal lamina surrounding the fiber and the sarcolemma. Therefore, it represents an invaluable experimental paradigm to study satellite cells from a variety of murine models. Here, we describe a re-adapted method to isolate intact and viable single myofibers from post-natal hindlimb muscles (Tibialis Anterior, Extensor Digitorum Longus, Gastrocnemius and Soleus). Following this protocol, we were able to study satellite cells from Lamin &#916;8-11 -/- mice, a severe EDMD murine model, at only 19 days after birth.
We detail the isolation procedure, as well as the culture conditions for obtaining a good amount of myofibers and their associated satellite-cells-derived progeny. When cultured in growth-factors rich medium, satellite cells derived from wild type mice activate, proliferate, and eventually differentiate or undergo self-renewal. In homozygous Lamin &#916;8-11 -/- mutant mice these capabilities are severely impaired.
This technique, if strictly followed, allows to study all processes linked to the myofiber-associated satellite cell even in early post-natal developmental stages and in fragile muscles.

Here, we propose a method to efficiently obtain single muscle fibers at early post-natal developmental stages from homozygous mutant Lamin &#916;8-11 mouse model, a very severe model for Emery-Dreifuss muscular dystrophy (EDMD).

Autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the LMNA gene, which encodes the A-type nuclear lamins, intermediate ...

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.

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, ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Functional Characterization and Visualization of Esophageal Fibroblasts Using Organoid Co-Cultures

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescence-activated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively high-throughput method for identifying and understanding esophageal stem cell niche components in vitro.

Organoid-fibroblast co-cultures provide a model to study the in vivo stem cell niche. Here, a protocol for esophageal organoid-fibroblast co-cultures is described. Additionally, whole mount imaging is used to visualize the fibroblast-organoid interaction.

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell ...

Isolation of Myofibroblasts from Mouse and Human Esophagus

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