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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+stromal+stem+cells+in+tissue+regeneration+and+wound+repair.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cells+of+the+intestinal+lamina+propria.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-Helper+2+Cytokines,+Transforming+Growth+Factor+beta1,+and+Eosinophil+Products+Induce+Fibrogenesis+and+Alter+Muscle+Motility+in+Patients+With+Eosinophilic+Esophagitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+cells+important+in+health+and+disease.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+subepithelial+myofibroblasts.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+cells+participate+in+the+murine+esophageal+mucosal+injury+response.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epimorphin+deletion+protects+mice+from+inflammation-induced+colon+carcinogenesis+and+alters+stem+cell+niche+myofibroblast+secretion.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+mouse+and+human+esophageal+epithelial+cells+in+3D+organotypic+culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+myofibroblasts+from+mouse+and+human+colon+tissue.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastroesophageal+reflux+disease-associated+esophagitis+induces+endogenous+cytokine+production+leading+to+motor+abnormalities.">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 consis...

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 border="0" cellpadding="0" cellspacing="0" width="1647">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:549px;">Name</td>
			<td style="width:549px;"></td>
			<td style="width:549px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture reagents</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HBSS&#160;</td>
			<td>Sigma Aldrich</td>
			<td>H6648</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dispase&#160;</td>
			<td>GIBCO, Invitrogen</td>
			<td>&#160;17105-041</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">collagenase XI&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C9407&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM&#160;</td>
			<td>GIBCO, Invitrogen&#160;</td>
			<td>11965-092</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">sorbitol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S1876</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FBS</td>
			<td>Sigma)</td>
			<td>F42442</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">transferrin&#160;</td>
			<td>Roche</td>
			<td>10-652-202-001</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin/EDTA&#160;</td>
			<td>Corning</td>
			<td>25-052-Cl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">epidermal growth factor&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E9644</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin/EDTA&#160;</td>
			<td>Corning</td>
			<td>&#160;25-052-Cl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reagents for immunostaining</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">goat serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse mAB to &#945;-SMA&#160;</td>
			<td>abcam</td>
			<td>ab7817</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit pAB to vimentin&#160;</td>
			<td>abcam</td>
			<td>ab45939</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cy2 conjugated Goat anti Rabbit&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-225-144</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI&#160;</td>
			<td>sigma-aldrich</td>
			<td>D8417</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD31 conjugated to eFluor 450&#160;</td>
			<td>ebiosciences</td>
			<td>48-0319-410</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD90 conjugated to APC&#160;</td>
			<td>ebiosciences&#160;</td>
			<td>17-0909-41&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Annexin V and 7AAD&#160;</td>
			<td>BD Pharmigen</td>
			<td>559763</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mouse Fc block for CD16/CD32&#160;</td>
			<td>BD Pharmigen</td>
			<td>2136662</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 ml tube&#160;</td>
			<td>eppendorf</td>
			<td>30108310</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Motic AE31 inverted microscope</td>
			<td>Motic AE31 inverted microscope</td>
			<td>Motic AE31 inverted microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuaire Biosafety Cabinet and Incubators</td>
			<td>Nuaire Biosafety Cabinet and Incubators</td>
			<td>Nuaire Biosafety Cabinet and Incubators</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4-well chamber slides&#160;</td>
			<td>Thermo Scientific</td>
			<td>177437</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eppendorf Centrifuge 5810R</td>
			<td>Eppendorf Centrifuge 5810R</td>
			<td>Eppendorf Centrifuge 5810R</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Olympus Vacuum-Driven Filter System&#160;</td>
			<td>Genesee Scientific&#160;</td>
			<td>25-227</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nikon Eclipse TE300 Fluorescent Microscope&#160;</td>
			<td>Nikon Eclipse TE300 Fluorescent Microscope (Tokyo, Japan)&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plates&#160;</td>
			<td>Corning</td>
			<td>3516</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T25 Flasks&#160;</td>
			<td>TRP&#160;</td>
			<td>90026</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T75 Flasks&#160;</td>
			<td>Corning</td>
			<td>43064</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dissection Scissors</td>
			<td>Dissection Scissors</td>
			<td>Dissection Scissors</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissection Forceps</td>
			<td>Dissection Forceps</td>
			<td>Dissection Forceps</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Single Tipped Q-Tips&#160;</td>
			<td>Kendall</td>
			<td>540500</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T75 Flasks</td>
			<td>Corning</td>
			<td>43064</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Metamorph software (Molecular Devices)</td>
			<td>Molecular Devices</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACSCAlibur&#160;</td>
			<td>BD Bioscience&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">FACSVerse</td>
			<td>BD Bioscience</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...

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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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Research

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 Genomic DNA from Mouse Tails

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

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 ...

Isolation of Microvascular Endothelial Tubes from Mouse Resistance Arteries

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic by-products, as exemplified by exercising skeletal muscle. Endothelial cells (ECs) line the intima of all resistance vessels and serve a key role in controlling diameter (e.g.&#160;endothelium-dependent vasodilation) and, thereby, the magnitude and distribution of tissue blood flow. The regulation of vascular resistance by ECs is effected by intracellular Ca2+ signaling, which leads to production of diffusible autacoids (e.g.&#160;nitric oxide and arachidonic acid metabolites)1-3 and hyperpolarization4,5 that elicit smooth muscle cell relaxation. Thus understanding the dynamics of endothelial Ca2+ signaling is a key step towards understanding mechanisms governing blood flow control. Isolating endothelial tubes eliminates confounding variables associated with blood in the vessel lumen and with surrounding smooth muscle cells and perivascular nerves, which otherwise influence EC structure and function. Here we present the isolation of endothelial tubes from the superior epigastric artery (SEA) using a protocol optimized for this vessel.
To isolate endothelial tubes from an anesthetized mouse, the SEA is ligated in situ to maintain blood within the vessel lumen (to facilitate visualizing it during dissection), and the entire sheet of abdominal muscle is excised. The SEA is dissected free from surrounding skeletal muscle fibers and connective tissue, blood is flushed from the lumen, and mild enzymatic digestion is performed to enable removal of adventitia, nerves and smooth muscle cells using gentle trituration. These freshly-isolated preparations of intact endothelium retain their native morphology, with individual ECs remaining functionally coupled to one another, able to transfer chemical and electrical signals intercellularly through gap junctions6,7. In addition to providing new insight into calcium signaling and membrane biophysics, these preparations enable molecular studies of gene expression and protein localization within native microvascular endothelium.

We present a preparation for visualizing and manipulating calcium signaling in native, intact microvascular endothelium. Endothelial tubes freshly isolated from mouse resistance arteries supplying skeletal muscle retain in vivo morphology and dynamic signaling within and between neighboring cells. Endothelial tubes can be prepared from microvessels of other tissues and organs.

The control of blood flow by the resistance vasculature regulates the supply of oxygen and nutrients concomitant with the removal of metabolic ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...