Name: isolation of cd 90+ fibroblast/myofibroblasts from human frozen gastrointestinal specimens
Uploaded: 2016-01-31T14:00:00
Description: Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

This work was supported by National Institute of Health (1R01DK103150-01A1, T32 DK007639, R01CA175803, K08CA125209) and the Institute for Translational Sciences at the University of Texas Medical Branch, and a Clinical and Translational Science Award (8UL1TR000071).

The protocol for obtaining discarded human tissue from surgical patients and the establishment of primary cultures was approved by the University of Texas Medical Branch and University of New Mexico Institutional Review Boards. The general requirements for the procurement of human tissue specimens is described below. Note that since MFs may be isolated from frozen tissue, biobanks and commercial vendors are also viable options.
1. Obtain Human Tissue
<ol>
	<li>Submit a protocol to obtain hum...

<ol>
	<li>Yamaguchi, H., 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+Fibroblasts+Mediate+Extracellular+Matrix+Remodeling+and+Invasion+of+Scirrhous+Gastric+Carcinoma+Cells.">Stromal Fibroblasts Mediate Extracellular Matrix Remodeling and Invasion of Scirrhous Gastric Carcinoma Cells.</a> PLoS ONE. 9, e85485 (2014).</li><li>Mullan, N., Hughes, K. R., Mahida, Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Human+Colonic+Myofibroblasts+Are+Resistant+to+Clostridium+difficile+Toxin+A-Induced,+but+Not+Toxin+B-Induced,+Cell+Death.">Primary Human Colonic Myofibroblasts Are Resistant to Clostridium difficile Toxin A-Induced, but Not Toxin B-Induced, Cell Death.</a> Infect Immun. 79, 1623-1630 (2011).</li><li>Powell, D. W., Pinchuk, I. V., Saada, J. I., Chen, X., Mifflin, R. 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R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+human+colonic+subepithelial+myofibroblasts+express+extracellular+matrix+proteins+and+cyclooxygenase-1+and+-2.">Adult human colonic subepithelial myofibroblasts express extracellular matrix proteins and cyclooxygenase-1 and -2.</a> Am J Physiol. 273, G1341-G1348 (1997).</li><li>Roncoroni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+fibroblasts+from+endoscopic+duodenal+biopsies+of+celiac+patients.">Isolation and culture of fibroblasts from endoscopic duodenal biopsies of celiac patients.</a> J Transl Med. 7 (40), (2009).</li><li>Pinchuk, I. V., 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+Induce+Th17+during+Helicobacter+pylori+Infection+and+in+the+Gastric+Tumor+Microenvironment.">Stromal Cells Induce Th17 during Helicobacter pylori Infection and in the Gastric Tumor Microenvironment.</a> PLoS ONE. 8, e53798 (2013).</li><li>Pinchuk, I. V., 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=Monocyte+chemoattractant+protein-1+production+by+intestinal+myofibroblasts+in+response+to+staphylococcal+enterotoxin+a:+relevance+to+staphylococcal+enterotoxigenic+disease.">Monocyte chemoattractant protein-1 production by intestinal myofibroblasts in response to staphylococcal enterotoxin a: relevance to staphylococcal enterotoxigenic disease.</a> J. Immunol. 178, 8097-8106 (2007).</li><li>Paulsson, J., Micke, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+relevance+of+cancer-associated+fibroblasts+in+human+cancer.">Prognostic relevance of cancer-associated fibroblasts in human cancer.</a> Sem Cancer Biol. 25, 61-68 (2014).</li><li>Valcz, G., Sipos, F., Tulassay, Z., Molnar, B., Yagi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+carcinoma-associated+fibroblast-derived+proteins+in+clinical+oncology.">Importance of carcinoma-associated fibroblast-derived proteins in clinical oncology.</a> J. Clin. Pathol. 67, 1026-1031 (2014).</li><li>Minonzio, G., 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=Frozen+adipose-derived+mesenchymal+stem+cells+maintain+high+capability+to+grow+and+differentiate.">Frozen adipose-derived mesenchymal stem cells maintain high capability to grow and differentiate.</a> Cryobiology. 69, 211-216 (2014).</li><li>Gargus, M., Niu, C., Shaker, A. <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+Myofibroblasts+from+Mouse+and+Human+Esophagus.">Isolation of Myofibroblasts from Mouse and Human Esophagus.</a> J. Vis. Exp. (95), e52215 (2015).</li><li>Khalil, H., Nie, W., Edwards, R. 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While this protocol was developed for the research application only, in the light of growing critical importance of stromal cells as a potential cancer prognostic biomarkers and therapeutic target, the ability to freeze &#34;functional&#34; biospecimens for later use offers a significant advantage. This represents a unique advantage for creation of clinical biorepository, which may serve as additional tools serving to advance the development of personalized medicine10, 11. Similar to findings previously report...

Myofibroblasts/fibroblasts (MFs) represent an abundant population of cells in gastrointestinal (GI) mucosa. These stromal cells are located just beneath the epithelium and form an interconnected network within mucosal lamina propria in the gut. MFs are not only responsible for the deposition of the extracellular matrix, but through paracrine regulation may influence electrolyte transport, restitution, and barrier function of the adjacent epithelium1, 2. Furthermore, MFs have been shown to play a key role in inflammation and tissue remodeling3, 4. Myofibroblasts are a critical part of the tumor microenvironment, where they are also known as cancer-associated fibroblasts, and contribute to the tumor cell growth and serve as a niche for cancer stem cells3. Emerging data suggests that MFs may also serve as local antigen presenting cells. Additionally, MFs function as important regulators of innate and adaptive immune responses, producing a variety of cytokines and growth factors5.
In healthy individuals, MFs cells are believed to differentiate from mesenchymal stem cells and express on their cell surface, the mesenchymal marker, CD903, 5. These cells are also positive for vimentin, but negative for epithelial and hematopoietic cell marker, EpCAM and CD45, respectively. Myofibroblasts are suggested to be an activated form of fibroblasts and can be distinguished from non-activated fibroblasts by the expression of &#945;-SMA5.
Over the past decade, multiple approaches for the isolation of myofibroblasts from human colonic mucosa have been published-mostly based on the method originally described by Mahida et al.5-8. While individual studies report isolation procedures from various gut mucosa, no universal protocol for the isolation of MFs from multiple areas of the GI tract (i.e., gastric, small and large intestinal mucosa) has been published. The protocol presented herein has been tested and successfully used for all three type of tissue mentioned above. . Furthermore, procedures for isolating MFs from frozen GI mucosa have not been reported.
Here, we present an optimized method, which is based on enzymatic digestion, and concurrently allows for the isolation of human gut mucosal MFs for culture and flow cytometry analysis in freshly-digested, single cell, mucosal preparations. This technique reliably yields primary cultures with an MF phenotype. Furthermore, the same methods can be used to isolate MFs from frozen specimens of gastric and small intestinal tissues&#160;as well. Isolation of myofibroblasts from fresh GI tissue has been previously described; however, the utilization of frozen specimens presents many benefits. Namely, researchers would be able to collect and store tissues from any number of collaborators across the world who have the capability to ship frozen tissue samples. Moreover, researchers may find the collection of discarded tissue from the operating room and/or endoscopy suite and immediate processing the tissue for isolation to conflict with their current experimentation schedule. Also, due to the unpredictable nature of surgery, tissue procurement may occur very late in the day, which will limit the time left for processing tissue. Freezing tissue for later processing will ameliorate these challenges.
Lastly, these methods have been successfully utilized in the isolation of colonic, gastric and small intestinal myofibroblasts in disease states such as colorectal carcinoma and inflammatory bowel diseases.

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			<td height="21" style="height:21px;width:323px;">MEM, 1X</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">MT-10-010-CV</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Hanks&#39; Balanced Salt Solution</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">H6648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sodium pyruvate</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">S8636</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Antibiotic-Antimycotic, 100X</td>
			<td style="width:111px;">Gibco</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Ciprofloxacin HCl</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">61-277-RF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">L-Glutamine, 100x</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">25-005-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">MEM Nonessential Amino Acids</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">25-025-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Dimethyl Sulfoxide (DMSO) Hybri-Max</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DL-Dithiothreitol solution (DTT)</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">646563</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">0.5M EDTA, pH 8.0</td>
			<td style="width:111px;">Cellgro</td>
			<td style="width:119px;">46-034-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">DNAse</td>
			<td style="width:111px;">Worthington</td>
			<td style="width:119px;">LS002139</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from&#160;Clostridium histolyticum, Type I</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1639</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from&#160;Clostridium histolyticum, Type II</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1764</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Collagenase from Clostridium histolyticum, Type IV</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">C5138</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Accumax cell dissociation solution</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">A7089</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">gentleMACS C Tubes</td>
			<td style="width:111px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-237</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">gentleMACS Dissociator</td>
			<td style="width:111px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Countess Automated Cell Counter</td>
			<td style="width:111px;">Invitrogen</td>
			<td style="width:119px;">C10227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Cell Strainer (70&#181;m)</td>
			<td style="width:111px;">Corning</td>
			<td style="width:119px;">352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PE Mouse Anti-Human Vimentin</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:119px;">562337</td>
			<td>Clone RV202</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">FITC Monoclonal Anti-Actin, &#945;-Smooth Muscle</td>
			<td style="width:111px;">Sigma-Aldrich</td>
			<td style="width:119px;">F3777</td>
			<td>Clone 1A4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">APC Mouse Anti-Human CD90</td>
			<td style="width:111px;">BD Biosciences</td>
			<td style="width:119px;">559869</td>
			<td>Clone 5E10</td>
		</tr>
	</tbody>
</table>

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.

Using this enzymatic digestion protocol, we have consistently been able to isolate and grow MF population gut CD90+ mucosal stromal cells from GI surgical specimens and biopsies (Figure 1). Visible MF colony proliferation could be observed on day 2 - 5 after seeding mononuclear cell suspensions into 6 well plates (Figure 1A-B). The MF primary cultures reach ~ 50 - 70 % confluence by day 7 - 11 (Figure 1C-D).
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Watch this Scientific Journal Video about Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens at JoVE.com

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

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 ...

The overall goal of this method is to isolate viable mucosal myofibroblasts and fibroblasts from frozen gastrointestinal tissue samples, originally obtained from biopsies or surgical specimens. The isolation of myofibroblasts and fibroblasts from properly frozen gastrointestinal tissue allows processing of fresh samples to be delayed. This facilitates the use of samples from different sources worldwide.If the tissue was preserved in freeze medium, place the cryovial in a 37 degree Celsius water bath until the tissue and medium are thawed. For four to five two square millimeter tissue fragments, add 10 milliliters of HBSS without calcium and magnesium, tightly close the lid, and wash the tissue by gently inverting the tube one time. Once the tissue has settled by gravity, carefully discard the supernatant, and wash the tissues again.Then, add 10 millilites of collagenase solution to the tube, and transfer the tissue suspension into a sterile DNase/RNase free tube with built-in rotors. Tightly close the rotor tube and attach it upside down onto the sleeve of a dissociator. Then, run the preset H-tumor zero one program.At the termination of the program, incubate the tissue slurry for 45 minutes at 37 degrees Celsius, under continuous 140 RPM rotation on a shaker. Then, return the tube to the dissociator, and run the H-tumor zero two program. After the second dissociation, add 50 microliters of DNA stock solution to the cell solution for 30 minutes at 37 degrees Celsius, under continuous 60 RPM rotation on the shaker.At the end of the second incubation, run the H-tumor zero three program. Now, filter the suspension through a sterile 70 micron cell strainer, into a 50 milliliter conical tube for centrifugation. Discard the supernatant, and wash the cell pellet in 25 milliliters of HBSS without calcium and magnesium two times, counting the cells after the first wash.For myofibroblast primary culture generation, resuspend the pellet in up to 4 x 10 to the 6th cells per three milliliters of medium, and seed them in 6 well cell culture-treated plates. Grow the cells at 37 degrees Celsius, and five percent carbon dioxide, changing the medium every two to three days until the formation of an 80 percent confluent myofibroblast monolayer. The cells are then ready to be passaged in T25 culture flasks.To analyze or sort the isolated myofibroblasts by flow cytometry, plate up to 2 x 10 to the 6th cells per two milliliters of fibroblast isolation medium, in a 24 well low binding plate for overnight incubation at 37 degrees Celsius and five percent carbon dioxide. Using this enzymatic digestion protocol, myofibroblasts and fibroblasts got CD-90 positive mucosal stromal cell populations, can be consistently isolated and grown from gastrointestinal surgical specimens and biopsies. While there is a slight decrease in the viability of the mucosal single cell preparation after freezing the surgical specimens, there is an increase in the efficiency of the myofibroblast and fibroblast culture generation due to a concomitant decrease in the bacterial and fungal concentration.After two passages, the myofibroblasts and fibroblasts are positive for CD90, the mesenchymal marker lineage marker, Vimentin, and smooth muscle actin, a marker of differentiated mesenchymal cells, as determined by both confocal microscopy and flow cytometric analysis. Once mastered, if performed properly, this technique can be completed in approximately two hours. While attempting this procedure, it is important to remember to allow the necessary time for the tissue to be fully enzymatically digested.Following this procedure, other methods, like flow cytometry, immunocytochemistry, and various functional assays may be performed. They may be used to determine the contribution of myofibroblasts and fibroblasts to gut mucosal homeostasis as well as to the pathology of acute and chronic inflammatory gastrointestinal disease. After watching this video, you should have a good understanding of how to isolate myofibroblasts and fibroblasts from frozen gastrointestinal tissue.Don't forget, unfixed human tissue can be contaminated by pathogenic microorganisms. Universal precautions while working with human biological samples should always be taken while performing this procedure.

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