Name: isolation of primary myofibroblasts from mouse and human colon tissue
Uploaded: 2013-10-12T19:00:00
Description: Watch this Scientific Journal Video about Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue at JoVE.com

This work was supported by National Institutes of Health Grant 5KO8DK085136-02 to JY.

1. Obtain Colon Tissue
Mouse
A protocol to perform animal experiments, detailing the rationale and objectives of the research, was submitted and approved by our institutional Office of Animal Research Oversight.
<ol>
	<li>Euthanize the mouse with inhaled isoflurane followed by cervical dislocation.</li>
	<li>Make a ventral midline incision, retract the small bowel away and identify the colon. Retract the bladder and uterus (if present) laterally and identify the pubi...

<ol>
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Cell Physiol. 297, C1576-C1587 (2009).</li><li>Valentich, J. D., Popov, V., Saada, J. I., Powell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+characterization+of+an+intestinal+subepithelial+myofibroblast+cell+line.">Phenotypic characterization of an intestinal subepithelial myofibroblast cell line.</a> Am. J. Physiol. 272, C1513-C1524 (1997).</li><li>Rodriguez Perez, C. E., Nie, W., Sinnett-Smith, J., Rozengurt, E., 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=TNF-alpha+potentiates+lysophosphatidic+acid-induced+COX-2+expression+via+PKD+in+human+colonic+myofibroblasts.">TNF-alpha potentiates lysophosphatidic acid-induced COX-2 expression via PKD in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 300, G637-G646 (2011).</li><li>Yoo, 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=TNF-alpha+induces+upregulation+of+EGFR+expression+and+signaling+in+human+colonic+myofibroblasts.">TNF-alpha induces upregulation of EGFR expression and signaling in human colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 302, G805-G814 (2012).</li><li>Mahida, Y. 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. 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Bowel Dis. 12, 153-165 (2006).</li><li>Hoang, B., Trinh, A., Birnbaumer, L., Edwards, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+MAPK-+and+PGE2-dependent+IL-11+production+in+Gialpha2-/-+colonic+myofibroblasts.">Decreased MAPK- and PGE2-dependent IL-11 production in Gialpha2-/- colonic myofibroblasts.</a> Am. J. Physiol. Gastrointest. Liver Physiol. 292, G1511-G1519 (2007).</li><li>Li, Z. B., Kollias, H. D., Wagner, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myostatin+directly+regulates+skeletal+muscle+fibrosis.">Myostatin directly regulates skeletal muscle fibrosis.</a> J. Biol. Chem. 283, 19371-19378 (2008).</li><li>Saada, J. I., 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=Subepithelial+myofibroblasts+are+novel+nonprofessional+APCs+in+the+human+colonic+mucosa.">Subepithelial myofibroblasts are novel nonprofessional APCs in the human colonic mucosa.</a> J. Immunol. 177, 5968-5979 (2006).</li><li>Lahar, N., 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+support+in+vitro+and+in+vivo+growth+of+human+small+intestinal+epithelium.">Intestinal subepithelial myofibroblasts support in vitro and in vivo growth of human small intestinal epithelium.</a> PLoS One. 6, e26898 (2011).</li></ol>

The general technique is similar when using either mouse or human colon. This technique can also be used to isolate myofibroblasts from the small intestine. Specific details for the isolation of myofibroblasts from 1. mouse and 2. human colon are discussed below.
1. Mouse Colon
Irrigation of the mouse colon is facilitated by attaching a 4 in, 16 G Popper blunt-end needle to one end of the colon. This also helps when cutting the colon lengthwise. You can accordion the c...

The regulation of gastrointestinal (GI) tract function involves complex, dynamic interactions between the mucosal layer and its underlying mesenchyme. These interactions normally serve to maintain homeostasis but may also lead to the development of disease states under pathologic conditions1. Myofibroblasts are a subpopulation of stromal cells located subjacent to the epithelial layer that communicate in a paracrine fashion with surrounding cell subpopulations to regulate a number of critical GI tract processes that include mucosal regeneration, repair, and fibrosis2.
The 18Co cell line is an example of a human colonic myofibroblast cell line that has been widely used to study myofibroblast function because it shares many of the characteristics of primary in situ myofibroblasts. These include the protein expression of a-smooth muscle actin (&#945;-SMA) and vimentin, as well as the expression of a number of cell surface receptors such as the epidermal growth factor receptor, or receptors for lysophosphatidic acid3,4. Because of these shared ultrastructural characteristics, as well as similarities in biologic function5, the 18Co cell line has been used extensively to study myofibroblast function in the context of many disease states such as inflammatory bowel disease or colorectal cancer3,6. However, there are inherent limitations and concerns when using a cell line. These include genotypic instability over time that differentiates cell lines from primary cells, altering the phenotype and biologic function of the cell, including growth rates and interactions with other cell populations. Cell lines also lack the normal components of the GI microenvironment (epithelial, stromal, and vascular components), which is a major limitation to its use. Therefore, the conclusions drawn from experimental cell line research requires further validation to reduce the risk of misinterpretation.
Primary myofibroblasts can be obtained from human and mouse colon tissue to confirm experimental findings identified in a cell line7. The method was originally described by Mahida, et al.8, and our protocol is one of many well-described techniques that can be used to isolate primary myofibroblasts from mouse and human colon9. For any mouse model of colonic disease, this technique can be used to isolate primary myofibroblasts to study how they interact with neighboring cells or contribute to both normal or pathologic GI processes10,11. This technique can be used to study how myofibroblasts contribute to mucosal healing, fibrosis, and the development of colorectal adenomas and carcinomas. Primary myofibroblasts can also be obtained from human colon tissue that has been resected at the time of operation for benign (inflammatory bowel disease, strictures, diverticulitis) or malignant conditions. Isolated primary cells from human and mouse colon tissue can be grown in cell culture and utilized over a limited number of passages.

Table of specific reagents and equipment:
<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>Dispase I</td>
 <td>Invitrogen/GIBCO</td>
 <td>#17105-041</td>
 <td></td>
 </tr>
 <tr>
 <td>Collagenase D</td>
 <td>Roche</td>
 <td>#1 088 858</td>
 <td></td>
 </tr>
 <tr>
 <td>4 in, 16 G Popper blunt-end needle</td>
 <td>Fisher</td>
 <td>#14-825-16K</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Once isolated, primary human myofibroblasts can be grown in cell culture and used over a limited number of passages (up to passage 4). These cells are characterized as being a-smooth muscle actin and vimentin positive, and desmin-negative (Figure 1A), consistent with intestinal subepithelial myofibroblasts5,7. They also have a characteristic stellate morphology (Figure 1B). Primary myofibroblasts can then be used to confirm experimental findings identified in a cell line. This...

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

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

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 ...

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