Name: preparation of 3d collagen gels and microchannels for the study of 3d interactions in vivo
Uploaded: 2016-05-09T14:00:00
Description: Watch this Scientific Journal Video about Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo at JoVE.com

The authors would like to acknowledge grant numbers UO1CA143069, R01CA142833, R01CA114462, RO1CA179556, T32-AG000213-24, and T32-GM008692-18 for funding this work. We also acknowledge Jeremy Bredfelt and Yuming Liu of LOCI for the development of and assistance with the CT-FIRE analysis.

1. Neutralization, Dilution and Polymerization of Collagen Solutions for 3D Investigation and Cellular Contraction Assays
<ol>
	<li>On ice in sterile tissue culture hood, neutralize collagen (1:1) with sterile, ice-cold 100 mM HEPES in 2x PBS, pH 7.4, in a 15 ml conical tube. Mix thoroughly with plastic pipette until solution is homogenous and mixing swirls are no longer visible. Be careful not to introduce air bubbles during the mixing process. Store briefly on ice.</li>
	<li>Dilute neutralized collagen to the appropr...

<ol>
	<li>Conklin, M. 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=Aligned+collagen+is+a+prognostic+signature+for+survival+in+human+breast+carcinoma.">Aligned collagen is a prognostic signature for survival in human breast carcinoma.</a> Am J Pathol. 178, 1221-1232 (2011).</li><li>Provenzano, P. P., 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=Collagen+reorganization+at+the+tumor-stromal+interface+facilitates+local+invasion.">Collagen reorganization at the tumor-stromal interface facilitates local invasion.</a> BMC Med. 4, 38 (2006).</li><li>Riching, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+collagen+alignment+limits+protrusions+to+enhance+breast+cancer+cell+persistence.">3D collagen alignment limits protrusions to enhance breast cancer cell persistence.</a> Biophys J. 107, 2546-2558 (2014).</li><li>Even-Ram, S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+in+3D+matrix.">Cell migration in 3D matrix.</a> Curr Opin Cell Biol. 17, 524-532 (2005).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+cell+migration:+a+multiscale+tuning+model.">Plasticity of cell migration: a multiscale tuning model.</a> J Cell Biol. 188, 11-19 (2010).</li><li>Petrie, R. J., Gavara, N., Chadwick, R. S., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonpolarized+signaling+reveals+two+distinct+modes+of+3D+cell+migration.">Nonpolarized signaling reveals two distinct modes of 3D cell migration.</a> J Cell Biol. 197, 439-455 (2012).</li><li>Cukierman, E., Pankov, R., Yamada, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+interactions+with+three-dimensional+matrices.">Cell interactions with three-dimensional matrices.</a> Curr Opin Cell Biol. 14, 633-639 (2002).</li><li>Wozniak, M. A., Desai, R., Solski, P. A., Der, C. J., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROCK-generated+contractility+regulates+breast+epithelial+cell+differentiation+in+response+to+the+physical+properties+of+a+three-dimensional+collagen+matrix.">ROCK-generated contractility regulates breast epithelial cell differentiation in response to the physical properties of a three-dimensional collagen matrix.</a> J Cell Biol. 163, 583-595 (2003).</li><li>Paszek, M. 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=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8, 241-254 (2005).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+density-induced+mechanoregulation+of+breast+cell+phenotype,+signaling+and+gene+expression+through+a+FAK-ERK+linkage.">Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage.</a> Oncogene. 28, 4326-4343 (2009).</li><li>Tlsty, T. D., Coussens, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+stroma+and+regulation+of+cancer+development.">Tumor stroma and regulation of cancer development.</a> Annu Rev Pathol. 1, 119-150 (2006).</li><li>Provenzano, P. P., 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=Collagen+density+promotes+mammary+tumor+initiation+and+progression.">Collagen density promotes mammary tumor initiation and progression.</a> BMC Med. 6, 11 (2008).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Trier, S. M., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+guidance+mediated+three-dimensional+cell+migration+is+regulated+by+Rho/ROCK-dependent+matrix+reorganization.">Contact guidance mediated three-dimensional cell migration is regulated by Rho/ROCK-dependent matrix reorganization.</a> Biophys J. 95, 5374-5384 (2008).</li><li>Guo, C., Kaufman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+and+magnetic+field+induced+collagen+alignment.">Flow and magnetic field induced collagen alignment.</a> Biomaterials. 28, 1105-1114 (2007).</li><li>Sung, K. E., 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=Control+of+3-dimensional+collagen+matrix+polymerization+for+reproducible+human+mammary+fibroblast+cell+culture+in+microfluidic+devices.">Control of 3-dimensional collagen matrix polymerization for reproducible human mammary fibroblast cell culture in microfluidic devices.</a> Biomaterials. 30, 4833-4841 (2009).</li><li>Lee, P., Lin, R., Moon, J., Lee, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+alignment+of+collagen+fibers+for+in+vitro+cell+culture.">Microfluidic alignment of collagen fibers for in vitro cell culture.</a> Biomed Microdevices. 8, 35-41 (2006).</li><li>Wozniak, M. A., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+three-dimensional+collagen+gels+to+study+mechanotransduction+in+T47D+breast+epithelial+cells.">Use of three-dimensional collagen gels to study mechanotransduction in T47D breast epithelial cells.</a> Biol Proced Online. 7, 144-161 (2005).</li><li>Gallagher, S., Winston, S. E., Fuller, S. A., Hurrell, J. G., Ausubel, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoblotting+and+immunodetection.">Immunoblotting and immunodetection.</a> Current protocols in molecular biology. , 18 (2008).</li><li>Liu, X., Harada, S., Ausubel, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+isolation+from+mammalian+samples.">DNA isolation from mammalian samples.</a> Current protocols in molecular biology. , 14 (2013).</li><li>Roeder, B. A., Kokini, K., Sturgis, J. E., Robinson, J. P., Voytik-Harbin, S. 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3D collagen gels are a valuable addition to our toolbox to understand how cells interpret and respond to their local microenvironment. This manuscript has provided a very basic protocol for embedding cells within a 3D collagen matrix, and to reproducibly generate matrices with random or aligned collagen fibers. Both protocols work as adaptable platforms where different collagen isoforms, crosslinkers, or other matrix proteins could potentially be added at the time of polymerization. It is also easy to modify the platform...

Many cellular processes have been extensively studied in 2 dimensions, thereby forming a collective knowledge of basic cell signaling mechanisms. These studies, however, neglect important cellular cues received from the structural and mechanical properties of the local cellular microenvironment and extracellular matrix (ECM). To better understand how cells interact within a physiological context, it is important to study them in 3-dimensional (3D) assays. The ECM for these 3D assays can either be cell-derived or reconstituted from purified proteins. Regardless of the source of the ECM, 3D matrix assays have proven to be invaluable for understanding how cells navigate and interact within the physiological world. For example, cells grown in 3D matrices display distinct modes of locomotion that depend on the mechanical nature of their surrounding ECM which are not observed in 2D experiments4-6. Moreover, cells cultured in 3D also have fewer and less pronounced stress fibers and focal adhesions than their counterparts grown on hard surfaces such as glass or plastic7. 
The importance of contextual 3D assays is not limited to cell migration, however. Some other cell signaling events can only be investigated through the use of 3D assays. During tissue and cell differentiation, the stiffness of the extracellular environment and ECM provides signals that can influence morphogenic events. For example, mammary epithelial tubulogenesis only occurs in low stiffness 3D matrices, but not in stiff matrices nor on 2D substrata8,9. When cultured within stiff 3D matrices, these same epithelial cells take on an aberrant phenotype with increased proliferation and cell membrane protrusions driven through altered FAK and ERK signaling10. Many other signaling pathways and cellular processes are known to be similarly affected by the stiffness of the local cellular environment, and these signaling cascades highlight the importance of investigating signaling events and cellular phenotype in the context of appropriate local mechanical properties of a 3D ECM. 
Collagen I is a particularly relevant protein to use for in vitro studies as it is the most abundant component of the ECM and is responsible for many of the mechanical properties of the cellular microenvironment. While it was originally thought of as merely a structural protein, its role is now known to be much more complex. Collagen fiber composition, architecture, orientation, density, and stiffness all provide a concentrated milieu of signaling information5. During the progression of certain diseases, such as chronic inflammation and tumorigenesis, the collagen network is dramatically remodeled2,11. More specifically in breast cancers, increased collagen deposition and tissue stiffness accompany and likely contribute to tumor progression. In these early tumors, the stiffened collagen network appears strained and more aligned, such that most of the fibers encapsulate the growing tumor2. As the tumor progresses, the collagen continues to reorganize, and regions of the fibrillar network become orientated perpendicular to the tumor boundary2,12. Perpendicular alignment serves as a prognostic biomarker where these patients have a poorer disease free progression and overall survival1. One explanation for this correlation is that the poor outcomes are a consequence of increased dissemination of tumor cells via persistent cell migration in aligned collagen networks3. 
To understand how cells specifically respond to alignment and organization that is observed in tumor progression, it is necessary to generate both random and aligned 3D collagen matrices for experimentation. There are three basic methodologies to induce alignment within fibrillar networks. The first technique utilizes a strain-inducing device where the collagen between two points is contracted or stretched to generate alignment. Fibers parallel to the axis of force are pulled taut while fibers perpendicular to the axis are compressed and buckled. While strain-induced techniques typically offer superb alignment, this approach requires bulky equipment that is not easily adaptable to many platforms3,13. Alternatively, cell-induced strain can be created by placing localized plugs of cells that subsequently contract and align the collagen13. This method has the problem of being variable, as many parameters may be subject to change. The second method utilizes magnetic beads and a magnetic field during polymerization to induce collagen alignment13,14. Good results can be obtained from this method with unsophisticated equipment, but it does require the use of antibodies or some other method to magnetize the polymer. Therefore, it can be somewhat expensive to use, and the stiffness of the collagen gel is potentially modified by the increased connections in the network. Moreover, the magnetic beads used in this process are often autofluorescent, which is problematic for imaging experiments. Lastly, alignment can be generated by PDMS microfluidic channels3,15,16. In this method, collagen alignment is achieved by flowing polymerizing collagen through small microfluidic channels. These microfluidic channels can be made in a multitude of designs, and are easily adaptable to many platforms. Moreover, they are very economical as very small quantities of collagen and other reagents are used due to their diminutive sizes.
Provided here is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. In addition, a more advanced technique, wherein PDMS microfluidic channels are used to control the organization and alignment of the collagen matrix is also provided. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a 3-dimensional, physiological microenvironment.

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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">High Concentration Rat tail Collagen</td>
			<td style="width:155px;">Corning</td>
			<td style="width:119px;">354249</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">SylGard184 elastomer kit</td>
			<td style="width:155px;">Corning</td>
			<td style="width:119px;">NC9285739</td>
			<td>Elastomer for PDMS channels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HEPES</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:119px;">BP310</td>
			<td>For HEPES neutralization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">KCl&#160;</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:119px;">BP366</td>
			<td>For HEPES neutralization buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:247px;">KH2PO4</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:119px;">BP362</td>
			<td>For HEPES neutralization buffer</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:247px;">Na2HPO4</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:119px;">S374</td>
			<td>For HEPES neutralization buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">NaCl</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:119px;">BP358</td>
			<td>For HEPES neutralization buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Levy Improved Neubauer Hemacytometer</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:119px;">15170-208</td>
			<td>cell counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">6-well non-tissue culture plate&#160;</td>
			<td style="width:155px;">Corning</td>
			<td style="width:119px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">50 mm glass bottom dish</td>
			<td style="width:155px;">MatTek</td>
			<td style="width:119px;">P50g-1.5-30-f</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Bel-Art Plastic Vacuum Desiccator</td>
			<td style="width:155px;">Bel-Art</td>
			<td style="width:119px;">F4200-2021</td>
			<td>Degassing chamber for PDMS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">transparency film&#160;</td>
			<td style="width:155px;">3M</td>
			<td style="width:119px;">pp2950</td>
			<td>Plastic film for pouring pdms channels</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:247px;">ThermoScientific CimaRec</td>
			<td style="width:155px;">ThermoScientific&#160;</td>
			<td style="width:119px;">HP141925</td>
			<td>Hot plate for curing PDMS microchannels</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:247px;">Vacuum regulator</td>
			<td style="width:155px;">Precision Medical</td>
			<td style="width:119px;">PM3100</td>
			<td>Vacuum regulator for collagen microchannels</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:247px;">8&#34; X 8&#34; rubber sheet&#160;</td>
			<td style="width:155px;">Amazon - Rubber-Cal</td>
			<td style="width:119px;">Silicone - 60A&#160;</td>
			<td>rubber sheet for pouring PDMS microchannel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">8&#34; X 8&#34; X .125&#34; acrylic sheet</td>
			<td style="width:155px;">Amazon&#160;</td>
			<td style="width:119px;">Plexiglass sheets</td>
			<td>for pouring PDMS microchannels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">10 lb weights</td>
			<td style="width:155px;">Amazon</td>
			<td style="width:119px;">CAP Barbell</td>
			<td>for pouring PDMS microchannels</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">15 ml Conical tubes</td>
			<td style="width:155px;">Fisher&#160;</td>
			<td align="right" style="width:119px;">352097</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">50 ml Conical tubes</td>
			<td style="width:155px;">Fisher&#160;</td>
			<td align="right" style="width:119px;">352098</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:247px;">Plastic pipets</td>
			<td style="width:155px;">Dot Scientific</td>
			<td style="width:119px;">229202B, 229206B, and 667225B</td>
			<td>2ml, 5ml, and 25ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">70%EtOH</td>
			<td style="width:155px;">Fisher</td>
			<td style="width:119px;">NC9663244</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of 3d collagen gels and microchannels for the study of 3d interactions in vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

While 3D assays can be done within the same stiffness of collagen gel, varying the gel stiffness can be used to determine how the cells will respond to mechanical changes in their cellular microenvironment. A stiff collagen hydrogel is defined as a gel where the embedded cells are unable to locally contract the surrounding collagen. The intrinsic contractility of different cell types is unique, and thus it is best to begin with a simple contractility curve to establish the collagen concen...

Watch this Scientific Journal Video about Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo at JoVE.com

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

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