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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tensile+mechanical+properties+of+three-dimensional+type+I+collagen+extracellular+matrices+with+varied+microstructure.">Tensile mechanical properties of three-dimensional type I collagen extracellular matrices with varied microstructure.</a> J Biomech Eng. 124, 214-222 (2002).</li><li>Bredfeldt, J. S., 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=Computational+segmentation+of+collagen+fibers+from+second-harmonic+generation+images+of+breast+cancer.">Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer.</a> J Biomed Opt. 19, 16007 (2014).</li><li>Bischel, L. L., Beebe, D. J., Sung, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+model+of+ductal+carcinoma+in+situ+with+3D,+organotypic+structure.">Microfluidic model of ductal carcinoma in situ with 3D, organotypic structure.</a> BMC Cancer. 15, 12 (2015).</li></ol>

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.

<table border="0" cellpadding="0" cellspacing="0" width="687">
	<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 ...

The overall goal of this procedure is to generate aligned and random 3D collagen matrices, for the purpose of observing cell migration or other cellular processes in the context of a 3D environment. This method is designed to capture collagen features that we find in real tissues, which can help answer the key questions such as the mechanisms that cells use to recognize an aligned or random extracellular matrix. The main advantage of this technique is that you can manipulate the organization of the 3D environment, image cells within and you can do this cost effectively with equipment that is commonly available.Generally, individuals new to this method will struggle because they don't mix the collagen solution and the neutralization buffer well enough and they draw the collagen through the channels inconsistently. Demonstrating this procedure will be Brian Burkel, who is a senior scientist in my laboratory. To begin, thoroughly mix the collagen and HEPES buffer prior to adding cells and media to ensure proper neutralization of the collagen.Then, add the cells and media to the neutralized collagen mixture and place it on ice as described in the accompanying text protocol. Pipette the ice cold mixture into a sterile non-tissue culture treated surface to minimize the attachment and growth of cells outside of the collagen gel. Use a pipette to evenly spread out the solution.Allow the gel to polymerize at room temperature for approximately 10 to 15 minutes. Next, cover the gels, and move them to a 37 degree Celsius incubator for an additional 45 to 60 minutes to finish polymerization. Upon polymerization, the gel will turn opaque.Once polymerized, add two to three milliliters of media. Then, release the gels from the sides of the well by running a 200 pipette around the perimeter of the well. Swirl the dish gently to release the gel from the bottom surface.In order to create PDMS Microchannels for aligning collagen matrices. First prepare an SuA silicon master, using standard soft lithography techniques described elsewhere. Next, use a craft stick to thoroughly mix 20 grams of elastomer base with 2 grams of curing agent in a disposable cup.Once well-mixed, degas the PDMS mixture by placing the disposable cup in a vacuum chamber under a vacuum pressure of 550 millimeters of mercury. Degas the mixture for approximately one to one and a half hours. While degassing the PDMS, prepare the silicon master by placing a clean sheet of transparency film onto a hot plate followed by the silicon master.Ensure that the micro channel mold faces up. After one to one and a half hours, remove the degassed PDMS from the vacuum chamber. Slowly pour the PDMS into the center of the master and allow gravity to spread it evenly.Next, carefully roll a second sheet of transparency film on top of the silicon master and PDMS to avoid air bubbles. Gently place a rubber sheet on top of the transparency, followed by a 1/8 inch sheet of acrylic, then, add the first of three ten pound weights on top of the acrylic sheet. Initially, the first weight will float, allow it to settle and stabilize before proceeding with the two remaining weight plates.When ready to continue, set a hot plate to 70 degrees Celsius and cure the PDMS for four hours. Once cured, allow the PDMS to cool for a minimum of one additional hour and then, carefully peel the top transparency film for the wafer. Finally, remove the channels with the forceps.Place the channels upside down on a new clean transparency film and clean all of the ports with using a circular motion with sharp forceps. Remove any fragments of PDMS that come loose. Next, place a piece of packing tape to the bench, sticky side up and set the PDMS channel side-down on top of the tape.Press down on the channels with the round end of a pair of forceps to ensure good contact. Remove the channels and repeat this process on both sides until all of the visible debris has been removed. Then, transfer the clean, prepped PDMS channels into a 50 milliliter conical tube filled with 70%ethanol.Vortex the channels at maximum speed for 30 seconds, and then replace the solution with fresh, 70%ethanol. Using aseptic techniques, transfer the PDMS channel side up, to a clean and sterile dish. Then, UV treat the channels until the ethanol has evaporated.Once dry, flip the channels over onto a cover slip or glass bottom dish and press them down onto the glass to make a good seal. Add a patch of sterile PDMS to cover and close the center part of the channels and allow the setup to dry thoroughly before proceeding. Next, place a 100 microliter droplet of a 10 micro grams per milliliter collagen solution on the top of a channel and draw it through the channel using a vacuum.Incubate the channels at 37 degrees Celsius for one hour and then transfer the channels, still filled with collagen coating solution into a refrigerator. Chill the set up for approximately for 15 to 30 minutes and then use an aspirator or pipette to remove the collagen coating solution from the channels. Begin by neutralizing the collagen solution, diluting it with cell media and incubating it on ice for 15 minutes.Then, place 120 to 150 microliters of the collagen solution into port A of a pre chilled channel. Next, attach a 25 milliliter pipette to a vacuum line and place it over port C.Use it to draw in the mixture through a single uniform motion, stopping once the collagen reaches the end. The single most important step to generating random matrices is to slowly draw the collagen through the channel.If it is drawn through too rapidly, it will result in increased alignment in the portion of the channel or throughout the entirety of the channel. Carefully remove any excess solution from the port regions and then place sterile PDMS patches over both ports, A and C.After two to three minutes, remove the center PDMS patch covering port B and add two to three micro litters of cells at a concentration of one to three million cells per milliliter into the center port. Allow the gel time to partially polymerize at room temperature for another 10 to 15 minutes and then transfer the constract to a 37 degree celsius incubator for an additional 15 to 30 minutes.Once polymerized, remove the PDMS port covers and add media to completely cover the channel. Here 50, 000 four T one cells were enbedded in either a 2 mg/ml or a 3.5 mg/ml collagen gel for 5 days. The four T cell one line is a highly contractile Med Aesthetic cancer line but it's only able to contract the high density gel by approximately 10%Comporable low density gels are contracted 57%during the same time frame.The degree of collagen alignment is modulated by the rate of collagen flow such that higher collagen flow rate yield better alignment. The use of narrower channels coupled with higher vacuum pressures enhances the alignment of the collagen network, while a wider channel used in conjunction with low vacuum pressures generates random matrices. Once mastered, pouring collagen gels or channels can be done in two and a half hours if performed properly.While attempting this procedure, it's important to remember to set up the components prior to starting. Once you begin working with the collagen, you need to work quickly. After it's development, this technique paved the way for researchers in the field of 3D cell biology, to explore cell adhesion, migration and invasion into three dimensional extracellular environments.After watching this video, you should have a good understanding of how to pour collagen gels of varying densities and how to generate, both random and aligned collagen matrices.

preparation-3d-collagen-gels-microchannels-for-study-3d-interactions

Research

JoVE Journal

Bioengineering

16.6K ビュー.  University of Wisconsin-Madison. The overall goal of this procedure is to generate aligned and random 3D collagen matrices, for the purpose of observing cell migration or other cellular processes in the context of a 3D environment. This method is designed to capture collagen features that we find in real tissues, which can help answer the key questions such as the mechanisms that cells use to recognize an aligned or random extracellular matrix. The main advantage of this technique is that you can manipulate the organization ...