Name: analysis of cell migration within a three-dimensional collagen matrix
Uploaded: 2014-10-05T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Cell Migration within a Three-dimensional Collagen Matrix at JoVE.com

This work was supported by the Fritz-Bender-Foundation, Munich, Germany

1. Preparation of Migration Chambers
<ol>
	<li>Prepare a paraffin wax/petroleum jelly (1:1) mix and heat until the mixture has melted. Using a paint-brush and draw 2-3&#160;layers of the paraffin wax/petroleum jelly (1:1) mix in the middle of the glass slide in accordance to Figures&#160;1B-1D. 
	NOTE: We are using common glass slides (76 x 26 x 1.0-1.5 mm (W/D/H))</li>
	<li>Apply the melted paraffin wax/petroleum jelly mix rapidly on the glass slide to avoid solidification while drawing. Ensure t...

The ability to migrate is a hallmark of tumor cells4. Without the ability to detach from the primary tumor and to migrate through the surrounding connective tissue tumor cells won&#8217;t be able to seed secondary lesions, which are the main cause of death of nearly all cancer patients. Because of this relationship many studies are focusing on cancer cell migration. The aim of these studies is the identification of novel target molecules and target pathways that efficiently block tumor cell migration, thereby ...

Like cell fusion (for an overview see&#160;1,2) cell migration is another biological phenomenon that is involved in a plethora of physiological processes including embryonic development, wound healing and immune responses (for review see&#160;3). However, the ability to migrate is also a prerequisite for tumor cells to metastasize (for review see&#160;3,4).
Cell migration is a complex, not yet fully understood process that is directed by the interplay of several signal transduction pathways initiated by various ligand (e.g., cytokines, chemokines, growth factors, hormones, extracellular matrix components) receptor (e.g., receptor tyrosine kinases, chemokine receptors, integrins) interactions5 ultimately causing the reorganization of the actin cytoskeleton concomitant with de- and reassembly of focal adhesion complexes and integrin-mediated signaling6.
To analyze cell migration several in vitro and in vivo cell migration assays have been developed in the past decades, including the Boyden chamber/transwell assay7, scratch assay/wound healing assay8-10, three-dimensional (3D) collagen matrix migration assay11 as well as intravital imaging/microscopy (for review see&#160;12). Each of these cell migration assays has pros and cons, e.g., concerning costs and need of equipment, handling or reliability of obtained data.
Both the Boyden chamber/transwell assay and the scratch assay/wound healing assay are easy, low-cost and well-developed assays to measure cell migration in vitro7-10. In the Boyden chamber/transwell assay cells are seeded on top of an insert containing pores (about 8 &#181;m in diameter) - the so-called upper compartment7. Optional, the insert could be coated with extracellular matrix components, e.g., fibronectin, collagen, etc., to mimic a more physiological environment. Likewise, endothelial cells could be grown on top of the insert, thereby mimicking an endothelial cell barrier13. Those cells that have passed through the pores during a defined time interval into the lower compartment harboring media and supplements, such as growth factors and chemokines, are used as a read-out to quantify cell migration (or extravasation).
In the scratch assay/wound healing assay cells are seeded in plates and are grown to confluency10. In dependence of the experimental setting plates could be pre-coated with extracellular matrix components, such as fibronectin. After creating a scratch/wound by scraping the cell monolayer single cells from each side of the scratch/wound can migrate into the gap, thereby filling/healing it10. The distance between the two sides of the scratch/wounds is determined in dependence of time and is used as a read-out for the migratory activity of the cells10. However, to discriminate between cell proliferation (which could also result in filling/healing of the scratch/wound) and cell migration it is recommended to combine the assay with time-lapse video microscopy and single cell tracking10.
However, both the Boyden chamber/transwell assay and scratch assay/wound healing assay, are rather imperfect concerning a physiological-like cellular environment. In the Boyden chamber/transwell assay cells have to migrate through a plastic pore, whereas in the scratch assay/wound healing assay cells are seeded on a two-dimensional pre-coated plastic plate. Likewise, it is well recognized that the migratory behavior differs markedly between a two-dimensional and 3D environment3. For instance, three-dimensional-matrix adhesions of fibroblasts differ from focal and fibrillar adhesions characterized on two-dimensional substrates in their content of &#945;5&#946;1 and &#945;v&#946;3 integrins, paxillin, other cytoskeletal components, and tyrosine phosphorylation of focal adhesion kinase14. Likewise, cells embedded within a 3D environment also displayed an altered migratory behavior15. Thus to analyze cell migration more accurately a migration assay is recommended allowing to measure the migration of single cells within a 3D physiological or physiological-like environment.
Intravital imaging/microscopy is the gold-standard for measuring cell migration within a 3D physiological context. This does not only belong to extracellular matrix-cell interactions, but also to the interactions among different cell types, such as tumor cells and endothelial cells during extravasation16 or lymphocyte trafficking within the lymph node17, which, to date, is possible due to improved fluorescence microscopy techniques, such as 2-photon confocal laser scanning microscopy, the use of vital fluorescent dyes and transgenic mouse strains expressing fluorescent proteins derivatives12,16,17. Additionally, intravital imaging/microscopy could be combined with manual and automated cell tracking18. However, because of the need of a 2-photon confocal laser scanning microscopy as well as animals (and appropriate transgenic animal models) intravital imaging/microscopy is a rather cost-intensive technique.
To overcome the limitations of the Boyden chamber/transwell assay and the scratch assay/wound healing assay and to analyze the migration of different cell types within a 3D environment the 3D collagen matrix migration assay was developed11,19. Thereby, migrating cells are embedded within a 3D collagen fiber network, which more resembles to the in vivo situation. Conjointly, due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes and leukocytes11,20, hematopoietic stem/progenitor cells21-24, and tumor cells5,25-29. In addition to single cells also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice30,31.
This protocol presents an overview about a simple, but powerful technique to analyze the migratory behavior of different cell types within a 3D environment &#8211; an in vitro method yielding in results that are close to the in vivo situation.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Name of Material/ Equipment</td>
			<td style="width:192px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Leica DM IL inverted microscope</td>
			<td style="width:192px;">Leica, Wetzlar, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Microscope stage heater</td>
			<td style="width:192px;">Distelkamp Electronic, Kaiserslautern, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">JVC C1431 video camera</td>
			<td style="width:192px;">JVC, Bad Vilbel, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Axis 241Q video server</td>
			<td style="width:192px;">Axis communication GmbH, Ismaning, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Mac G5 Computer</td>
			<td style="width:192px;">Apple Macintosh</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">iMac</td>
			<td style="width:192px;">Apple Macintosh</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">FileMaker Pro</td>
			<td style="width:192px;">FileMaker GmbH, Unterschlei&#223;heim, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Multi-camera video surveillance software(Security Spy)</td>
			<td style="width:192px;">Bensoftware, London, UK</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Runtime Revolution Media 2.9.0</td>
			<td style="width:192px;">RunRev Ltd., Edinburgh, UK</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Paraffin</td>
			<td style="width:192px;">Applichem GmbH, Darmstadt, Germany</td>
			<td style="width:119px;">A4264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Petrolatum jelly</td>
			<td style="width:192px;">local drug store</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Purecol (liquid collagen)</td>
			<td style="width:192px;">Nutacon BV, Leimuiden, The Netherlands</td>
			<td style="width:119px;"></td>
			<td>contains 2.9-3.3 mg/ml bovine collagen (95% collagen type I, 5% collagen type IV)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">10x MEM</td>
			<td style="width:192px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">M0275</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">7.5% Sodium Bicarbonate solution</td>
			<td style="width:192px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">S8761</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">EGF</td>
			<td style="width:192px;">Sigma Aldrich, Taufkirchen, Germany</td>
			<td style="width:119px;">E9644</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">U73122</td>
			<td style="width:192px;">Merck Millipore, Darmstadt, Germany</td>
			<td align="right" style="width:119px;">662035</td>
			<td>dissolve first in CHCL3; reconstitute in DMSO just prior to use</td>
		</tr>
	</tbody>
</table>

analysis of cell migration within a three-dimensional collagen matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

The used 3D-collagen matrix migration assay combined with time-lapse video-microscopy and computer-assisted cell tracking allows for the determination of various cell migration parameters including both population-based parameter (e.g., mean locomotory activity) and single cell-based parameters (e.g., time of active movement, speed, distance migrated). An example of the obtained cell tracking data sets, data processing and data presentation are given in Figure 2. A cell tracking data fi...

Watch this Scientific Journal Video about Analysis of Cell Migration within a Three-dimensional Collagen Matrix at JoVE.com

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

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