The authors greatly acknowledge the PICT IBiSA platform at Institut Curie (CNRS UMR144). This work was funded by grants from: the European Research Council to A-M.L-D (Strapacemi 243103), the Association Nationale pour la Recherche (ANR-09-PIRI-0027-PCVI), the InnaBiosant&#233; foundation (Micemico) to M.P. and A-M.L-D and the ERC Strapacemi young investigator grant to A-M.L-D.

Important note: This protocol assumes that the mold containing the shape for the desired microchannels has been already made. Further information on the preparation of the mold has been already published10. This protocol also assumes that bone marrow DCs culture is known.
1. Chip Fabrication
<ol>
	<li>Mix PDMS oil and curing agent at a weight ratio 10:1 in a plastic cup. Mix both compounds thoroughly.</li>
	<li>Cast the mix over the mold bearing microchannels. The...

<ol>
	<li>Lauffenburger, D. A., Horwitz, A. F. <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:+a+physically+integrated+molecular+process.">Cell migration: a physically integrated molecular process.</a> Cell. 84 (3), 359-369 (1996).</li><li>L&#228;mmermann, T., 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=Rapid+leukocyte+migration+by+integrin-independent+flowing+and+squeezing.">Rapid leukocyte migration by integrin-independent flowing and squeezing.</a> Nature. 453 (7191), 51-55 (2008).</li><li>Schor, S. L., Allen, T. D., Harrison, C. 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The authors have nothing to disclose.

Here we describe a device composed of microchannels as a method to study the migratory properties of large number of cells in single experiments. This experimental system mimics the confined environmental constraints found in tissues by endogenous migratory cells. However, by forcing migration in a single dimension, it facilitates automatic cell tracking and the extraction of measurables (Figure 5). We also show that our device is compatible with fluorescence microscopy and can therefore be adapted to st...

Migration is a complex cellular function that is important for many physiological processes in multicellular organisms, including development, immune responses, and tissue regeneration. In addition, certain pathological situations such as tumor invasion and metastasis rely on cell motility1. For these reasons, cell migration has become a major field of study in the context of both fundamental and translational research. In vivo, most tissues are characterized by a rich extracellular matrix and high cell density. Cell migration therefore, under physiological conditions, occurs in a complex confined environment. Classically, most likely due to historical reasons and technical limits, cell migration has been studied in flat 2D systems that do not reproduce many of the environment properties found in tissues, such as confinement. Moreover, factors as cell adhesion, that are essential for motility in 2D, have been recently showed to not be necessarily required for migration in vivo or inside gels, suggesting that the mechanisms that rule cell locomotion in 2D and in other environments are distinct2. Several systems have been developed to mimic the complex properties of tissues, the most famous being collagen gels, which aim at recapitulating the properties of the extracellular matrix composition3. Here we propose microchannels as a simple complementary method that allows the study cell migration in one dimension under a confined environment.
In this system cells migrate along microchannels into which they enter spontaneously. Migratory cells then acquire the shape of the channels, adopting a tubular geometry that most likely reinforces their polarity. The linear movement of the cells in the channels allows automatic cell tracking and the extraction of quantitative parameters from experiments. From the technical point of view, this system is easy and flexible. The coating of the channel walls can be manipulated, the size and the shape of the channels can be adapted, and a large number of cells can be analyzed in single experiments. This system can be also scaled-up to perform medium range screen analysis of molecules involved in cell motility. The protocol described here has been standardized using dendritic cells (DC) as a cellular model. These cells are key to the immune system as they participate in the initiation and maintenance of specific immune responses4. In vitro, DCs have been shown to spontaneously migrate in confined environments and are therefore a good model to study cell motility in microchannels5,6. Importantly, this system can be extended to analyze migration of any other motile cell type as T lymphocytes, neutrophils, or tumor cells7-9.

<table border="0" cellpadding="0" cellspacing="0" width="947">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:273px;">PolyDimethylSiloxane (PDMS)</td>
			<td style="width:131px;">GE Silicones</td>
			<td style="width:139px;">RTV615</td>
			<td style="width:405px;">Package of 90% silicone base and 10% curing agent</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Core sample cutter</td>
			<td>Ted Pella Int.</td>
			<td>Harris Uni-Core&#160;</td>
			<td>Diameter 2,5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass-bottom dish</td>
			<td>WPI</td>
			<td>Fluorodish FD 35-100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasonic cleaner</td>
			<td>Branson Ultrasonics</td>
			<td>Branson 200</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC 32 G</td>
			<td>For small samples (35 dishes). A bigger version is also available</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fibronectin from bovine plasma</td>
			<td>Sigma Aldrich</td>
			<td>F0895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PolyLysine grafted PEG (Pll-g-PEG)</td>
			<td>Susos</td>
			<td>PLL(20)-g[3.5]-PEG(5)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Y27632</td>
			<td>TOCRIS</td>
			<td>1254</td>
			<td></td>
		</tr>
	</tbody>
</table>

study of cell migration in microfabricated channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.

In each experiment the surface of the PDMS is coated with a molecule adapted to the interest of the study. Figure 2 shows channels coated with a fluorescent molecule, PLL-g-PEG, before and after washing (step 2.4). Such an experiment allows the control of the homogeneity of the coating in the channels.
After cell loading, video microscopy can be performed to follow cell migration. Figure 3A shows an example of DC migrating in microchannels at a density appropr...

Watch this Scientific Journal Video about Study of Cell Migration in Microfabricated Channels at JoVE.com

Study of Cell Migration in Microfabricated Channels

A quantitative method to study spontaneous migration of cells in a one-dimensional confined microenvironment is described. This method takes advantage of microfabricated channels and can be used to study migration of large number of cells under different conditions in single experiments.

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which ...

study-of-cell-migration-in-microfabricated-channels

Research

JoVE Journal

Bioengineering

11.8K visualizações.  Institut Curie.  Um método quantitativo para estudar a migração espontânea de células em um microambiente confinado unidimensional é descrito. Este método tira vantagem de canais microfabricados e pode ser utilizado para estudar a migração de grande número de células em condições diferentes nas experiências individuais. 