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 total height must be between 0.5-1 cm.</li>
	<li>Remove air bubbles in a vacuum jar bell during 1 hr.</li>
	<li>Harden PDMS in the mold by placing the latter in an oven for 2 hr at 65 &#176;C.</li>
	<li>Once the PDMS is at room temperature, cut a large piece around the structure with a surgical blade and peel it off from the mold (Figure 1A).</li>
	<li>Drill holes where cells will be injected (typically 2 mm) and resize the PDMS with a surgical blade to fit the size to glass-bottom dishes in which migration will be assessed (Figure 1B). Before proceeding, remove residual dust from the dish using lens cleaning paper. This favors the binding of PDMS to glass described in step 1.10.</li>
	<li>Clean the resized PDMS chip containing the channels by sticking and peeling adhesive tape on the structure sides.</li>
	<li>Sonicate the PDMS pieces 30 sec in 70% ethanol to remove dusts and small PDMS particles. Dry them quickly afterwards by blowing clean air.</li>
	<li>Activate the PDMS (structures upwards) and culture dishes by air (or oxygen) plasma treatment during 30 sec at 300 mTorr.</li>
	<li>Place both activated surfaces in contact to permanently stick the PDMS to the substrate. If needed, use metallic forceps to press slightly on top of the PDMS to force the contact between the polymer and the glass of the dish (Figure 1C).</li>
	<li>Incubate the chip in the oven at 65 &#176;C for 1 hr to strengthen the binding.</li>
</ol>
2. Coating of Microchannels
<ol>
	<li>Activate the whole structure by air plasma at 300 mTorr for 1 min. This will promote the entry of liquid into the channels at the next step.</li>
	<li>Fill rapidly the entry holes of the chip with fibronectin at 10 &#956;g/ml in water. Other substrates such as PEG may be used to modify cell adherence to the channel walls. Pay attention that the liquid spreads throughout the entire structure. This can be easily checked by eye under regular light or using a regular bright field microscope. 
	NOTE: In very small structures, in which diffusion is harder, entry of liquid in the channels can be forced by placing the structure in a vacuum jar bell during at least 15 min. To verify the efficiency of the coating fluorescent substrates can be used (Figure 2).</li>
	<li>Incubate 1 hr at room temperature to allow adsorption of the fibronectin or any other coating substrate to the walls of the channels.</li>
	<li>Wash the structures 3x with PBS to remove the nonbound substrate.</li>
	<li>After washing, proceed to Protocol 3 or store the chip at 4 &#176;C for a later use (24 hr maximum).</li>
</ol>
3. Cell Loading
<ol>
	<li>Remove the PBS from the plate and fill the microsystem with cell medium. Let incubate for 1 hr to saturate the channels with the medium. 
	NOTE: For experiments involving drugs like molecule inhibitors, it is advised to preincubate the channels with a medium containing the drug at the right concentration. Moreover, some drugs tend to solubilize in the PDMS structure which reduces the effective concentration. Some solutions have been proposed to counteract this issue11-12.</li>
	<li>Remove floating DCs and recover semi-adherent cells by flushing with culture medium. Count cells using a hemocytometer. 
	NOTE : For nucleus imaging, a Hoechst 33342 staining can be achieved beforehand by incubating 2 x 106 cells during 30 min with the dye at 200 ng/ml in complete medium. Wash twice by centrifugation to remove excess of dye before continuing the protocol.</li>
	<li>Centrifuge the cells at 300 x g during 5 min (time and speed can be different depending on the cell type) and discard the medium. Dilute the pellet to reach a concentration of 20 x 106 cells/ml.</li>
	<li>Remove the excess of medium from the plate and empty the entry holes in the PDMS structure using a micropipette. Fill the entries with 5 &#181;l of cell solution to reach an amount of 1 x 105 cells in each entry hole. 
	NOTE: High cell density is required to favor the contact of the cells with the channels. Low cell density may result in low number of cells inside channels and failure of the experiment.</li>
	<li>Incubate the microchip for 30 min at 37 &#176;C in the incubator. Add 2 ml of complete medium to the experiment dish. 
	NOTE: The whole PDMS structure must be covered in order to avoid drying of the cells during the experiment. If 2 ml is not enough, add more medium to completely cover the PDMS structure.</li>
</ol>
4. Imaging
<ol>
	<li>Clean the external bottom surface of the dishes with lens cleaning tissue before placing the plates under the microscope.</li>
	<li>To analyze migration in large number of cells, use 10X magnification and wide field illumination in a CO2 and temperature equipped video-microscope (Figure 3). To facilitate cell tracking, Hoechst staining and UV light can be used (see step 3.2). Migration can be also analyzed using confocal microscopy in order to get higher resolution of cellular structures during migration.</li>
	<li>For time-lapse microscopy&#160;at 10X, choose a time frequency according to the expected cell speed (typically 2 min for dendritic cells migrating with a speed of 5 &#956;m/min). 
	Note: The protocol described here does not include the analysis of the cell migration parameters. A few points are listed in the discussion to advise the readers on how to proceed to extract information from time-lapse movies.</li>
</ol>

<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. J. <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+through+three-dimensional+gels+of+native+collagen+fibres:+collagenolytic+activity+is+not+required+for+the+migration+of+two+permanent+cell+lines.">Cell migration through three-dimensional gels of native collagen fibres: collagenolytic activity is not required for the migration of two permanent cell lines.</a> J. Cell. Sci. 41, 159-175 (1980).</li><li>Steinman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decisions+about+dendritic+cells:+past,+present,+and+future.">Decisions about dendritic cells: past, present, and future.</a> Annu. Rev. Immunol. 30, 1-22 (2012).</li><li>Faure-Andr&#233;, G., 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=Regulation+of+dendritic+cell+migration+by+CD74,+the+MHC+class+II-associated+invariant+chain.">Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain.</a> Science. 322 (5908), 1705-1710 (2008).</li><li>Renkawitz, 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=Adaptive+force+transmission+in+amoeboid+cell+migration.">Adaptive force transmission in amoeboid cell migration.</a> Nat. Cell Biol. 11 (12), 1438-1443 (2008).</li><li>Jacobelli, 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=Confinement-optimized+three-dimensional+T+cell+amoeboid+motility+is+modulated+via+myosin+IIA-regulated+adhesions.">Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA-regulated adhesions.</a> Nat. Immunol. 11 (10), 953-961 (2010).</li><li>Irimia, D., Charras, G., Agrawal, N., Mitchinson, T., Toner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polar+stimulation+and+constrained+cell+migration+in+microfluidic+channels.">Polar stimulation and constrained cell migration in microfluidic channels.</a> Lab Chip. 7 (12), 1783-1790 (2007).</li><li>Moreau, H., 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=Dynamic+in+situ+cytometry+uncovers+T+cell+receptor+signaling+during+immunological+synapses+and+kinapses+in+vivo.">Dynamic in situ cytometry uncovers T cell receptor signaling during immunological synapses and kinapses in vivo.</a> Immunity. 37 (2), 351-363 (2012).</li><li>Heuz&#233;, M. L., Collin, O., Terriac, E., Lennon-Dum&#233;nil, A. M., Piel, 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+confinement:+a+micro-channel-based+assay.">Cell migration in confinement: a micro-channel-based assay.</a> Methods Mol. Biol. 769, 415-434 (2011).</li><li>Ren, K., Zhao, Y., Su, J., Ryan, D., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+method+for+modifying+poly(dimethylsiloxane)+to+be+airtight+and+resistive+against+absorption+of+small+molecules.">Convenient method for modifying poly(dimethylsiloxane) to be airtight and resistive against absorption of small molecules.</a> Anal. Chem. 82 (14), 5965-5971 (2010).</li><li>Ren, K., Dai, W., Zhou, J., Su, J., Wu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-Teflon+microfluidic+chips.">Whole-Teflon microfluidic chips.</a> PNAS. 108 (20), 8162-8166 (2011).</li><li>Maiuri, 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=The+first+World+Cell+Race.">The first World Cell Race.</a> Curr Biol. 22 (17), 673-675 (2012).</li></ol>

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

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Bioengineering

11.8K 조회수.  Institut Curie.  일차원 국한 미세 환경에서 세포의 자발적인 이동을 연구하는 정량적 방법이 설명된다. 이 방법은 미세 채널을 활용하고 번의 실험에서 서로 다른 조건 하에서 세포의 다수의 이동을 연구하기 위해 이용 될 수있다. 

비디오: Study of Cell Migration in Microfabricated Channels

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Creating Adhesive and Soluble Gradients for Imaging Cell Migration with Fluorescence Microscopy

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.

A method for the assembly of adhesive and soluble gradients in a microscopy chamber for live cell migration studies is described. The engineered environment combines antifouling surfaces and adhesive tracks with solution gradients and therefore allows one to determine the relative importance of guidance cues.

Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as ...

Simultaneously Capturing Real-time Images in Two Emission Channels Using a Dual Camera Emission Splitting System: Applications to Cell Adhesion

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to investigate mechanisms governing cell adhesion under dynamic flow conditions. For instance, cancer cells labeled with multiple fluorophores can be perfused over a potentially reactive substrate to model mechanisms of cancer metastasis. However, multi-channel single camera systems and color cameras exhibit shortcomings in image acquisition for real-time live cell analysis. To overcome these limitations, we used a dual camera emission splitting system to simultaneously capture real-time image sequences of fluorescently labeled cells in the flow chamber. Dual camera emission splitting systems filter defined wavelength ranges into two monochrome CCD cameras, thereby simultaneously capturing two spatially identical but fluorophore-specific images. Subsequently, psuedocolored one-channel images are combined into a single real-time merged sequence that can reveal multiple target molecules on cells moving rapidly across a region of interest.

Dual camera emission splitting systems for two-color fluorescence microscopy generate real-time image sequences with exceptional optical and temporal resolution, a requirement of certain live cell assays including parallel plate flow chamber adhesion assays. When software is employed to merge images from simultaneously acquired emission channels, pseudocolored image sequences are produced.

Multi-color immunofluorescence microscopy to detect specific molecules in the cell membrane can be coupled with parallel plate flow chamber assays to ...

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of the Drosophila larva makes it an attractive model organism for live imaging studies. However, an important challenge for live imaging techniques is to noninvasively immobilize and position an animal on the microscope. This protocol presents a simple and easy to use method for immobilizing and imaging Drosophila larvae on a polydimethylsiloxane (PDMS) microfluidic device, which we call the 'larva chip'. The larva chip is comprised of a snug-fitting PDMS microchamber that is attached to a thin glass coverslip, which, upon application of a vacuum via a syringe, immobilizes the animal and brings ventral structures such as the nerve cord, segmental nerves, and body wall muscles, within close proximity to the coverslip. This allows for high-resolution imaging, and importantly, avoids the use of anesthetics and chemicals, which facilitates the study of a broad range of physiological processes. Since larvae recover easily from the immobilization, they can be readily subjected to multiple imaging sessions. This allows for longitudinal studies over time courses ranging from hours to days. This protocol describes step-by-step how to prepare the chip and how to utilize the chip for live imaging of neuronal events in 3rd instar larvae. These events include the rapid transport of organelles in axons, calcium responses to injury, and time-lapse studies of the trafficking of photo-convertible proteins over long distances and time scales. Another application of the chip is to study regenerative and degenerative responses to axonal injury, so the second part of this protocol describes a new and simple procedure for injuring axons within peripheral nerves by a segmental nerve crush.

Using Microfluidics Chips for Live Imaging and Study of Injury Responses in Drosophila Larvae

Drosophila larvae are an attractive model system for live imaging due to their translucent cuticle and powerful genetics. This protocol describes how to utilize a single-layer PDMS device, called the &#39;larva chip&#39; for live imaging of cellular processes within neurons of 3rd instar Drosophila larvae.

Live imaging is an important technique for studying cell biological processes, however this can be challenging in live animals. The translucent cuticle of ...

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.

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 ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Distinctive Capillary Action by Micro-channels in Bone-like Templates can Enhance Recruitment of Cells for Restoration of Large Bony Defect

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not occur. With conventional templates, the absence of internal micro-channels results in the lack of cell infiltration, distribution, and inhabitance deep inside the templates. Hence, a highly porous and uniformly interconnected trabecular-bone-like template with micro-channels (biogenic microenvironment template; BMT) has been developed to address these obstacles. The novel BMT was created by innovative concepts (capillary action) and fabricated with a sponge-template coating technique. The BMT consists of several structural components: inter-connected primary-pores (300-400 &#181;m) that mimic pores in trabecular bone, micro-channels (25-70 &#181;m) within each trabecula, and nanopores (100-400 nm) on the surface to allow cells to anchor. Moreover, the BMT has been documented by mechanical test study to have similar mechanical strength properties to those of human trabecular bone (~3.8 MPa)12.
The BMT exhibited high absorption, retention, and habitation of cells throughout the bridge-shaped (&#928;) templates (3 cm height and 4 cm length). The cells that were initially seeded into one end of the templates immediately mobilized to the other end (10 cm distance) by capillary action of the BMT on the cell media. After 4 hr, the cells homogenously occupied the entire BMT and exhibited normal cellular behavior. The capillary action accounted for the infiltration of the cells suspended in the media and the distribution (active migration) throughout the BMT. Having observed these capabilities of the BMT, we project that BMTs will absorb bone marrow cells, growth factors, and nutrients from the periphery under physiological conditions.
The BMT may resolve current limitations via rapid infiltration, homogenous distribution and inhabitance of cells in large, volumetric templates to repair massive skeletal defects.

A step-by-step generic process to create a bone-like template with engineered micro-channels is presented. High absorption and retention capabilities of the template are demonstrated by capillary action via micro-channels.

Without an active, thriving cell population that is well-distributed and stably anchored to the inserted template, exceptional bone regeneration does not ...

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.

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.

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