The authors acknowledge Clemson University's Creative Inquiry program and NSF CBET1254609 for providing funding for this project.

1. Producing a Microchannel Chamber to Create a Concentration Gradient
<ol>
	<li>Cutting an acrylic mold piece
	<ol>
		<li>Obtain a piece of acrylic of the desired width. Typically use 1/16-inch-thick acrylic sheets. Thicker sheets are difficult to cut well and very thin sheets do not have the requisite mechanical strength, causing them to break or warp during the process.</li>
		<li>Create a CAD file with the desired shape of the acrylic piece to produce a cavity within the polydimethylsiloxane (PDMS)...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+devices+fabricated+in+poly+(dimethylsiloxane)+for+biological+studies.">Microfluidic devices fabricated in poly (dimethylsiloxane) for biological studies.</a> Electrophoresis. 24 (21), 3563-3576 (2003).</li><li>Lin, F., 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=Generation+of+dynamic+temporal+and+spatial+concentration+gradients+using+microfluidic+devices.">Generation of dynamic temporal and spatial concentration gradients using microfluidic devices.</a> Lab Chip. 4 (3), 164-167 (2004).</li><li>Darby, I., Hewitson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+Differentiation+in+Wound+Healing+and+Fibrosis.">Fibroblast Differentiation in Wound Healing and Fibrosis.</a> Int Rev Cytol. 257, 143-179 (2007).</li><li>Thampatty, B. P., Wang, J. H. -. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+to+study+fibroblast+migration.">A new approach to study fibroblast migration.</a> Cell Motil Cytoskel. 64, 1-5 (2007).</li><li>Discher, D., Mooney, D., Zandstra, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+Factors,+Matrices,+and+Forces+Combine+and+Control+Stem+Cells.">Growth Factors, Matrices, and Forces Combine and Control Stem Cells.</a> Science. 324, 1673-1677 (2009).</li><li>Zengel, 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=&#181;-Slide+Chemotaxis:+A+new+chamber+for+long-term+chemotaxis+studies.">&#181;-Slide Chemotaxis: A new chamber for long-term chemotaxis studies.</a> BMC Cell Biol. , (2011).</li><li>Chen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boyden+Chamber+Assay.">Boyden Chamber Assay.</a> Methods Molec Biol. 2, 15-22 (2005).</li><li>Safferling, K., 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=Wound+healing+revised:+A+novel+reepithelialization+mechanism+revealed+by+in+vitro+and+in+silico+models.">Wound healing revised: A novel reepithelialization mechanism revealed by in vitro and in silico models.</a> JCB. 203 (4), 691-709 (2013).</li><li>Saadi, 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=Generation+of+stable+concentration+gradients+in+2D+and+3D+environments+using+a+microfluidic+ladder+chamber.">Generation of stable concentration gradients in 2D and 3D environments using a microfluidic ladder chamber.</a> Biomed Microdevices. 9, 627-635 (2007).</li><li>Cammer, M., Cox, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotactic+Responses+by+Macrophages+to+a+Directional+Source+of+a+Cytokine+Delivered+by+a+Micropipette.">Chemotactic Responses by Macrophages to a Directional Source of a Cytokine Delivered by a Micropipette.</a> Methods Mol Biol. , 125-135 (2014).</li><li>Zicha, D., Dunn, G. A., Brown, 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=A+new+direct-viewing+chemotaxis+chamber.">A new direct-viewing chemotaxis chamber.</a> J Cell Sci. 99 (4), 769-775 (1991).</li><li>Wells, C. M., Ridley, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+cell+migration+using+the+Dunn+chemotaxis+chamber+and+time-lapse+microscopy.">Analysis of cell migration using the Dunn chemotaxis chamber and time-lapse microscopy.</a> Methods Mol Biol. , 31-41 (2005).</li><li>Liang, C. C., Park, A. Y., Guan, J. 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A., 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=On-chip+open+microfluidic+devices+for+chemotaxis+studies.">On-chip open microfluidic devices for chemotaxis studies.</a> Microsc. Microanal. 18 (04), 816-828 (2012).</li><li>Adler, M., Polinkovsky, M., Gutierrez, E., Groisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+oxygen+gradients+with+arbitrary+shapes+in+a+microfluidic+device.">Generation of oxygen gradients with arbitrary shapes in a microfluidic device.</a> Lab Chip. 10 (3), 388-391 (2010).</li><li>Huang, Y., Agrawal, B., Sun, D., Kuo, J. S., Williams, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics-based+devices:+New+tools+for+studying+cancer+and+cancer+stem+cell+migration.">Microfluidics-based devices: New tools for studying cancer and cancer stem cell migration.</a> Biomicrofluidics. 5 (1), 013412 (2011).</li><li>Taylor, A. M., Rhee, S. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+chambers+for+cell+migration+and+neuroscience+research.">Microfluidic chambers for cell migration and neuroscience research.</a> Microfluid Tech: Rev Protoc. , 167-177 (2006).</li><li>Tang, S. K., Whitesides, G. M., Fainman, Y., Lee, L., Psaltis, D., Yang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Basic microfluidic and soft lithographic techniques. Optofluidics: Fundamentals, Devices and Applications. , (2010).</li><li>Taylor, A. 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=Microfluidic+multicompartment+device+for+neuroscience+research).">Microfluidic multicompartment device for neuroscience research).</a> Langmuir. 19 (5), 1551-1556 (2003).</li><li>Aidelberg, G., Goldshmidt, Y., Nachman, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Microfluidic+Device+for+Studying+Multiple+Distinct+Strains.">A Microfluidic Device for Studying Multiple Distinct Strains.</a> JoVE. (69), (2012).</li></ol>

Our microchannel chamber may be used for a multitude of purposes, including determining the cell migration rate in response to growth factors and chemoattractants and measuring the diffusion rate of a drug from a polymer matrix. It is possible to utilize our microchannel chamber to grow cells and place a chemoattractant at one end of the chamber. The cells grow in response to the chemoattractant, and the cell migration can be quantified by taking time-lapse images that may be analyzed in order to determine the cell migra...

In order for vital processes such as wound healing, immune responses, and embryonic development to occur, cell migration must take place. Cell migration involves the interaction between cells and neighboring cells, the extracellular matrix, and soluble chemical cues (attractants or repellants). As an example, in the process of wound healing, fibroblasts play an integral role in fibrogenesis and wound contraction, where the cells are recruited to the site of injury to synthesize collagen in order to form the extracellular matrix3. Numerous mechanisms behind the migration of fibroblasts to a wound site have been studied, and they include different mechanical, physical, electrical, and chemotactic factors4. Fibroblasts respond especially well to different concentration gradients of growth factors. These different growth factors work together to optimize tissue regeneration5. While observing the chemotactic response of fibroblasts to growth factor concentrations, one can study the pattern of directional migration of fibroblasts and how they orient themselves around physical obstacles in order to reach their destination. Therefore, the goals of this study were to first develop a system in which fibroblast growth could be tracked under guidance by physical barriers and to secondly model the growth of fibroblasts as they navigate through the system.
Currently, the Boyden chamber assay is the most widely used system to measure the migration of cells6. The Boyden chamber consists of a two-chamber multi-well plate where each well may contain medium with or without chemoattractants7. A filter membrane provides a porous interface between the two chambers in each well; this creates a barrier so that cells cannot pass through unless it is by active migration. Typically, for the Boyden chamber, a chemoattractant is added to the lower chamber, and the system is allowed to equilibrate to form a gradient between the upper and lower wells4. One problem with the Boyden chamber assay is that steep gradients end up forming along a single axis perpendicular with the surface of the membrane. This causes the difference in the chemoattractant concentration between the upper and lower wells to be a lower than what was originally expected. Due to this constraint, the Boyden chamber assay makes it hard to correlate specific cell responses with particular gradient characteristics, such as the slope and the concentration difference. Without these measurements, it is hard to study multi-gradient signal integration.
To address some of the constraints of the traditional Boyden chamber assay, microfluidic assays have been developed to form customizable concentration gradients1. Standard methods for creating microfluidic systems require clean rooms for lithographic techniques. These techniques can be difficult to learn especially in a standard classroom setting. Thus, we have designed a chamber system for measuring cell migration that can be made without using a clean room. Using our system, the wells of the assay can be adjusted to a preferred size, and a linear concentration gradient of customized slope can be produced. This allows for accurate measurement of chemotaxis from random movement. The design is an inexpensive and easy-to-use system to model cell growth in response to different chemical stimuli.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Sigma-Aldrich</td>
			<td>761036</td>
			<td>poly(dimethylsiloxane) 2-part kit including silicone elastomer base and silicone elastomer curing agent</td>
		</tr>
		<tr>
			<td>Acrylic Sheets</td>
			<td>US Plastic</td>
			<td>44200</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes&#160;</td>
			<td>Falcon&#160;</td>
			<td>25373-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein isothiocyanate&#8211;dextran</td>
			<td>Sigma-Aldrich</td>
			<td>FD20s-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose, Type I, Low EEO</td>
			<td>Sigma-Aldrich</td>
			<td>A6013-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Fisher Scientific</td>
			<td>11965092</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serium</td>
			<td>Fisher Scientific</td>
			<td>16000036</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Fisher Scientific</td>
			<td>15140148</td>
			<td>Cell media components</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>BP24384</td>
			<td></td>
		</tr>
		<tr>
			<td>EVOS XL Cell Imaging System</td>
			<td>Thermo Fisher Scientific</td>
			<td>AME3300</td>
			<td>Instrument used for taking time-lapse images</td>
		</tr>
		<tr>
			<td>Versa LASER</td>
			<td>Universal Laser Systems, Inc.&#160;</td>
			<td>Model number VLS2.30</td>
			<td>Laser cutter used for cutting plastic</td>
		</tr>
	</tbody>
</table>

a customizable chamber for measuring cell migration

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between cells, the extracellular matrix, and soluble and non-soluble chemical factors (e.g., chemoattractants). Standard methods for measuring the migration of cells, such as the Boyden chamber assay, work by counting cells on either side of a divider. These techniques are easy to use; however, they offer little geometric modification for different applications. In contrast, microfluidic devices can be used to observe cell migration with customizable concentration gradients of soluble factors1,2. However, methods for making microfluidics based assays can be difficult to learn.
Here, we describe an easy method for creating cell culture chambers to measure cell migration in response to chemical concentration gradients. Our cell migration chamber method can create different linear concentration gradients in order to study cell migration for a variety of applications. This method is relatively easy to use and is typically performed by undergraduate students.
The microchannel chamber was created by placing an acrylic insert in the shape of the final microchannel chamber well into a Petri dish. After this, poly(dimethylsiloxane) (PDMS) was poured on top of the insert. The PDMS was allowed to harden and then the insert was removed. This allowed for the creation of wells in any desired shape or size. Cells may be subsequently added to the microchannel chamber, and soluble agents can be added to one of the wells by soaking an agarose block in the desired agent. The agarose block is added to one of the wells, and time-lapse images can be taken of the microchannel chamber in order to quantify cell migration. Variations to this method can be made for a given application, making this method highly customizable.

Figure 2 shows the movement of the cell front across the channel in response to a gradient of fetal bovine serum placed at the opposite end of the channel from where the cells are plated. The cell front is shown at 48 h (Figure 2A), 72 h (Figure 2B), 96 h (Figure 2C), and 120 h (Figure 2D) post plating. The movement of the cell front was tracked with these time-lapse images, and the migration distance and...

Watch this Scientific Journal Video about A Customizable Chamber for Measuring Cell Migration at JoVE.com

A Customizable Chamber for Measuring Cell Migration

This protocol details a customizable method to measure cell migration in response to chemoattractants that may also be used to determine the diffusion rate of a drug out of a polymer matrix.

Cell migration is a vital part of immune responses, growth, and wound healing. Cell migration is a complex process that involves interactions between ...

The overall goal of this procedure is to provide a simple way to study the effects of chemotactic and chemorepulsive agents on cell migration. This method can help answer key questions in the cell interaction fields such as the effects of different growth factors on wound healing. The main advantage of this technique is that it is easily customizable for different applications and simple to use for researchers of all skill levels.Demonstrating the procedure will be Aniqa Chowdhury, an undergraduate student, and Tyler Harvey, a graduate student from my laboratory. To begin, create a CAD file with the desired shape of the micro-channel. Adjust the channel's width and length to achieve the required gradient.Here, 2.254 centimeter squares are connected by a 0.127 centimeter wide channel. Next, obtain a piece of acrylic that is the desired thickness in order to create the proper depth of the microchannel. An acrylic sheet width of about one sixteenth of an inch is ideal as thicker sheets are difficult to cut and very thin sheets can break or warp during the process.Import the CAD file into a laser cutting apparatus and place the acrylic piece on the cutting surface. Then cut out the chamber, according to the manufacturer's protocol. Place the freshly cut barbell shaped acrylic cutout in a small Petri dish and cover it until it is needed.In a weigh boat, add ten parts of an elastomer base, with one part of the curing agent and stir using a micro pipette for five minutes. Transfer the mixture into a vacuum chamber, and pull a vacuum for 30 minutes to remove any trapped air bubbles from the PDMS. When finished, turn off the vacuum and remove the weigh boat.Pour the degassed PDMS on top of a barbell shaped acrylic cutout in a small Petri dish. Make sure that the PDMS completely covers the insert. Allow the PDMS to cure for at least 18 hours at room temperature, then carefully cut the PDMS around the acrylic piece with a scalpel and remove the insert.In a standard cell culture cabinet, place the microchannel chamber in a Petri dish, feature side up and completely cover it with 70%ethanol. Then shut the hood and turn on the UV light. Expose the chamber to UV light for one to two hours.Following exposure, open the laminar flow hood and wait 15 minutes for the flow hood to re-establish flow. Next, remove the 70%ethanol and wash the chambers twice with sterile PBS. Use one milliliter PBS for each wash.Following the final wash, remove the PBS and allow the chambers to dry overnight with the lids off. Harvest the cell type to be tested and add 100 microliters of the cell suspension to 400 microliters of the 0.4%trypan blue and mix gently. Apply 100 microliters of this solution to the hemocytometer by gently filling both chambers under the glass cover slip.Use a microscope with a 10x objective to focus on the grid on the hemocytometer. Then use a hand tally counter to count the live, unsustained cells within all four sets of 16 squares on the hemocytometer. Divide the total cell number by four and multiply this number by 50, 000 to get the number of cells per milliliter of cell suspension.Based on this calculation, add 2500 cells and 100 microliters of media to one well in each chamber. Allow the cells to sit in the chamber for 60 minutes then add additional media. To begin, add agarose to water and heat the solution in a microwave for one minute, or until the agarose has completely dissolved and the solution is clear.Allow the solution to cool for 15 to 30 seconds before pouring into a Petri dish. To remove any bubbles, place the Petri dish into a vacuum chamber and allow the agarose to solidify while under a vacuum for 30 minutes. Once solidified, cut a two millimeter by two millimeter by two millimeter cube of agarose with a scalpel from the Petri dish.Completely submerge the cube in a solution containing the desired concentration of the chemo tactic factor, and soak the agarose block overnight at room temperature. Next place the agarose block containing the chemo tactic factor at the far end of the microchannel chamber. Utilize a marker, such as polystyrene microbeads or a microscopic defect in the chamber.To identify the relative starting location of the cells. Use the imaging software to take time lapsed images of the moving cell front every hour for 12 hours. The series of images shown here documents the movement of a cell front across the channel in response to a gradient of fetal bovine serum placed at the opposite end of the channel.The migration distance was measured based on the distance of the growth front from a microscopic defect in the device. From these measurements, the average cell front velocity was found to be 13.4 micrometers per hour. Once mastered, this technique can be completed in four hours, with an additional 18 hours of wait time for PDMS to cure, and an additional 12 hours for the time lapse imaging.After it's development, this technique paved the way for researchers of different skill levels, including undergraduate students with little lab experience to easily explore wound healing and cell migration in vitro. While attempting this procedure, it's important to remember to carefully remove the acrylic insert from the PDMS mold in order to ensure successful production of a microchannel chamber. After watching this video, you should have a good understanding of how to produce a simple microchannel chamber for studying cell migration in response to different chemical cues.Following this procedure, other cues can be studied such as multiple soluble chemical gradients, substrates, fluid sheer and electrical fields in order to study the effect they have on cell migration. Don't forget that working with laser cutters can be extremely hazardous and precautions such as obtaining proper training, and using personal protective equipment should always be taken while performing this procedure.

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Research

JoVE Journal

Biology

10.5K ビュー.  Clemson University. The overall goal of this procedure is to provide a simple way to study the effects of chemotactic and chemorepulsive agents on cell migration. This method can help answer key questions in the cell interaction fields such as the effects of different growth factors on wound healing. The main advantage of this technique is that it is easily customizable for different applications and simple to use for researchers of all skill levels.Demonstrating the procedure will be Aniqa Chowdhury, an ...