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). Adjust the channel width and length to achieve the desired gradient relevant to individual experimental protocols.
		<ol>
			<li>Here, use an acrylic piece of the following dimensions: two squares (0.254 cm x 0.254 cm) connected by a 0.127 cm wide channel (0.254 cm long) (Figure 1).</li>
		</ol>
		</li>
		<li>Import the CAD file into the laser cutting apparatus and place the acrylic piece in a laser cutter according to the manufacturer&#39;s protocol. 
		NOTE: Alternatively, 3D-print the piece from the CAD design. The advantage of this method is that alternate materials can be used for the mold; however, the resolution and reproducibility can be decreased with 3D printing compared to laser cutting.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/55264/55264fig1.jpg" /> 
Figure 1: Computer-Aided Design (CAD) Representation of Microchannel Chamber. This image depicts the CAD drawing of the acrylic insert needed to create the microchannel chamber. 1A) Top view of CAD drawing, 1B) Side view of CAD drawing with dimensions in inches, 1C) Top view of CAD drawing with dimensions in inches <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55264/55264fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Pouring the polydimethylsiloxane (PDMS)
	<ol>
		<li>In a weigh boat, mix 10 parts by weight of commercial Elastomer Base (e.g. Sylgard 184) with 1 part by weight of commercial Elastomer Curing Agent (e.g. Sylgard 194). Typically, mix 10 g of Elastomer Base to 1 g of Elastomer Curing Agent.</li>
		<li>Stir to combine the base and curing agent with a micropipette for 5 min.</li>
		<li>Set up a vacuum bell and place the weigh boat with PDMS in the vacuum for 30 min to remove trapped air bubbles.</li>
		<li>Turn off the vacuum and remove the weigh boat. Pour the PDMS on top of the acrylic cut-out in a small Petri dish. Make sure that the PDMS completely covers the insert.</li>
		<li>Allow the PDMS to cure overnight (at least 18 h) at room temperature.</li>
	</ol>
	</li>
	<li>Removing the insert from PDMS
	<ol>
		<li>After curing the PDMS, remove the insert by carefully cutting the PDMS around the acrylic piece with a scalpel.</li>
	</ol>
	</li>
</ol>
2. Plating the Cells in a Microchannel Chamber
<ol>
	<li>Sterilizing the chamber for plating cells. 
	NOTE: While PDMS can be sterilized using ethylene oxide or other techniques, a quick UV treatment is fast, inexpensive, and sufficient for cell culture studies. The short UV treatment duration did not impair the structure or mechanical integrity of the PDMS piece.
	<ol>
		<li>In a standard cell culture cabinet, completely cover the chamber with 70% ethanol.</li>
		<li>Put the Petri dish in laminar flow hood with the lids off.</li>
		<li>Shut the hood and turn on the UV light. Expose to UV light for 1-2 h.</li>
		<li>Open the laminar flow hood and wait 15 min to reestablish flow.</li>
		<li>Remove the 70% ethanol.</li>
		<li>Wash the chambers twice with sterile phosphate buffered saline (PBS). Use 1 mL of PBS for each wash. Remove PBS and allow chambers to dry overnight with the lids off. 
		NOTE: Alternatively, instead of using a laminar flow hood, use a UV chamber box for sterilization if one is available. Make a UV box by placing a UV light source in the back of a cardboard box lined with aluminum foil. The aluminum foil reflects the light to allow for even illumination of the sample.</li>
	</ol>
	</li>
	<li>Plating Cells in Chamber
	<ol>
		<li>Count cells using Trypan blue and a hemocytometer.
		<ol>
			<li>Add 100 &#181;L of the cell suspension to 400 &#181;L of 0.4% Trypan blue and mix gently. Apply 100 &#181;L of this solution to the hemocytometer by gently filling both chambers under the glass coverslip.</li>
			<li>Use a microscope with a 10X objective to focus on the grid on the hemocytometer. Use a hand tally counter to count the live unstained cells within one set of 16 squares on the hemocytometer.</li>
			<li>Count cells in all 4 sets of 16 squares. Take the average cell count from the 4 sets of 16 squares and multiply by 5x104. This final number is the number of cells/mL in the cell suspension.</li>
		</ol>
		</li>
		<li>Add 2,500 cells to one well in each chamber. This translates to a near-confluent cell density of ~30,000/cm2.</li>
		<li>Allow cells to sit in the chamber for 60 min before adding media (Dulbecco&#39;s Modified Eagle&#39;s Medium, 10% fetal bovine serum, 1% penicillin-streptomycin).</li>
	</ol>
	</li>
</ol>
3. Dextran Soaked Agarose Blocks
<ol>
	<li>Creating an agarose block
	<ol>
		<li>Pour 3% agarose into the 3D printed mold (2 mm x 2 mm x 2 mm).</li>
		<li>Allow the agarose to solidify in a vacuum chamber for 30 min to remove any bubbles.</li>
		<li>Remove the agarose block from the mold. 
		NOTE: Alternatively, pour 3% agarose into small Petri dish at a thickness of 2 mm. Cut a 2 mm x 2 mm x 2 mm block using a scalpel or other cutting tool. This is not as precise as the mold system but it can be a little easier to do.</li>
	</ol>
	</li>
	<li>Completely submerge the agarose block in a solution of the desired concentration of the chemotactic factor. To assess the concentration gradient formation, flush the chamber first with fluorescent dextran. The concentration and molecular weight of dextran should match that of a soluble factor or drug of interest.</li>
	<li>Soak the agarose block overnight.</li>
</ol>
4. Time-lapse of the Dextran Diffusion to Assess the Soluble Factor Concentration Gradient
<ol>
	<li>Place the dextran soaked agarose block in a small square well of the microchannel chamber.</li>
	<li>Place the microchannel chamber in a cell imaging system or use another fluorescent microscope with a time-lapse capability. Begin the time-lapse according to manufacturer&#39;s instructions. 
	NOTE: Results shown are taken with GFP filter, 50% light, 4/10 pH, and 4X magnification.</li>
</ol>
5. Time-lapse of Cell Growth
<ol>
	<li>Plate cells in the microchannel chamber at one end of the chamber. Here, use 3T3 fibroblasts. Add 2,500 cells to one well in the microchannel chamber. Count cells as described in 2.2.1.
	<ol>
		<li>Allow cells to sit in the chamber for 60 min before adding media (Dulbecco&#39;s Modified Eagle&#39;s Medium, 10% fetal bovine serum, 1% penicillin-streptomycin). Keep the microchannel chamber with plated cells at 37 &#176;C in 5% CO2. Cell migration through the channel can be viewed with a microscope.</li>
		<li>Add a marker or use a microscopic defect in the chamber as a marker in order to serve as a starting place to quantify the distance the cell front moves.</li>
	</ol>
	</li>
	<li>Establish a gradient by placing an agarose gel (8 mm3 block) containing the concentrated growth factor, chemo-attractant, drug, or other factor being tested (here, 40% FBS is being used as an example) at one end of the microchannel chamber.</li>
	<li>Take time-lapse images of the moving cell front. Here, the results show images of the cell front that were taken every 12 h using an imaging system.</li>
	<li>Use pictures of the cell front to quantify cell growth rate. Calculate the growth rate by first using the MATLAB polyfit function (roipoly) to create a polygonal mask defined by the cell front, the channel walls, and the reference marker. The dimensions of this mask yield the migration distance of the front from which average cell front velocity is calculated. Other studies have utilized a similar method8. 
	NOTE: Compare the edge of the cell growth front on each frame to the concentration of the soluble factor at that same distance. The soluble factor concentration gradient is calculated using a 1D diffusion model approximation.</li>
	<li>Take fluorescent images of the cells using Phalloidin and DAPI staining.
	<ol>
		<li>Fix cells before staining with Phalloidin.
		<ol>
			<li>Warm 4% paraformaldehyde at 37 &#176;C.</li>
			<li>Add enough 4% paraformaldehyde to cover cells. Keep cells in the paraformaldehyde for 10 min.</li>
			<li>Rinse twice with PBS for 15 min each time. Use enough PBS to cover the cells.</li>
		</ol>
		</li>
		<li>Remove PBS from cells and add enough permeabilizing solution (0.1% Triton X-100 in PBS) to cover cells. Leave solution for 10 min.</li>
		<li>Wash twice with PBS for 5 min each time. Use enough PBS to cover the cells.</li>
		<li>Remove PBS from cells and add blocking solution (1% bovine serum albumin in PBS) for 20 min.</li>
		<li>Remove blocking solution and wash 2 times with PBS for 5 min each time. Use enough PBS to cover the cells.</li>
		<li>From this point, protect cells from light with aluminum foil when not being used. Remove PBS from cells and add Phalloidin 488 diluted 1:50 in PBS for 1 h.</li>
		<li>Wash 2 times in PBS for 5 min for each wash. Use enough PBS to cover the cells. Then remove PBS from cells.</li>
		<li>Add DAPI (4&#39;,6-diamidino-2-phenylindole) diluted 1:1,000 in PBS to cells for 15 min.</li>
		<li>Remove DAPI and wash cells twice with PBS for 5 min for each wash.</li>
		<li>Remove last wash and add enough PBS to cover cells. Cells can now be imaged with a fluorescence microscope.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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 migration rate. Representative results of this method are shown in Figure 3, where the growth rate was determined to be 13.4 &#181;m/h using the MATLAB polyfit function.
Another usage of our microchannel chamber is to measure the diffusion rate of a drug out of a polymer matrix. A fluorescently tagged dextran of a similar molecular weight to the drug of interest may be used to model the diffusion of the drug of interest. It is important to note that the samples must be handled carefully as a large amount of convection will disrupt the gradient. The representative results of this method are shown in Figure 4. An advantage of this model in comparison to the Boyden chamber assay is that it is more applicable to adherent cell types because of the flat surface; whereas, the cells must fall through a small tube in the Boyden chamber before adhering. Another limitation of the Boyden chamber assay is that it is an endpoint assay; thus, chemotaxis cannot be visually observed. In contrast, with our method, chemotaxis is observed through the time-lapse images9. The use of micropipettes containing chemoattractants have also been used a method of observing cell migration. However, the main drawback of this method is low throughput10. The Dunn chemotaxis chamber is another assay that is used in conjunction with time-lapse imaging like the method detailed here11,12. The chamber consists of two concentric circles carved on the face of the glass slide in order to create an inner and outer well, and a bridge separates the two wells. The chemoattractant is placed in the outer well, and the cells are allowed to migrate from the inner well to the outer well. This migration is quantified using time-lapse images that are analyzed using image analysis software. Similar to the Boyden assay, one of the limitations of the Dunn chamber assay is that it cannot be easily modified to create custom soluble factor concentration gradients.
Another simple technique for assessing cell migration is an in vitro scratch13. This method is fast and inexpensive as it only requires creating a scratch in the cell monolayer and capturing of time-lapse images of cell migration close to the previously created scratch. However, the major disadvantage of this method is that it is not suitable to measure responses to a chemical factor concentration gradient. Our method offers the potential to create a chemical gradient and to quantify cell migration relative to the chemical gradient. Our method may be combined with the scratch method as a scratch may be created in the cell monolayer, while a chemotactic factor is present in one of the wells in the microchannel chamber. This allows for the quantification and modeling of wound healing. Another potential application of our device is to determine the response of cancer cells to agents that inhibit cell growth.
Other microfluidics based systems have also been used for cell migration studies14,15. These studies utilize lithographic techniques with clean rooms to create master molds in silicon and/or laser machining to create microfluidic channels in silicon wafers. Silicon wafer micro machining can be expensive in comparison to our method that utilizes cheaper materials (PDMS and acrylic) to manufacture a microchannel chamber. Our device overcomes the limitation of other PDMS based microfluidic devices in which it was not possible to create small 3D channels15. With our method, we have successfully created PDMS based device with small diameter channels. Other studies have used a PDMS-based microfluidic device with two layers of microchannels and a multi-channel gas mixer that is computer controlled in order to create arbitrary gradients of oxygen to study the migration of cells under different gradients of oxygen16. There is potential for our device to be used in a similar fashion with the addition of a gas mixing chamber at one end of the microchannel chamber.
Microfluidic devices are often made using soft lithographic techniques; these can be adapted to create cell migration chambers. These methods involve printing a mask with channel patterns with computer-aided design (CAD). These masks are then projected on a master mold coated with a photosensitive resist through optical lithography. After the master mold is made, PDMS is poured on the master and allowed to harden in order to create a PDMS mold that can be used for microfluidic studies17,18,19. Chemoattractant or chemorepulsive compounds are placed in the chamber in high concentration in one of the reservoirs to create gradients17. While these techniques are similar to our set up, they can be expensive due to the high cost of creating the master mold for the system and the use of clean rooms. In addition, these methods can be difficult for students to learn in a class based setting. The inserts used in our set-up to create the channels can be created with a variety of methods, including 3D printing and laser cutting. Thus, our system allows for the creation of a wide variety of molds for relatively low cost particularly as 3D printing becomes more affordable and widely available.
PDMS based microfluidic devices for measuring cell migration and growth have been used for a wide range of applications. For instance, in neuroscience research studies20, devices were fabricated using soft lithography techniques in which the PDMS component was placed on a tissue culture dish or glass in order to form two compartments that were separated by a physical barrier with micron-sized grooves to allow neurons to grow across the compartments. Time-lapse images were taken to show neurons crossing the barrier. The method of micropatterning used to produce this device is complex, while the production method for our device is simple and can be used by researchers at all levels. Another study also used an easy low-cost method for producing a similar PDMS microfluidic device using a master mold created from scotch tape in order to create an impression in the PDMS to produce microchannels. An advantage of our approach over the one described in this method is that the channels in our method are fabricated in one step, while this protocol requires adhesion between two PDMS layers which creates problems with medium flow to the cells 21.
In conclusion, our microchannel chamber method is customizable and may be used to obtain different results. The method allows for the creation of a concentration gradient in order to suit the user&#39;s needs. Furthermore, our device offers an advantage over existing devices due to its relatively low cost and ease of use for researchers at all levels.

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><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&amp;nbsp;</td><td>Falcon&amp;nbsp;</td><td>25373-041</td><td></td></tr><tr><td>Fluorescein isothiocyanate&amp;ndash;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&#039;s Modified Eagle&#039;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.&amp;nbsp;</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 growth rate were calculated by the MATLAB polyfit function. From this, the average cell front velocity was found to be 13.4 &#181;m/h (Figure 3). Figure 4 shows the fluorescent gradient established by placing a dextran soaked agarose block at one end of the channel and allowing dextran to diffuse through the channel for 12 h. A time-lapse image was taken at every hour, and the fluorescence across the distance of the channel was measured and plotted as shown in Figure 4 and compared to a 1D diffusion model.
<img alt="Figure 2" src="/files/ftp_upload/55264/55264fig2.jpg" /> 
Figure 2: Cell Front Movement. Time-lapse images of the cell front in the microchannel chamber moving through the channel. The cell front marked in orange lines, and the migration distance is included. This migration distance was measured from the marking on the plate to the front of the cell front as shown. A 1 mm scale bar is shown in white. A) 48 h post plating, B) 72 h post plating, C) 96 h post plating, D) 120 h post plating. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55264/55264fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55264/55264fig3.jpg" /> 
Figure 3: Growth Rate Calculation. Cell front movement tracked with time-lapse pictures. Growth rate was calculated using the MATLAB polyfit function (roipoly) to yield average cell front velocity of 13.4 &#181;m/h. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55264/55264fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55264/55264fig4.jpg" /> 
Figure 4: Fluorescent Gradient with Dextran. Fluorescent intensity measured across the distance of the channel where each line represents the gradient at a particular hour. ImageJ was used to calculate the fluorescence intensity. This can be done using the Analyze | Measure tool. First, choose Set Measurements under Analyze and select intensity. Then, choose Measure under Analyze in order to get average pixel intensity. This can be plotted versus distance in microns as shown in this figure. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55264/55264fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

a-customizable-chamber-for-measuring-cell-migration

Nanyang Technological University

Research

JoVE Journal

Biology

10.6K Views.  Clemson University.  Dit protocol Gegevens een aanpasbare werkwijze celmigratie als reactie op chemoattractanten die ook kunnen worden gebruikt om de diffusiesnelheid van een geneesmiddel te bepalen uit een polymeermatrix meten. 

Video: A Customizable Chamber for Measuring Cell Migration

Assessment of Endothelial Cell Migration After Exposure to Toxic Chemicals

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels &#8211; either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention.
We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC.

Investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of certain illnesses and to potentially identify novel strategies for therapeutic intervention. The following protocol describes techniques to assess cell migration that have been adapted for the investigation of EEC.

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms ...

Developmental Biology

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

Cancer Research

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

Medicine

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 ...

Bioengineering

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

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.

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 ...

Real-Time Quantitative Measurement of Tumor Cell Migration and Invasion Following Synthetic mRNA Transfection

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell migration and invasion is essential for understanding the mechanisms of tumor cell infiltration into neighboring healthy tissues and metastasis. Previously, it was demonstrated that gene knockdown followed by the impedance-based real-time measurement of tumor cell migration and invasion enables the identification of the genes required for tumor cell migration and invasion. Recently, the mRNA vaccines against SARS-CoV-2 have increased interest in using synthetic mRNA for therapeutic purposes. Here, the method using synthetic mRNA was revised to study the effect of gene overexpression on tumor cell migration and invasion. This study demonstrates that elevated gene expression with synthetic mRNA transfection followed by impedance-based real-time measurement may help identify the genes that stimulate tumor cell migration and invasion. This method paper provides important details on the procedures for examining the effect of altered gene expression on tumor cell migration and invasion.

Many upregulated genes stimulate tumor cell migration and invasion, leading to poor prognosis. Determining which genes regulate tumor cell migration and invasion is critical. This protocol presents a method for investigating the effects of the increased expression of a gene on the migration and invasion of tumor cells in real time.

Tumor cells are highly motile and invasive and display altered gene expression patterns. Knowledge of how changes in gene expression regulate tumor cell ...

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and epithelial wound healing. Applying a direct current electric field to cultured cells in vitro induces directional cell migration, or galvanotaxis. The 2-dimensional galvanotaxis method we demonstrate here is modified with custom-made poly(vinyl chloride) (PVC) chambers, glass surface, platinum electrodes and the use of a motorized stage on which the cells are imaged. The PVC chambers and platinum electrodes exhibit low cytotoxicity and are affordable and re-useable. The glass surface and the motorized microscope stage improve quality of images and allow possible modifications to the glass surface and treatments to the cells. We filmed the galvanotaxis of two non-tumorigenic, SV40-immortalized prostate cell lines, pRNS-1-1 and PNT2. These two cell lines show similar migration speeds and both migrate toward the cathode, but they do show a different degree of directionality in galvanotaxis. The results obtained via this protocol suggest that the pRNS-1-1 and the PNT2 cell lines may have different intrinsic features that govern their directional migratory responses.

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

A Customizable Chamber for Measuring Cell Migration

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