This work is supported by the New York State Office of Science, Technology and Academic Research (NYSTAR) (in the form of a Center for Advanced Technology grant), and by grants from the Nanobiotechnology Center (NBTC), an STC Program of the National Science Foundation under Agreement No. ECS-9876771, and the American Brain Tumor Association and the New York Academy of Medicine (JAB).

Procedure
Coating slides with fibronectin
Prior to the assembling of the microfluidic device, sterilize glass slides with fibronectin. Coat the slides in a biohood to maintain sterility. Align a&nbsp;sterile PDMS spacer&nbsp;along the edges of the slide and mark the side that is facing up with a permanent marker. Pipette 1 ml of a 5 &mu;g/ml solution of fibronectin (Sigma) inside the PDMS spacer&nbsp;over the entirety of the glass slide. Leave the slides undisturbed for an hour, then dry completely using an N2 gun. The slides can be stored at 4oC for later experiments.
Assembling the device
The entire device assembly protocol is done in a biohood under sterile conditions, using the following procedures:
<ol>
<li>Clean the silicon master with the microchannels patterned on it by wiping it down with 70% ethanol and drying with the N2 gun. Place a sterile PDMS spacer of 1 mm height using forceps around the relief features of the master. </li>
<li>Prepare the agarose gel used in the device by weighing 0.3 g of agarose powder (Fisher Scientific) and 10 ml of CO2-independent media (Invitrogen) in a 50 ml beaker. Stir the mixture with a spatula and heat in a microwave for 20 seconds on HIGH. If undissolved granules are present, heat the mixture for another 10 seconds and swirl the hot mixture to get rid of the last remaining granules. Repeat the process with a heating time of 8 seconds, and then 5 seconds.</li>
<li>Quickly pour&nbsp;the agarose solution onto the silicon master surrounded by the PDMS spacer and immediately cover the agarose mixture with a sterile glass slide. Apply a&nbsp;constant, gentle pressure to the slide for about 2 minutes to ensure that the agarose will gel to the same constant 1 mm height as the PDMS spacer. </li>
<li>After the agarose gels, peel off the patterned agarose gel with the PDMS spacer from the silicon master and transfer to a fibronectin coated glass slide with the microchannels in the agarose gel facing the slide.&nbsp;Use a&nbsp;16 gauge syringe tip to punch holes into the reservoir regions of the microchannels for inlet and outlet accesses. Add 500 &mu;l of CO2-independent media to the patterned agarose gel on the slide to prevent desiccation of the microchannels.</li>
<li>Wash the acrylic manifold used in the device with 70% ethanol and rinse with DI water. Align the holes in the manifold with the holes punched into the agarose gel with the microchannels. Then, align the&nbsp;manifold with the gel, PDMS spacer, and the fibronectin slide with the metal frame of the device. Place screws into device and tighten to the point of resistance using a screwdriver. This is done to ensure that the device is not over-tightened to the extent of deforming the microchannels in the agarose. Tighten the screws in a clockwise order to ensure that the gel inside the device does not shift. Test the device for leaks and blockages in the microchannels using a 1ml syringe. The syringe is fitted with Tygon tubing with a gel-loading pipette tip at the end of the tubing to inject CO2-independent media in the microchannels. A microchannel is deemed successfully formed when media is observed to only exit from the outlet of the microchannel when media is injected into the corresponding inlet. Now, the device is ready for use. </li>
</ol>
Preparing and Seeding the cells in the device
When the cells reach 70% confluency (cell density around 100,000 cells/ml), they are prepared to be seeded into the device.
Subject the cells to two DPBS (Invitrogen) washes.&nbsp;&nbsp;Add 200 &mu;l of trypsin-EDTA (Invitrogen) to detach the cells. Transfer the cells to a 15 ml centrifuge tube containing 5ml of M2 media containing serum to inactivate the trypsin. Centrifuged&nbsp; the growth media with the cells at 1000 RPM for 5 minutes. Aspirated off the supernatant and resuspend the pellet of cells 5 ml of DPBS. Centrifuge the DPBS cells again under the same conditions. Aspirated off the supernatant again, and resuspend the pellet of cell in 500 &mu;l of CO2-independent media.
Seed the resulting suspension of cells into the center microchannel of the device via gravity-driven flow. Add the inlet and outlet of the center microchannel with 60 &mu;l of the cell suspension and 20 &mu;l of CO2-independent medium, respectively. Observe the center microchannel under a brightfield microscopy to look for cell adhesion. The cells usually adhere after 10 minutes. After the cells adhere to the channel bottom at a proper density, pipeted out the remaining cell suspension in the inlet and the media in the outlet. Place 60 &mu;l of CO2-independent media in both inlet and outlet of the center channel. Place PDMS covers over the center inlet and outlet to minimize evaporation of media from the center channel.
Imaging the cells in the device
Prior to the assembly of the device, thread the sterile peristaltic tubing through the weather station of the microscope (Olympus X81) and connect with the peristaltic pump (Watson-Marlow 205U/CA). Flush PBS through the tubing at a rate of 4 RPM. After the assembly of the device, prepare appropriate concentrations of epidermal growth factor (EGF) (Sigma) solutions in the CO2-independent media in a 15 ml centrifuge tubes. Vortex the EGF solutions for 5 seconds and connect with the tubing. Connect the tubings marked with corresponding EGF concentrations to the device. It takes about an hour for the EGF to diffuse across the channel ridge and establish a steady chemical gradient.
An imaging software, Slidebook, is used to take a set of images at every 10 minutes for about 5 hours in an experimental run. The x-y automated stage is programmed such that two sub-areas (each with an area of 400&mu;m x400&mu;m) of the same center channel, and a total of three center channels are imaged at a given time point. Cells in different center channels (typically 3 in one run) have different EGF concentrations, as marked on the tubing.
Materials and Equipment
Cell lines: C17.2 cells are NSCs derived from the external germinal layer of postnatal mouse cerebellum 6, 7. Using this cell line, we have developed two other murine stem cell lines using retrovirus transfection method. These two stably transfected cell lines are (1) wtEGFR -- that over-expresses the human wild type EGFR, and (2) &Delta;EGFR - that is a constitutively active EGFR mutant. For ease of detection, retroviruses carry a backbone with the gene of interests followed by an internal site for ribosomal binding (IRES) and the Green fluorescent protein (GFP).
Cell culture: The mouse C17.2 NSCs and their variants were grown in a 6-well plate in M2 media (DMEM (Invitrogen) with 10% fetal bovine serum (FBS) (Invitrogen), 5% horse serum (HS) (Invitrogen), and 1% penicillin/streptomycin (Invitrogen).) Cells were maintained at 37oC in 80% air and 5% CO2. 
Microfluidic device: A plastic manifold, a PDMS spacer, and a stainless frame for holding the device in place.
Imaging
An inverted fluorescent microscope (Olympus IX-81) is used, in connection with an intensified CCD camera (Orca, Hamamatsu). The microfluidic device is mounted on an automated x-y-z stage, and the stage is enclosed by an environmental chamber that maintains a temperature of 37oC. There are usually 3-4 devices on the same chip that will be filmed at the same time point using the automated x-y-z stage.
Flow maintaining
A peristaltic pump with 8 parallel lines (Watson-Marlow 205U/CA) auto is used to maintain flows in all of the sink and source channels.
&nbsp;
<img src="/files/ftp_upload/old/674_2008_9_29_6/674_Figure-01.jpg" alt="Figure 1" width="573" height="414" /> Fig. 1 Motility of three NSC strains (parental, wtEGFR, and DEGFR) in various EGF concentrations All the trajectories are taken from tracking a movie of 5 hours (except for wtEGF 100ng/ml, 4hour), and the origin of all the tracks were repositioned at (0,0) for comparison purpose. 
&nbsp;
&nbsp;<img src="/files/ftp_upload/old/674_2008_9_29_7/674_Figure-02.jpg" alt="Figure 2" width="576" height="546" />
 Fig. 2 Correlation of EGFR expression level and motility (a) two images of wtEGF NSCs on the surface of a microfluidic channel with 100ng/ml EGF. The left image is a fluorescent image, and the right is a transmitted bright field image. The scale bar is 100&micro;m, and the Channel width is 400 &micro;m. Cells are separated into two populations by the brightness of the GFP expressed. Cells in the blue circles are dimmer, thus have less EGFR expression level; and cells in red circles are brigher, thus have higher EGFR expression level. (b) Trajectories of cells with low and high EGFR expression level. These trajectories are taken from a four hour-long movie.

<ol>
	<li>Blow, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics:+in+search+of+a+killer+application.">Microfluidics: in search of a killer application.</a> Nature Methods. 4, 665-670 (2007).</li><li>Shing-Yi Cheng, S. H., Wassweman, M. a. x., Archer, S. h. i. v. a. u. n., Shuler, M. i. c. h. a. e. l. . L., Wu, M. i. n. g. m. i. n. g. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hydrogel-based+microfluidic+device+for+the+studies+of+directed+cell+migration.">A hydrogel-based microfluidic device for the studies of directed cell migration.</a> Lab-on-a-chip. 7, 763-769 (2007).</li><li>Pedersen, M. W., Tkach, V., Pedersen, N., Berezin, V., Poulsen, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+a+naturally+occurring+constitutively+active+variant+of+the+epidermal+growth+factor+receptor+in+mouse+fibroblasts+increases+motility.">Expression of a naturally occurring constitutively active variant of the epidermal growth factor receptor in mouse fibroblasts increases motility.</a> Int J Cancer. 108, 643-653 (2004).</li><li>Ayuso-Sacido, A., Graham, C., Greenfield, J. P., Boockvar, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+duality+of+EGFR+signaling+and+neural+stem+cell+phenotype:+Cell+enhancer+or+cell+transformer.">The duality of EGFR signaling and neural stem cell phenotype: Cell enhancer or cell transformer.</a> Current Stem Cell Research and Therapy. 1, 231-238 (2006).</li><li>Eisenbach, M., Constantinou, C., Aloni, H., Shinitzky, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repellents+for+Escherichia+coli+operate+neither+by+changing+membrane+fluidity+nor+by+being+sensed+by+periplasmic+receptors+during+chemotaxis.">Repellents for Escherichia coli operate neither by changing membrane fluidity nor by being sensed by periplasmic receptors during chemotaxis.</a> J Bacteriol. 172, 5218-5224 (1990).</li><li>Snyder, E. Y., Deitcher, D. L., Walsh, C., Arnold-Aldea, S., Hartwieg, E. A., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+neural+cell+lines+can+engraft+and+participate+in+development+of+mouse+cerebellum.">Multipotent neural cell lines can engraft and participate in development of mouse cerebellum.</a> Cell. 68, 33-51 (1992).</li><li>Snyder, E. Y., Yoon, C., Flax, J. D., Macklis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+neural+precursors+can+differentiate+toward+replacement+of+neurons+undergoing+targeted+apoptotic+degeneration+in+adult+mouse+neocortex.">Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex.</a> Proc Natl Acad Sci U S A. 94, 11663-11668 (1997).</li></ol>

Fig. 1 shows trajectories of three NSC strains, the parental cells, the wtEGFR and the &Delta;EGFR (control). Each set of trajectories were obtained from a 5 hours long movie. For parental cells, cell motility peaked when EGF concentration was ~ 10ng/ml; for wtEGFR cells, cell motility peak shifted to 100ng/ml or above; for &Delta;EGFR cells, the cell motility was independent of EGF concentration as expected.

The wtEGF cells with 100ng/ml were the most motile cells, they moved about 1.5 times faster than the parental cells with 10ng/ml. This indicates that (1) over expression of EGFR enhances cells motility; (2) the number of EGF ligand-receptor biding events has a direct impact on the cell motility. &Delta;EGFR cell line is a constitutively active EGFR mutant. This mutant appears in ~ 60% of human glioblastoma. It has a truncated extracellular domain that becomes a constitutive active ligand-independent kinase. The intensity of the autophosphorylation is small (approximately 10%) compared with ligand activated wild-type EGFR (wtEGFR) 4. This underlies the observation that &Delta;EGF cells have slightly higher motility than the parental and wtEGFR (in the absence of EGF), but the motility is kept a constant in various EGF concentrations.

Since the wtEGFR cells carried same copies of GFP (green fluorescent protein) and EGFR, the fluorescent intensity of the wtEGFR cells reveal the EGFR expression levels. This allows us to directly correlate the EGFR protein expression level (green fluorescent intensity) with the cell motility. Fig. 2 shows the trajectories of two cell population, the cells in red circle are brighter, thus has higher EGFR expression level; and the cells in blue circle are dinner, thus has lower EGFR expression level. The results again demonstrate that the higher the EGFR expression level, the more motile these cells are.

Traditionally, motility of NSCs has been determined using a Boyden chamber assay, where the percentage of cells migrating through a membrane is evaluated5. This is a population based assay, where cell behavior can not be assessed individually. The microfluidic device shown here presents an opportunity to study cell motion at a single cell level, and in a well controlled chemical environment. It allows one, for the first time, to correlate directly the EGFR protein expression level with the NSCs motility phenotype in living cells. The small size of the device is especially useful for experiments where cells sources are limited, such as the human stem cells.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Dulbecco's Modified Eagle Medium (DMEM) (1X)</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>11971-025</td>
<td>&nbsp;</td>
<tr>
<td>CO2-independent media</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>18045-088</td>
<td>A non-HEPES proprietary medium suitable for supporting cell growth for a variety of epithelial, fibroblast, and lymphoid cell lines in atmospheric conditions, contains no L-glutamine</td>
</tr>
<tr>
<td>Trypsin-EDTA</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25200-072</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>14190-144</td>
<td>Phosphate-buffered saline, without calcium or magnesium</td>
</tr>
<tr>
<td>Pen/Strep</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>Contains 10,000 units of penicillin (base) and 10,000 g of streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline</td>
</tr>
<tr>
<td>FBS</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>10437-028</td>
<td>Fetal Bovine Serum, Qualified</td>
</tr>
<tr>
<td>Horse Serum</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>16050-130</td>
<td>Horse Serum, EIA tested (negative) serum</td>
</tr>
<tr>
<td>Fibronectin</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>F0895</td>
<td>Fibronectin from human plasma, also called cold insoluble globulin</td>
</tr>
<tr>
<td>EGF</td>
<td>Reagent</td>
<td>Sigma-Aldrich</td>
<td>E4127</td>
<td>Epidermal Growth Factor from murine submaxillary gland, lyophilized</td>
</tr>
<tr>
<td>Agarose</td>
<td>Reagent</td>
<td>FisherScientific</td>
<td>BP1356-500</td>
<td>&nbsp;</td>		</tbody>
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assessing neural stem cell motility using an agarose gel-based microfluidic device

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is largely due to the difficulty with which one can create a cell-compatible and steady microenvironment using conventional microfabrication techniques and materials. We address this problem via the introduction of a novel microfabrication material, agarose gel, as the base material for the microfluidic device. Agarose gel is highly malleable, and permeable to gas and nutrients necessary for cell survival, and thus an ideal material for cell-based assays. We have shown previously that agarose gel based devices have been successful in studying bacterial and neutrophil cell migration2. In this report, three parallel microfluidic channels are patterned in an agarose gel membrane of about 1mm thickness. Constant flows with media/buffer are maintained in the two side channels using a peristaltic pump. Cells are maintained in the center channel for observation. Since the nutrients and chemicals in the side channels are constantly diffusing from the side to center channel, the chemical environment of the center channel is easily controlled via the flow along the side channels. Using this device, we demonstrate that the movement of neural stem cells can be monitored optically with ease under various chemical conditions, and the experimental results show that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells. Motility of neural stem cells is an important biomarker for assessing cells aggressiveness, thus tumorigenic factor3. Deciphering the mechanism underlying NSC motility will yield insight into both disorders of neural development and into brain cancer stem cell invasion.

Watch this Scientific Journal Video about Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device at JoVE.com

Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical.

While microfluidic technology is reaching a new level of maturity for macromolecular assays, cell-based assays are still at an infant stage1. This is ...

assessing-neural-stem-cell-motility-using-an-agarose-gel-based

Nanyang Technological University

Research

JoVE Journal

Biology

11.6K Views.  Cornell University. We demonstrate that the over expression of epidermal growth factor receptors (EGFR) enhances the motility of neural stem cells(NSCs) using a novel agarose gel based microfluidic device. This technology can be readily adaptable to other mammalian cell systems where cell sources are scarce, such as human neural stem cells, and the turn around time is critical. 

Video: Assessing Neural Stem Cell Motility Using an Agarose Gel-based Microfluidic Device

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Fabrication of a Microfluidic Device for the Compartmentalization of Neuron Soma and Axons

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for culturing neurons. Previously, a silicon wafer was patterned with the design for the neuron microfluidic device using SU-8 and photolithography to create a master mold, or what we simply refer to as a "master". Next, we pour the silicon polymer PDMS on top of the master which is then cured by heating the PDMS to 80&deg;C for 1 hour. The PDMS forms a negative mold of the device. The PDMS is then carefully cut and lifted away from the master. Holes are punched where the reservoirs will be and the excess PDMS trimmed away from the device. Nitrogen is used to blow away any excess debris from the device. At this point the devices are now ready for use and can either bonded to corning No. 1 cover glass with a plasma sterilizer/cleaner or can be reversibly bound to the cover glass by simply placing the device on top of the cover glass. The reversible bonding of the device to glass is covered in a separate video and requires first that the device be sterilized either with 70% ethanol or by autoclaving. Plasma treating sterilizes the devices so no further treatment is necessary. It is, however, important, when plasma-treating the devices, to add liquid to the devices within 10 minutes of the plasma treatment while the surfaces are still hydrophilic. Waiting longer than 10 minutes to add liquid to the device makes it difficult for the liquid to enter the device. The neuron devices are typically plasma-bound to cover glass and 0.5 mg/ml poly-L-lysine (PLL) in pH 8.5 borate buffer is immediately added to the device. After a minimum of 3 hours incubating with PLL, the devices are washed with dH2O water a minimum of 3 times with at least 15 minutes between each wash. Next, the water is removed and fresh media is added to the device. At this point the device is ready for use. It is important to remember at this point to never remove all the media from the device. Always leave media in the main channel.

In this video we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to farbricate a microfluidic device for culturing neurons.

In this video, we demonstrate the technique of soft lithography with polydimethyl siloxane (PDMS) which we use to fabricate a microfluidic device for ...

Counting Human Neural Stem Cells

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously dissected and prepared. The E18 cortex is, upon dissection, immediately dissociated into individual neurons. It is possible to store E18 cortex in Hibernate E buffer containing B27 at 4&deg;C for up to a week before the dissociation is performed. However, there will be a drop in cell viability. Typically we obtain our E18 Cortex fresh. It is transported to the lab in ice cold Calcium free Magnesium free dissection buffer (CMFM). Upon arrival, trypsin is added to the cortex to a final concentration of 0.125%. The cortex is then incubated at 37&deg;C for 8 minutes. DMEM containing 10% FBS is added to the cortex to stop the reaction. The cortex is then centrifuged at 2500 rpm for 2 minutes. The supernatant is removed and 2 ml of Neural Basal Media (NBM) containing 2% B27 (vol/vol) and 0.25% Glutamax (vol/vol) is added to the cortex which is then re-suspended by pipetting up and down. Next, the cortex is triturated with previously fire polished glass pipettes, each with a successive smaller opening. After triturating, the cortex is once again centrifuged at 2500 rpm for 2 minutes. The supernatant is then removed and the cortex pellet re-suspended with 2 ml of NBM containing B27 and Glutamax. The cell suspension is then passed through a 40 um nylon cell strainer. Next the cells are counted. The neurons are now ready for loading into the neuron microfluidic device.

In this video we demonstrate the preparation of E18 Cortical Rat Neurons.

In this video, we demonstrate the preparation of E18 cortical rat neurons. E18 cortical rat neurons are obtained from E18 fetal rat cortex previously ...

Cell Capture Using a Microfluidic Device

Non-plasma Bonding of PDMS for Inexpensive Fabrication of Microfluidic Devices

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond devices to the Corning No. 1 cover glass.  This could be due, perhaps, to a plasma cleaner not being available.  In other instances, it may be desirable to remove the device from the glass after the culturing of neurons for certain types of microscopy or for immunostaining, though it is not necessary to remove the device for immunostaining since the neurons can be stained in the device.  Some researchers, however, still prefer to remove the device.  In this case, reversible bonding of the device to the cover glass makes that possible.  There are some disadvantages to non-plasma bonding of the devices in that not as tight of a seal is formed.  In some cases axons may grow under the grooves rather than through them.  Also, because the glass and PDMS are hydrophobic, liquids do not readily enter the device making it necessary at times to force media and other reagents into the device.  Liquids will enter the device via capillary action, but it takes significantly longer as compared to devices that have been plasma bonded.  The plasma cleaner creates temporary hydrophilic charges on the glass and device that facilitate the flow of liquids through the device after bonding within seconds.  For non-plasma bound devices, liquid flow through the devices takes several minutes.  It is also important to note that the devices to be used with non-plasma bonding need to be sterilized first, whereas plasma treated devices do not need to be sterilized prior to use because the plasma cleaner will sterilize them.

In this video we demonstrate how to use the neuron microfluidic device without plasma bonding.

In this video, we demonstrate how to use the neuron microfluidic device without plasma bonding.  In some cases it may be desirable to reversibly bond ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.