This paper is based on work supported by the National Health Research Institutes (NHRI) in Taiwan under the Innovative Research Grant (IRG) (EX105-10523EI), the Taiwan Ministry of Science and Technology (MOST 103-2221-E-001-001-MY2, 104-2221-E-001-015-MY3, 105-2221-E-001-002-MY2), the Academia Sinica Thematic Project (AS-105-TP-A06), and the Research Program in Nanoscience and Nanotechnology. The authors would like to thank Ms. Rachel A. Lucas for proofreading the manuscript.

1. Fabrication of the Microfluidic Device
NOTE: The entire microfluidic device is fabricated using the soft lithography replica molding process13.
<ol>
	<li>Fabrication of molds for the PDMS layers in the microfluidic device
	<ol>
		<li>Design the microfluidic channel patterns using commercially available illustration or drawing software. Submit the file to a company for high-resolution transparency photomask printing14.</li>
		<li>Clean silicon wafers with acetone (&#8805;99.5%), isopropyl alcohol (IPA; &#8805;99.9%), and buffered oxide etch (BOE; 6:1 NH4F.HF). Rinse with deionized (DI) water and dehydrate the wafer with a nitrogen gun.</li>
		<li>Spin coat approximate 20 g of negative tone photoresist, SU-8 2050, onto the wafers at 500 rpm for 15 sec and then 2,000 rpm for 30 sec. 
		&#8203;NOTE: The spin coating condition should yield SU-8 2050 photoresist layers with a thickness of approximately 75 &#181;m on the wafers after optical lithography.</li>
		<li>Soft bake the wafers on a 65 &#176;C hotplate for 3 min and then at 95 &#176;C for 9 min. After the soft bake, expose the wafers using a mask aligner with the designed transparency masks under UV light; the total exposure energy should be about 300 mJ/cm2. Post-exposure bake (PEB) the wafers at 65 &#176;C for 2 min and then at 95 &#176;C for 7 min.</li>
		<li>After the PEB, immerse the wafers in SU-8 developer with strong agitation or in an ultrasonic bath (37 kHz and 180 W effective ultrasonic power) for 7 min. Rinse the wafer with acetone and IPA to remove the residual SU-8.</li>
		<li>Place the wafer and 100 &#181;l of silane (1H,1H,2H,2H-perfluorooctyltrichlorosilane) into a 6 cm diameter Petri dish in a desiccator connected with a diaphragm vacuum pump for wafer surface silanization in order to prevent undesired bonding. Turn on the pump for 15 min, turn it off, and seal the desiccator with a vacuum for 30 min.</li>
		<li>Take the silanized wafers out of the desiccator and tape them to 15 cm diameter Petri dishes for the following soft lithography process:</li>
	</ol>
	</li>
	<li>Fabrication and assembly of PDMS layers
	<ol>
		<li>Preparation of the PDMS pre-polymer
		<ol>
			<li>Mix PDMS monomer (base) and the curing agent at a 10:1 ratio (v/v). Degas the mixture in the desiccator setup for 60 min.</li>
		</ol>
		</li>
		<li>Fabrication of the PC-embedded top layer
		<ol>
			<li>Transfer 2 g of the PDMS pre-polymer onto the mold with the top-layer fluidic channel patterns to make a thin layer of PDMS. Place the mold in the desiccator setup to degas the PDMS for 60 min.</li>
			<li>Place the mold overnight in a 60 &#176;C oven for PDMS curing. Make sure that the mold is on a horizontal plane.</li>
			<li>Cool down the mold to room temperature. Pour an additional 13 g of PDMS pre-polymer onto the mold and degas the PDMS in the desiccator setup for 60 min.</li>
			<li>Slowly embed a 1 mm thick PC film into the fresh PDMS layer as a gas diffusion barrier; expel any bubbles if needed.</li>
			<li>Put the mold overnight in the 60 &#176;C oven, and make sure that it is on a horizontal plane.</li>
			<li>Cool down the cured PDMS to room temperature. Cut the device with a scalpel to an area of approximately 5.5 x 5 cm2, which can cover all channel patterns, and peel off the PDMS slab from the mold.</li>
			<li>Punch holes for inlets and outlets using a 2 mm-diameter biopsy punch. Store the fabricated top PDMS-PC layer away from ambient dust for later assembly.</li>
		</ol>
		</li>
		<li>Fabrication of the bottom PDMS layer
		<ol>
			<li>Pour 11 g of the PDMS pre-polymer onto the mold for the bottom layer. Place the mold in the desiccator to degas the PDMS for 60 min. Keep the mold overnight in a 60 &#176;C oven to cure the PDMS. Make sure that it is lying on a horizontal plane.</li>
			<li>Cool down the PDMS to room temperature. Cut the device to an area of approximately 5.5 x 5 cm2, which can cover all channel patterns, and peel it off of the mold. Store the fabricated bottom PDMS layer away from ambient dust for later assembly.</li>
		</ol>
		</li>
		<li>Fabrication of the PDMS membrane
		<ol>
			<li>Spin coat approximate 4 g of PDMS pre-polymer onto a silanized blank wafer at 100 rpm for 90 sec and then 3,000 rpm for 4 sec. Bake the spin-coated wafer in a 60 &#176;C oven overnight.</li>
		</ol>
		</li>
		<li>Assembly of the device
		<ol>
			<li>Place the fabricated PDMS-PC top layer and the wafer with the spin-coated PDMS membrane in an O2 plasma surface treatment machine with the bonding surfaces facing up. Treat the PDMS surfaces with 90 W of O2 plasma for 40 sec.</li>
			<li>Bond the top layer onto the PDMS membrane right after the O2 plasma surface treatment. Place a weight (about 600 g) on top of the bonded layers and put them in a 60 &#176;C oven overnight to promote bonding.</li>
			<li>Cool down the bonded layers to room temperature and cut the membrane bonded to the top layer with a scalpel to an area of approximately 5.5 x 5 cm2, which can cover all channel patterns. Peel off the bonded structure from the silicon wafer and punch holes at the inlets and outlet of the chemical gradient channel using a 2 mm-diameter biopsy punch.</li>
			<li>Place the membrane-bonded top layer and the fabricated PDMS bottom layer in the O2 plasma surface treatment machine, with the bonding surfaces facing up, to activate the PDMS surfaces using the plasma at 90 W for 40 sec.</li>
			<li>Attach the top and bottom layers together for bonding immediately after the surface treatment. Place a weight (about 600 g) on top of the entire bonded device and put it in a 60 &#176;C oven overnight.</li>
			<li>Take the entire fabricated device out of the oven and cool it down to room temperature.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Microfluidic Cell Migration Assay
NOTE: In this paper, we use a commonly used cell line, adenocarcinomic human alveolar basal epithelial cell (A549), and a chemokine, stromal cell derived factor (SDF-1&#945;), as examples. For researchers working on other cells and chemokines, please adjust the experimental processes accordingly.
<ol>
	<li>Day 0: Cell preparation
	<ol>
		<li>Culture the stock of A549 cells in complete growth medium containing DMEM F12 L-glutamine substitute, 10% (v/v) fetal bovine serum (FBS), and 1% (v/v) antimicrob-antimycotic solution. Sub-culture by dissociation with 0.25% trypsin-EDTA.</li>
		<li>Prepare cell suspensions for the experiments by centrifuging dissociated cells at 140 x g for 3 min at room temperature. For the microfluidic device experiments, count the cells using a hemocytometer and seed at least 1 x 106 cells in a T75 flask with 10 ml of complete growth medium. 
		&#8203;NOTE: All cell culture processes are performed in an incubator with a 37 &#176;C and 5% CO2 atmosphere.</li>
	</ol>
	</li>
	<li>Day 1: Device preparation
	<ol>
		<li>Place the device into the O2 plasma machine and treat it with O2 plasma at 90 W for 40 sec to render the microfluidic channel surfaces hydrophilic.</li>
		<li>Immediately after the surface treatment, install two 14 G blunt needles by directly inserting the needles into the 2 mm diameter holes punched into the PDMS layer at the chemical gradient channel inlets. Make sure that the needles do not block the microfluidic channels. Draw 0.8 ml of ddH2O using a 1 ml syringe with a blunt 14 G needle. Inject the ddH2O into the chemical gradient channel from the outlet until the water flows out from both needles at the inlets.</li>
		<li>Prepare 1 ml of 50 &#181;g/ml fibronectin solution by diluting fibronectin stock with Dulbecco&#39;s Phosphate-Buffered Saline (DPBS).</li>
		<li>Infuse the fibronectin solution from the outlet of the chemical gradient channel using a 1 ml syringe with a blunt 14 G needle until the solution flows out from both needles at the inlets.</li>
		<li>Incubate the entire device overnight in a conventional cell culture incubator.</li>
	</ol>
	</li>
	<li>Day 2: Cell seeding and microscopy imaging
	<ol>
		<li>Cell seeding
		<ol>
			<li>Aspirate the medium from the cells that were cultured since day 0 and wash the cells with 5 ml of DPBS 2 times. Aspirate the DPBS, add 2 ml of trypsin-EDTA, and incubate the cells in the incubator for 5 min to detach the cells from the flask surface.</li>
			<li>Wait until the cells are detached and suspended in the solution and add 8 ml of serum-free medium (DMEM F12 + 1% (v/v) antimicrob-antimycotic solution) to the flask. Transfer all of the liquid into a 15 ml conical tube and centrifuge it at 140 x g for 5 min under room temperature.</li>
			<li>Aspirate the supernatant after centrifuging and add an appropriate amount of the serum-free medium to make the final cell density 1 x 106 cells/ml. Take out the microfluidic device prepared on Day 1. Count the cells using a hemocytometer or another automatic cell counting instrument.</li>
			<li>Inject serum-free medium from the outlet of the chemical gradient channel using a 1 ml syringe with a blunt 14 G needle until the medium flows out from both needles at the inlets. Inject 200 &#181;l of the cell suspension from the outlet of the chemical gradient channel.</li>
			<li>Observe the cell culture chamber in the device under a microscope to confirm that the cells have been introduced to the microfluidic channel. Place the device into a humid container (e.g., a plastic box with ddH2O inside) and keep it in a cell culture incubator for 5 hr to promote the adhesion of the cells onto the device surface.</li>
		</ol>
		</li>
		<li>Preparation of reagents for chemical gradient generation
		<ol>
			<li>Prepare the chemical (SDF-1&#945;) at the desired concentration (100 ng/ml) in serum-free medium. Draw the serum-free medium with and without the chemical into two separate 3 ml syringes connected to high-purity tubing. Set up the syringes on a syringe pump with a flow rate of 1 &#181;l/min for later use.</li>
		</ol>
		</li>
		<li>Preparation of the reagents for oxygen gradient generation
		<ol>
			<li>Make 15 ml of 1 M NaOH solution and 15 ml of 200 mg/ml pyrogallol solution. Draw the NaOH and pyrogallol solutions into two separate 15 ml syringes connected to high-purity PTFE tubing and high-purity tubing, respectively. Set up the syringes on a syringe pump with flow rate of 5 &#181;l/min for later use.</li>
		</ol>
		</li>
		<li>Microfluidic device setup
		<ol>
			<li>After the 5 hr incubation, take the entire device out and place it on a 15 cm diameter Petri dish. Fix the device in the Petri dish using adhesive putty. Transfer the Petri dish to a live cell imaging microscope in an incubator.</li>
			<li>Connect the tubing from the syringes for the chemical gradient generation to the inlets of the chemical gradient channel. Connect the outlet to tubing leading to a waste reservoir. Turn on the syringe pump with the syringes for chemical gradient generation.</li>
			<li>Connect the high-purity tubing from the syringes containing NaOH and pyrogallol to the inlets of the oxygen gradient channel. Connect tubing to the outlet to collect the waste. Turn on the syringe pump with the syringes for oxygen gradient generation.</li>
			<li>Add about 15 ml of ddH2O to the Petri dish to keep the device moisturized. Set up the live cell imaging microscope for time-lapsed image capture, and take an image every 15 min.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Day 3: Data collection and analysis
	<ol>
		<li>Transfer the captured image files to a computer. Analyze the images using an open source image analysis software, ImageJ, with an open source plugin for Manual Tracking (https://imagej.nih.gov/ij/plugins/track/track.html) and a free Chemotaxis Tool software (http://ibidi.com/xtproducts/en/Software-and-Image-Analysis/Manual-Image-Analysis/Chemotaxis-and-Migration-Tool)15,16.</li>
	</ol>
	</li>
</ol>
3. Characterization of the Gradients
NOTE: The chemical and oxygen gradients can be characterized before or after the cell experiments.
<ol>
	<li>Numerical simulation of the chemical gradient
	<ol>
		<li>Estimate the laminar flow nature of microfluidics using computational fluidic dynamics (CFD) simulation.</li>
	</ol>
	</li>
	<li>Experimental characterization of the oxygen gradient
	<ol>
		<li>Prepare an oxygen-sensitive fluorescent dye, tris(2,2&#8242;-bipyridyl) ruthenium(III) chloride hexahydrate, solution in water at a concentration of 5 mg/ml.</li>
		<li>Draw the oxygen-sensitive dye solution into two separate 3 ml syringes connected to high-purity tubing. Set up the syringes on a syringe pump with flow rate of 1 &#181;l/min (identical to the flow rates used for the chemical gradient generation).</li>
		<li>Connect the tubing from the syringes to the inlets of the chemical gradient channel. Connect the outlet to tubing leading to a waste reservoir. Turn on the syringe pump with the syringes for chemical gradient generation.</li>
		<li>Measure the fluorescence intensity using an inverted fluorescence microscope with the excitation light passing through a 470 &#177; 20 nm optical filter. Collect the emission light through a 515 nm long-pass emission filter using a cool CCD camera.</li>
		<li>Connect the high-purity tubing from the syringes containing NaOH and pyrogallol to the inlets of the oxygen gradient channel. Connect tubing to the outlet to collect the waste. Turn on the syringe pump with the syringes for oxygen gradient generation.</li>
		<li>Collect the fluorescence images of the oxygen-sensitive dye flowing in the cell culture chamber.</li>
		<li>Stop the flow for oxygen gradient generation and disconnect all the tubing to the oxygen generation channel. Connect high-purity tubing from the gas cylinders to the outlet of the oxygen gradient channel.</li>
		<li>Collect the fluorescence images of the oxygen-sensitive dye flowing in the cell culture chamber when flowing air, pure nitrogen, and pure oxygen into the tubing connected to the oxygen gradient channel.</li>
		<li>Estimate the oxygen gradients by analyzing the fluorescence images using the Stern-Volmer equation according to References 10 and 11.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Phillips, J. E., Burns, K. L., Le Doux, J. M., Guldberg, R. E., Garc&#237;a, 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=Engineering+graded+tissue+interfaces.">Engineering graded tissue interfaces.</a> Proc. Natl. Acad. Sci. USA. 105 (34), 12170-12175 (2008).</li><li>Wang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+signaling+mechanisms+underlying+polarity+and+chemotaxis.">The signaling mechanisms underlying polarity and chemotaxis.</a> Cold Spring Harb. Perspect Biol. 1 (4), 1-16 (2009).</li><li>Oh, S. H., Ward, C. L., Atala, A., Yoo, J. J., Harrison, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+generating+scaffolds+for+enhancing+engineering+tissue+survival.">Oxygen generating scaffolds for enhancing engineering tissue survival.</a> Biomaterials. 30 (5), 757-762 (2009).</li><li>Decaris, M. L., Lee, C. I., Yoder, M. C., Tarantal, A. F., Leach, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+oxygen+microenvironment+on+the+proangiogenic+potential+of+human+endothelial+colony+forming+cells.">Influence of the oxygen microenvironment on the proangiogenic potential of human endothelial colony forming cells.</a> Angiogenesis. 12, 303-311 (2009).</li><li>Chung, B. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cell+growth+and+differentiation+in+a+gradient-generating+microfluidic+device.">Human neural stem cell growth and differentiation in a gradient-generating microfluidic device.</a> Lab Chip. 5 (4), 401-406 (2005).</li><li>Harris, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+-+a+key+regulatory+factor+in+tumor+growth.">Hypoxia - a key regulatory factor in tumor growth.</a> Nat. Rev. Cancer. 2 (1), 38-47 (2002).</li><li>Allen, J. W., Bhatia, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+steady-state+oxygen+gradients+in+vitro:+application+to+liver+zonation.">Formation of steady-state oxygen gradients in vitro: application to liver zonation.</a> Biotechnol. Bioeng. 82 (3), 253-262 (2003).</li><li>McCord, A. M., Jamal, M., Shankavaram, U. T., Lang, F. F., Camphausen, K., Tofilon, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologic+oxygen+concentration+enhances+the+stem-like+properties+of+CD133++human+glioblastoma+cells+in+vitro.">Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro.</a> Mol. Cancer Res. 7 (4), 489-497 (2009).</li><li>Lo, J. F., Sinkala, E., Eddington, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+gradients+for+open+well+cellular+cultures+via+microfluidic+substrates.">Oxygen gradients for open well cellular cultures via microfluidic substrates.</a> Lab Chip. 10 (18), 2394-2401 (2010).</li><li>Chang, C. 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=A+polydimethylsiloxane-polycarbonate+hybrid+microfluidic+device+capable+of+generating+perpendicular+chemical+and+oxygen+gradients+for+cell+culture+studies.">A polydimethylsiloxane-polycarbonate hybrid microfluidic device capable of generating perpendicular chemical and oxygen gradients for cell culture studies.</a> Lab Chip. 14 (19), 3762-3772 (2014).</li><li>Chen, Y. 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=Generation+of+oxygen+gradients+in+microfluidic+devices+for+cell+culture+using+spatially+confined+chemical+reactions.">Generation of oxygen gradients in microfluidic devices for cell culture using spatially confined chemical reactions.</a> Lab Chip. 11 (21), 3626-3633 (2011).</li><li>Peng, C. C., Liao, W. H., Chen, Y. H., Wu, C. Y., Tung, Y. 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+microfluidic+cell+culture+array+with+various+oxygen+tensions.">A microfluidic cell culture array with various oxygen tensions.</a> Lab Chip. 13 (16), 3239-3245 (2013).</li><li>Xia, Y., Whitesides, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography.">Soft Lithography.</a> Ann. Rev. Mate. Sci. 28, 153-184 (1998).</li><li>Friend, J., Yeo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+microfluidic+devices+using+polydimethylsiloxane.">Fabrication of microfluidic devices using polydimethylsiloxane.</a> Biomicrofluidics. 4 (2), 026502 (2010).</li><li>Cordelières, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual Tracking. , (2005).</li><li>Asano, Y., Horn, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Instructions Chemotaxis and Migration Tool 2.0. , (2013).</li><li>Chen, Y. H., Peng, C. C., Cheng, Y. J., Wu, J. G., Tung, Y. C. <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+nitric+oxide+gradients+in+microfluidic+devices+for+cell+culture+using+spatially+controlled+chemical+reactions.">Generation of nitric oxide gradients in microfluidic devices for cell culture using spatially controlled chemical reactions.</a> Biomicrofluidics. 7 (6), 064104 (2013).</li></ol>

The authors declare that they have no competing financial interests.

The most critical steps to fabricate the PDMS microfluidic device with an embedded PC thin film are: (1) expelling all the bubbles when inserting the PC film into the PDMS pre-polymer while fabricating the PDMS-PC top layer and (2) making sure all the PDMS curing processes are performed on a well-leveled horizontal plane. For cell migration experiments, the most critical steps are: (1) eliminating the bubbles within the microfluidic device, tubing, and syringe pumps during the experiments; (2) ensuring that the microfluidic device is placed on a well-leveled horizontal plane during live cell imaging for the cell migration observation; and (3) keeping the device moisturized by adding ddH2O to the Petri dish during the experiments and making sure that the water is not dried out.
In order to successfully fabricate the PDMS-PC hybrid microfluidic device without delamination, it is critical to remove all bubbles during the PC film insertion. The PC film can be slowly inserted from an angle (about 15 to 30 degrees away from the PDMS pre-polymer surface) to prevent bubbles generation during the insertion of the PC film into the PDMS pre-polymer. If necessary, the entire PDMS pre-polymer with the embedded PC film can be placed in the desiccator connected to the vacuum pump for 10 min to expel the trapped bubbles. If the PC film floats up after the vacuum process, a pipette tip can be used to press the PC film down onto the cured PDMS layer. Repeat the vacuum and press processes if necessary.
For the cell experiments, a lack of air bubbles is critical for the microfluidic cell culture. Make sure that no air bubbles are introduced into the entire microfluidic setup (including syringe pumps, tubing, and the microfluidic device) when making connections. If air bubbles are created within the microfluidic setup due to the decrease of gas solubility under the elevated temperature of the experiments inside the incubator, all the experimental components (including the syringes and tubing) and reagents (including the growth medium, pyrogallol, and NaOH) can be placed into the incubator beforehand (at least 20 min prior to usage) to minimize the temperature variation. Syringe pumps often generate heat from the operation of the motors within the pumps. It is usually acceptable to operate syringe pumps inside incubators; however, do check the incubator temperature during the experiments. If the temperature elevates during the experiments, additional cooling procedures need to be carried out. Several feasible cooling methods can be employed, such as placing a box of ice into the incubator, reducing the number of syringe pumps placed inside the incubator, or using an incubator with a force cooling system.
The PDMS-PC microfluidic cell culture device developed in this paper is capable of reliably generating perpendicular chemical and oxygen gradients for cell migration studies. The limitation of the developed device is that the generated oxygen gradient profiles depend on the balance between oxygen flux, driven by chemical reaction scavenging, and oxygen diffusion from the ambient atmosphere, through the device, and into the medium. As a result, the oxygen gradient profiles cannot be arbitrarily controlled inside the device. Compared to existing microfluidic cell culture platforms, the device developed in this paper is the first one capable of performing cell culture studies under combinations of chemical and oxygen gradients. The entire device can be fabricated using the conventional soft lithography replica molding process, without tedious alignment and expensive instrumentation. The gradients can be numerically simulated and experimentally characterized to provide more physiological microenvironment-like conditions for in vitro cell studies. By using a spatially confined chemical reaction method with a PC film as a gas diffusion barrier, the oxygen gradient can be generated without using pressurized gas cylinders and sophisticated gas flow control units. In addition, the setup requires only small amounts of chemicals (less than 10 ml per day) to maintain the oxygen gradients. Since the oxygen tension control is confined locally around the microfluidic channel, and does not disturb the ambient oxygen concentration, the entire setup can be placed inside a conventional cell culture incubator without additional temperature, humidity, and CO2 control instrumentation. As a result, the developed device has great potential to be practically used in biological labs.
Due to technical limitations, cellular behaviors under oxygen tensions have seldom been studied in the existing literature. With the help of the device developed in this paper, cell culture under oxygen gradients can be performed in a facile manner that greatly promotes cell studies under oxygen gradients. Furthermore, a similar operation principle can be applied to generate other physiologically relevant gaseous gradients, such as carbon dioxide and nitric oxide, for in vitro cell culture studies17. These capabilities demonstrate that the PDMS-PC microfluidic device shows great potential for various cell culture applications, including drug testing and cell proliferation and migration assays, to advance in vitro cell culture studies.

The spatial distribution of soluble factors and oxygen tension can regulate a number of important cellular functions in vivo1,2,3,4. In order to better investigate their effect on cells, an in vitro cell culture platform capable of stably generating chemical and oxygen gradients is highly desired. Various soluble factors play key roles in biological activities and affect cellular behavior. Recently, due to the advancement of microfluidic techniques, a number of microfluidic devices capable of stably generating chemical gradients have been developed to study cell migration5. Furthermore, several studies have also revealed the necessity of oxygen tension for in vitro cell cultures6,7,8. However, the control of oxygen tension for cell culture mainly relies on direct chemical addition for oxygen scavenging or cell incubators with pressurized gas cylinders. Direct chemical addition alters the cell culture medium and affects the cellular responses. Oxygen control incubators require special incubator design, precise gas flow control, and a large volume of nitrogen gas in order to achieve hypoxia conditions. Moreover, it is infeasible to control the spatial distribution of oxygen using this setup, which retards the cellular behavior study under various oxygen tensions and gradients. To overcome these limitations, a number of microfluidic devices have been developed to generate oxygen gradients for cell culture applications9. However, most of them are operated using pressurized gases, which may cause evaporation and bubble generation problems. Therefore, they often require sophisticated instrumentation and may not be reliable for long-term cell culture studies.
To overcome the challenges and to further study the interactions between chemical and oxygen gradients for cell migration, we developed a microfluidic cell culture device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film10. The device is composed of two microfluidic channel layers separated by a PDMS membrane. The top layer is a PDMS-PC layer for oxygen gradient generation; the bottom layer is made of PDMS for chemical gradient generation and cell culture. The device can simultaneously generate perpendicular chemical and oxygen gradients without using gas cylinders and sophisticated flow control systems. In the device, PDMS provides great optical transparency, gas permeability, and biological compatibility for cell culture and imaging. The embedded PC film serves as a gas diffusion barrier for efficient oxygen tension control. In the microfluidic channel, we used a series of serpentine-shaped channels to generate chemical gradients. The design has been broadly exploited to generate chemical gradients in microfluidic devices for various applications due to its reliability and easy experimental setup. Furthermore, the chemical gradient profiles can be designed by varying the channel geometries beforehand with numerical simulation. For oxygen gradient generation, we took advantage of the spatially confined chemical reaction method that was previously developed in our lab10,11,12. The oxygen can be scavenged from the designated areas without nitrogen purging. For practical usage in biological labs, the entire experimental setup is compatible with conventional cell culture incubators. By integrating these methods, we can simultaneously generate stable chemical and oxygen gradients without bulk gas cylinders and sophisticated instrumentation in order to study cell migration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 ml Syringe</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>302104</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tube</td>
			<td>Smartgene</td>
			<td>6011-000</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml Syringe</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>302151</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Centrifuge Tube</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>Falcon 352096</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm Petri-Dish</td>
			<td>Dogger Science</td>
			<td>DP-43151</td>
			<td></td>
		</tr>
		<tr>
			<td>1H,1H,2H,2H-Perfluorooctyltrichlorosilane</td>
			<td>Alfa Aesar, Ward Hill, MA</td>
			<td>78560-45-9</td>
			<td></td>
		</tr>
		<tr>
			<td>3 ml Syringe</td>
			<td>Becton-Dickinson, Franklin Lakes, NJ</td>
			<td>302118</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;&#39; Silicon Dummy Wafer</td>
			<td>Wollemi Technical, Taoyuan, Taiwan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>ECHO Chemical, Miaoli, Taiwan</td>
			<td>AH3102-000000-72EC</td>
			<td></td>
		</tr>
		<tr>
			<td>AG Double Expose Mask Aligner</td>
			<td>M&#38;R Nano Technology, Taoyuan, Taiwan</td>
			<td>AG500-4D-D-V-S-H</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimyotic solution</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>GIBCO 15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy punch</td>
			<td>Miltex, Plainsboro, NJ</td>
			<td>33-31</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt needle</td>
			<td>JensenGlobal, Santa Barbara, CA</td>
			<td>Gauge 14</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-Line Hemocytometer</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>Z359629</td>
			<td>for cell counting</td>
		</tr>
		<tr>
			<td>Buffered Oxide Etch</td>
			<td>ECHO Chemical, Miaoli, Taiwan</td>
			<td>PH3101-000000-72EC</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Incubator</td>
			<td>Caron, Marietta, OH</td>
			<td>6016-1</td>
			<td></td>
		</tr>
		<tr>
			<td>COMSOL Multiphysics</td>
			<td>COMSOL, Burlington, MA</td>
			<td>Ver. 4.3b</td>
			<td>for numerical simulation of chemical gradients in the device</td>
		</tr>
		<tr>
			<td>D-PBS</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>GIBCO 14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Desicattor</td>
			<td>A-VAC Industries, Anaheim, CA</td>
			<td>35.10001.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/ F12+GlutaMax-1</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>GIBCO 10565-018</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>GIBCO 10082</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin from Human Plasma</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>F2006</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Fluorescence Microscope</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td>DMIL LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl Alcohol (IPA)</td>
			<td>ECHO Chemical, Miaoli, Taiwan</td>
			<td>CMOS112-00000-72EC</td>
			<td></td>
		</tr>
		<tr>
			<td>JuLi Smart Fluorescence Cell Imager</td>
			<td>NanoEnTek, Seoul, Korea</td>
			<td>DBJ01B</td>
			<td></td>
		</tr>
		<tr>
			<td>Mechanical Convention Oven</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>Lindberg Blue M MO1450C</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Showa Chemical Industry, Tokyo, Japan</td>
			<td>1943-0150</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasma tretment system</td>
			<td>Nordson MARCH, Concord CA</td>
			<td>PX-250</td>
			<td>for oxygen plasma surface treatment</td>
		</tr>
		<tr>
			<td>Polycarbonate (PC) film</td>
			<td>Quantum Beam Technologies, Tainan Taiwan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimehtylsiloxane (PDMS)</td>
			<td>Dow Corning, Midland, MI</td>
			<td>SYLGARD 184</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyrogallol</td>
			<td>Alfa Aesar, Ward Hill, MA</td>
			<td>A13405</td>
			<td></td>
		</tr>
		<tr>
			<td>Removable Adhesive Putty</td>
			<td>3M</td>
			<td>860</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorvall Legend Mach 1.6R Tabletop Centrifuge</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spin Coater</td>
			<td>ELS Technology, Hsinchu, Taiwan</td>
			<td>ELS 306MA</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 2050</td>
			<td>MicroChem, Westborough, MA</td>
			<td>SU-8 2050</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 Developer</td>
			<td>MicroChem, Westborough, MA</td>
			<td>Y020100</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Feather, Osaka, Japan</td>
			<td>5005093</td>
			<td>for PDMS cutting</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Chemyx, Houston, TX</td>
			<td>Fusion 400</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Cell Culture Flask</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>Nunc 156367</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Solution, 0.4%</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>ThermoFisher Scientific,Waltham, MA</td>
			<td>GIBCO 25200</td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon PTFE Tubing</td>
			<td>Saint-Gobain Performance Plastics, Akron, OH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tygon Tubing</td>
			<td>Saint-Gobain Performance Plastics, Akron, OH</td>
			<td>621</td>
			<td></td>
		</tr>
	</tbody>
</table>

polydimethylsiloxane-polycarbonate microfluidic devices for cell migration studies under perpendicular chemical and oxygen gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

Fabricated PDMS-PC hybrid microfluidic cell culture device. Fig. 1 shows a photo and an illustration of the microfluidic device. The bottom layer contains four levels of serpentine-shaped channels to generate solutions from reagents introduced from two separated inlets with six different mixing ratios. Theoretically, the six different mixing ratios are 1:0, 4:1, 3:2, 2:3, 1:4, and 0:1 (left:right) between the two solutions introduced from the inlets. The chemical gradients constructed by the six different mixing-ratio solutions can be generated in the cell culture chamber, located downstream. The top and bottom layers are separated by a PDMS membrane. In the top layer, the reagents for the oxygen scavenging chemical reaction are introduced into the microfluidic channel from two separate inlets. The reagents are mixed with each other for the reaction immediately before flowing on top of the cell culture chamber to scavenge the oxygen from the bottom channel, without direct chemical contact. The embedded PC film, with a smaller gas diffusion coefficient compared to PDMS, acts as a diffusion barrier that makes oxygen scavenging more efficient. Oxygen gradually diffuses back to the cell culture chamber through the PDMS in the downstream area to form an oxygen gradient along the flow direction. Since the oxygen scavenging chemical reaction is spatially confined, only local oxygen tensions are affected. As a result, the device can be utilized in a conventional cell incubator without altering its global oxygen tension. In the migration experiments, cells are seeded inside the cell culture chamber for observation. The growth medium and chemical reagents are introduced to the device using syringe pumps with precisely controlled flow rates.
Characterization of chemical and oxygen gradients generated inside the device. Due to the laminar flow nature of microfluidics, flow behaviors can be predicted using computational fluidic dynamics (CFD) simulation. In this paper, we constructed a 3D model and performed the simulation using a commercially available multiphysics modeling software. Fig. 2(a) shows a comparison between experimentally characterized fluorescein concentration profiles across the width of the cell culture chamber based on fluorescence intensity measurements and the numerical simulation results. The agreement between the experimental and simulation results suggests that the CFD model can well estimate the chemical gradients generated inside the device. Fig. 2(b) plots the simulated SDF-1&#945; gradient generated in the cell culture chamber. Fig. 3 shows the oxygen gradient characterization results by flowing the oxygen-sensitive fluorescence dye inside the cell culture chamber before the cell experiments. The result indicates that an oxygen gradient, ranging from about 1 to 16%, can be established using the aforementioned protocol.
Cell migration results. As a demonstration, we performed A549 cell migration studies under 4 combinations of chemokine (SDF-1&#945;) and oxygen gradients: (1) no chemokine and no oxygen gradients as a control, (2) with a chemokine gradient and without an oxygen gradient, (3) with an oxygen gradient and without a chemokine gradient, and (4) with both chemokine and oxygen gradients. Fig. 4 shows the photo of the entire experimental setup. The experiments were all performed in a conventional cell culture incubator with the entire setup (including the microfluidic devices, syringe pumps, and live cell imaging microscopes) placed inside it. The cell migration results are shown in Fig. 5. Fig. 5(a) shows the images collected during the experiments using the live cell imaging analyzer, and Fig. 5(b) and (c) plots the cell migration trajectories and average movements under the four combinations analyzed by the ImageJ software with the plugins. The results show that the average cell migration distance in the control approaches zero, which suggests random movement of the cells in the experiment. In contrast, with only the chemokine gradient, the average movement of the cells is towards the left, where the SDF-1&#945; concentration is higher. The results suggest the SDF-1&#945; chemotaxis behavior of A549 cells, which has been previously reported. In the experiment with only oxygen gradients, the average movement of the cells is upwards, where the oxygen tension is lower. More interestingly, in the experiment with perpendicular chemokine and oxygen gradients, the average movement of the cells is upwards and without any obvious movement in the horizontal direction (chemokine gradient direction).
<img alt="Figure 2" src="/files/ftp_upload/55292/55292fig1.jpg" /> 
Figure 1: Fabricated PDMS-PC microfluidic cell culture device. (a) The experimental photo of the fabricated device capable of reliably generating perpendicular chemical and oxygen gradients for cell migration studies. The chemical gradient channel is filled with blue and yellow food dyes to demonstrate the gradient generation inside the cell culture chamber. The oxygen gradient channel is filled with red food dye. The scale bar is 1 cm. (b) The schematic of the microfluidic device. The top layer is fabricated using PDMS with an embedded PC layer as a gas diffusion barrier for efficient oxygen gradient control inside the cell culture chamber. (c) The master molds for the fabrication of the top and bottom layers. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55292/55292fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55292/55292fig2.jpg" /> 
Figure 2: Chemical gradient inside the microfluidic cell culture device. (a) Numerically simulated and experimentally characterized fluorescein concentration gradient inside the cell culture chamber across the width of the cell culture chamber (Y direction). The similarity between the simulated and experimentally measured gradients indicates that the simulation can well predict the chemical gradient. The figure inset shows the three-dimensional (3D) model constructed for the simulation. (b) Numerical simulation result of the SDF-1&#945; chemokine gradient across the width of the cell culture chamber for cell migration studies. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55292/55292fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55292/55292fig3.jpg" /> 
Figure 3: Oxygen gradient inside the microfluidic cell culture device. Experimentally measured oxygen gradients within the cell culture chamber along the flow direction. The gradients were estimated using the oxygen-sensitive fluorescence dye and image analysis. The gradients, from left to right of the chamber, are characterized, and the results show consistent gradient profiles across the width of the chamber.
<img alt="Figure 4" src="/files/ftp_upload/55292/55292fig4.jpg" /> 
Figure 4: Photos of the experimental setup. The entire setup, including microfluidic devices, syringe pumps, and a live cell imaging microscope, is placed inside a conventional cell culture incubator for optimized cell culture conditions during the experiments. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55292/55292fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55292/55292fig5.jpg" /> 
Figure 5: Cell migration study results under perpendicular SDF-1&#945;&#8203; and oxygen gradients. (a) Images captured before and after the 12-h cell migration study. The cell migration paths can be analyzed from the captured time-lapsed images using the live cell imaging microscope. (b) The cell migration paths and the analyzed average migration movement from the captured images under 4 different gradient combinations: no gradient, only chemokine gradient, only oxygen gradient, and both chemokine and oxygen gradients. The images were captured every 15 min. The scale bar is 250 &#181;m. (c) Plots of the average cell migration distances in the perpendicular (oxygen gradient) and horizontal (chemokine gradient) directions under four different gradient combinations. The data are expressed as the mean &#177; SD, obtained from three independent experimental sets, and 10 cells were analyzed in each experiment. The statistical significantly different (unpaired Student&#39;s t-test, p &#60; 0.01) results are designated by different letters (a and b). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55292/55292fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients at JoVE.com

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...

polydimethylsiloxane-polycarbonate-microfluidic-devices-for-cell

Research

JoVE Journal

Bioengineering

14.1K Views.  Academia Sinica. The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

Video: Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

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 ...

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

Microfabrication of Nanoporous Gold Patterns for Cell-material Interaction Studies

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, biomedical device coatings, and drug delivery tools. Nanoporous gold (np-Au), produced by a nano-scale self-assembly process, is a relatively new material that exhibits large effective surface area, high electrical conductivity, and catalytic activity. These properties have made np-Au an attractive material to scientific community. Most studies on np-Au employ macro-scale specimens and focus on fundamental science of the material and its catalytic and sensor applications. The macro-scale specimens limit np-Au's potential in miniaturized systems, including biomedical devices. In order to address these issues, we initially describe two different methods to micropattern np-Au thin films on rigid substrates. The first method employs manually-produced stencil masks for creating millimeter-scale np-Au patterns, while the second method uses lift-off photolithography to pattern sub-millimeter-scale patterns. As the np-Au thin films are obtained by sputter-deposition process, they are compatible with conventional microfabrication techniques, thereby amenable to facile integration into microsystems. These systems include electrically-addressable biosensor platforms that benefit from high effective surface area, electrical conductivity, and gold-thiol-based surface bioconjugation. We describe cell culture, immunostaining, and image processing techniques to quantify np-Au's interaction with mammalian cells, which is an important performance parameter for some biosensors. We expect that the techniques illustrated here will assist the integration of np-Au in platforms at various length-scales and in numerous applications, including biosensors, energy storage systems, and catalysts.

We report on techniques to micropattern nanoporous gold thin films via stencil printing and photolithography, as well as methods to culture cells on the microfabricated patterns. In addition, we describe image analysis methods to characterize morphology of the material and the cultured cells using scanning electron and fluorescence microscopy techniques.

Nanostructured materials with feature sizes in tens of nanometers have enhanced the performance of several technologies, including fuel cells, biosensors, ...

Millifluidics for Chemical Synthesis and Time-resolved Mechanistic Studies

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the first, synthesis of ultra-small copper nanoclusters is described. The second example provides their utility for investigating time resolved kinetics of chemical reactions by analyzing gold nanoparticle formation using in situ X-ray absorption spectroscopy. The final example demonstrates continuous flow catalysis of reactions inside millifluidic channel coated with nanostructured catalyst.

Millifluidic devices are utilized for controlled synthesis of nanomaterials, time-resolved analysis of reaction mechanisms and continuous flow catalysis.

Procedures utilizing millifluidic devices for chemical synthesis and time-resolved mechanistic studies are described by taking three examples. In the ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Layer-by-layer Collagen Deposition in Microfluidic Devices for Microtissue Stabilization

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D culture of cells in collagen type I gels helps to stabilize cell morphology and function, which is necessary for creating microfluidic tissue models in microdevices. Translating traditional 3D culture techniques for tissue culture plates to microfluidic devices is often difficult because of the limited channel dimensions. In this method, we describe a technique for modifying native type I collagen to generate polycationic and polyanionic collagen solutions that can be used with layer-by-layer deposition to create ultrathin collagen assemblies on top of cells cultured in microfluidic devices. These thin collagen layers stabilize cell morphology and function, as shown using primary hepatocytes as an example cell, allowing for the long term culture of microtissues in microfluidic devices.

The creation of functional microtissues within microfluidic devices requires the stabilization of cell phenotypes by adapting traditional cell culture techniques to the limited spatial dimensions in microdevices. Modification of collagen allows the layer-by-layer deposition of ultrathin collagen assemblies that can stabilize primary cells, such as hepatocytes, as microfluidic tissue models.

Although microfluidics provides exquisite control of the cellular microenvironment, culturing cells within microfluidic devices can be challenging. 3D ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Designing Microfluidic Devices for Studying Cellular Responses Under Single or Coexisting Chemical/Electrical/Shear Stress Stimuli

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other physical or chemical stimuli. They have been commonly used for in vitro cell studies by providing in vivo like surroundings. Especially, how cells response to chemical gradients, electrical fields, and shear stresses has drawn many interests since these phenomena are important in understanding cellular properties and functions. These microfluidic chips can be made of glass substrates, silicon wafers, polydimethylsiloxane (PDMS) polymers, polymethylmethacrylate (PMMA) substrates, or polyethyleneterephthalate (PET) substrates. Out of these materials, PMMA substrates are cheap and can be easily processed using laser ablation and writing. Although a few microfluidic devices have been designed and fabricated for generating multiple, coexisting chemical and electrical stimuli, none of them was considered efficient enough in reducing experimental repeats, particular for screening purposes. In this report, we describe our design and fabrication of two PMMA-based microfluidic chips for investigating cellular responses, in the production of reactive oxygen species and the migration, under single or coexisting chemical/electrical/shear stress stimuli. The first chip generates five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 in the culture regions, together with a shear stress gradient produced inside each of these areas. The second chip generates the same relative concentrations, but with five different electric field strengths created within each culture area. These devices not only provide cells with a precise, controllable micro-environment but also greatly increase the experimental throughput.

Micro-fabricated devices integrated with fluidic components provide an in vitro platform for cell studies mimicking the in vivo micro-environment. We developed polymethylmethacrylate-based microfluidic chips for studying cellular responses under single or coexisting chemical/electrical/shear stress stimuli.

Microfluidic devices are capable of creating a precise and controllable cellular micro-environment of pH, temperature, salt concentration, and other ...