the spatial distribution of soluble factors and oxygen tension can regulate a number of important cellular functions in vivo1234 . in order to better investigate their effect on cells , an in vitro cell culture platform capable of stably generating chemical and oxygen gradients 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 cultures678 . 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 . 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 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 lab101112 . 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 . note : the entire microfluidic device is fabricated using the soft lithography replica molding process13 . fabrication of molds for the pdms layers in the microfluidic device 
 design the microfluidic channel patterns using commercially available illustration or drawing software . submit the file to a company for high - resolution transparency photomask printing14.clean silicon wafers with acetone ( 99.5% ) , isopropyl alcohol ( ipa ; 99.9% ) , and buffered oxide etch ( boe ; 6:1 nh4f.hf ) . rinse with deionized ( di ) water and dehydrate the wafer with a nitrogen gun.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 . note : the spin coating condition should yield su-8 2050 photoresist layers with a thickness of approximately 75 m on the wafers after optical lithography.soft bake the wafers on a 65 c hotplate for 3 min and then at 95 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 / cm . post - exposure bake ( peb ) the wafers at 65 c for 2 min and then at 95 c for 7 min.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.place the wafer and 100 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.take the silanized wafers out of the desiccator and tape them to 15 cm diameter petri dishes for the following soft lithography process : 
  design the microfluidic channel patterns using commercially available illustration or drawing software . clean silicon wafers with acetone ( 99.5% ) , isopropyl alcohol ( ipa ; 99.9% ) , and buffered oxide etch ( boe ; 6:1 nh4f.hf ) . rinse with deionized ( di ) water and dehydrate the wafer with a nitrogen gun . 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 . note : the spin coating condition should yield su-8 2050 photoresist layers with a thickness of approximately 75 m on the wafers after optical lithography . soft bake the wafers on a 65 c hotplate for 3 min and then at 95 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 / cm . post - exposure bake ( peb ) the wafers at 65 c for 2 min and then at 95 c for 7 min . 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 . place the wafer and 100 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 . take the silanized wafers out of the desiccator and tape them to 15 cm diameter petri dishes for the following soft lithography process : 
 fabrication and assembly of pdms layers 
 preparation of the pdms pre - polymer mix pdms monomer ( base ) and the curing agent at a 10:1 ratio ( v / v ) . fabrication of the pc - embedded top layer 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.place the mold overnight in a 60 c oven for pdms curing . make sure that the mold is on a horizontal plane.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.slowly embed a 1 mm thick pc film into the fresh pdms layer as a gas diffusion barrier ; expel any bubbles if needed.put the mold overnight in the 60 c oven , and make sure that it is on a horizontal plane.cool down the cured pdms to room temperature . cut the device with a scalpel to an area of approximately 5.5 x 5 cm , which can cover all channel patterns , and peel off the pdms slab from the mold.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 . fabrication of the bottom pdms layer pour 11 g of the pdms pre - polymer onto the mold for the bottom layer . make sure that it is lying on a horizontal plane.cool down the pdms to room temperature . cut the device to an area of approximately 5.5 x 5 cm , which can cover all channel patterns , and peel it off of the mold . fabrication of the pdms membrane 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 . assembly of the device 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.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 c oven overnight to promote bonding.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 cm , 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.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.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 c oven overnight.take the entire fabricated device out of the oven and cool it down to room temperature . preparation of the pdms pre - polymer mix pdms monomer ( base ) and the curing agent at a 10:1 ratio ( v / v ) . mix pdms monomer ( base ) and the curing agent at a 10:1 ratio ( v / v ) . fabrication of the pc - embedded top layer 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.place the mold overnight in a 60 c oven for pdms curing . make sure that the mold is on a horizontal plane.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.slowly embed a 1 mm thick pc film into the fresh pdms layer as a gas diffusion barrier ; expel any bubbles if needed.put the mold overnight in the 60 c oven , and make sure that it is on a horizontal plane.cool down the cured pdms to room temperature . cut the device with a scalpel to an area of approximately 5.5 x 5 cm , which can cover all channel patterns , and peel off the pdms slab from the mold.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 . 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 . place the mold overnight in a 60 c oven for pdms curing . pour an additional 13 g of pdms pre - polymer onto the mold and degas the pdms in the desiccator setup for 60 min . slowly embed a 1 mm thick pc film into the fresh pdms layer as a gas diffusion barrier ; expel any bubbles if needed . put the mold overnight in the 60 c oven , and make sure that it is on a horizontal plane . cut the device with a scalpel to an area of approximately 5.5 x 5 cm , which can cover all channel patterns , and peel off the pdms slab from the mold . 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 . fabrication of the bottom pdms layer pour 11 g of the pdms pre - polymer onto the mold for the bottom layer . make sure that it is lying on a horizontal plane.cool down the pdms to room temperature . cut the device to an area of approximately 5.5 x 5 cm , 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 . 
  pour 11 g of the pdms pre - polymer onto the mold for the bottom layer . cut the device to an area of approximately 5.5 x 5 cm , 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 . fabrication of the pdms membrane 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 . 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 . assembly of the device 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.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 c oven overnight to promote bonding.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 cm , 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.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.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 c oven overnight.take the entire fabricated device out of the oven and cool it down to room temperature . 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 . 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 c oven overnight to promote bonding . 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 cm , 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 . 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 . place a weight ( about 600 g ) on top of the entire bonded device and put it in a 60 c oven overnight . take the entire fabricated device out of the oven and cool it down to room temperature . 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 ) , as examples . for researchers working on other cells and chemokines , day 0 : cell preparation 
 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.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 10 cells in a t75 flask with 10 ml of complete growth medium . note : all cell culture processes are performed in an incubator with a 37 c and 5% co2 atmosphere . 
  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 . 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 10 cells in a t75 flask with 10 ml of complete growth medium . note : all cell culture processes are performed in an incubator with a 37 c and 5% co2 atmosphere . day 1 : device preparation 
 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.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 . 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.prepare 1 ml of 50 g / ml fibronectin solution by diluting fibronectin stock with dulbecco 's phosphate - buffered saline ( dpbs).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.incubate the entire device overnight in a conventional cell culture incubator . 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 . 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 . 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 . prepare 1 ml of 50 g / ml fibronectin solution by diluting fibronectin stock with dulbecco 's phosphate - buffered saline ( dpbs ) . 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 . day 2 : cell seeding and microscopy imaging 
 cell seeding 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.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.aspirate the supernatant after centrifuging and add an appropriate amount of the serum - free medium to make the final cell density 1 x 10 cells / ml . take out the microfluidic device prepared on day 1 . count the cells using a hemocytometer or another automatic cell counting instrument.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 l of the cell suspension from the outlet of the chemical gradient channel.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 . preparation of reagents for chemical gradient generation prepare the chemical ( sdf-1 ) 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 l / min for later use . preparation of the reagents for oxygen gradient generation 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 l / min for later use . microfluidic device setup after the 5 hr incubation , take the entire device out and place it on a 15 cm diameter petri dish . transfer the petri dish to a live cell imaging microscope in an incubator.connect the tubing from the syringes for the chemical gradient generation to the inlets of the chemical gradient channel . turn on the syringe pump with the syringes for chemical gradient generation.connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . turn on the syringe pump with the syringes for oxygen gradient generation.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 . cell seeding 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.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.aspirate the supernatant after centrifuging and add an appropriate amount of the serum - free medium to make the final cell density 1 x 10 cells / ml . take out the microfluidic device prepared on day 1 . count the cells using a hemocytometer or another automatic cell counting instrument.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 l of the cell suspension from the outlet of the chemical gradient channel.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 . 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 . 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 . aspirate the supernatant after centrifuging and add an appropriate amount of the serum - free medium to make the final cell density 1 x 10 cells / ml . take out the microfluidic device prepared on day 1 . 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 l of the cell suspension from the outlet of the chemical gradient channel . 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 . preparation of reagents for chemical gradient generation prepare the chemical ( sdf-1 ) 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 l / min for later use . prepare the chemical ( sdf-1 ) 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 l / min for later use . preparation of the reagents for oxygen gradient generation 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 l / min for later use . 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 l / min for later use . microfluidic device setup after the 5 hr incubation , take the entire device out and place it on a 15 cm diameter petri dish . transfer the petri dish to a live cell imaging microscope in an incubator.connect the tubing from the syringes for the chemical gradient generation to the inlets of the chemical gradient channel . turn on the syringe pump with the syringes for chemical gradient generation.connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . turn on the syringe pump with the syringes for oxygen gradient generation.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 . after the 5 hr incubation , take the entire device out and place it on a 15 cm diameter petri dish . connect the tubing from the syringes for the chemical gradient generation to the inlets of the chemical gradient channel . connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . 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 . day 3 : data collection and analysis 
 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)1516 . 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)1516 . note : the chemical and oxygen gradients can be characterized before or after the cell experiments . numerical simulation of the chemical gradient 
 estimate the laminar flow nature of microfluidics using computational fluidic dynamics ( cfd ) simulation . 
  estimate the laminar flow nature of microfluidics using computational fluidic dynamics ( cfd ) simulation . experimental characterization of the oxygen gradient 
 prepare an oxygen - sensitive fluorescent dye , tris(2,2-bipyridyl ) ruthenium(iii ) chloride hexahydrate , solution in water at a concentration of 5 mg / ml.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 l / min ( identical to the flow rates used for the chemical gradient generation).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.measure the fluorescence intensity using an inverted fluorescence microscope with the excitation light passing through a 470  20 nm optical filter . collect the emission light through a 515 nm long - pass emission filter using a cool ccd camera.connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . turn on the syringe pump with the syringes for oxygen gradient generation.collect the fluorescence images of the oxygen - sensitive dye flowing in the cell culture chamber.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.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.estimate the oxygen gradients by analyzing the fluorescence images using the stern - volmer equation according to references 10 and 11 . prepare an oxygen - sensitive fluorescent dye , tris(2,2-bipyridyl ) ruthenium(iii ) chloride hexahydrate , solution in water at a concentration of 5 mg / ml . 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 l / min ( identical to the flow rates used for the chemical gradient generation ) . turn on the syringe pump with the syringes for chemical gradient generation . measure the fluorescence intensity using an inverted fluorescence microscope with the excitation light passing through a 470  20 nm optical filter . collect the emission light through a 515 nm long - pass emission filter using a cool ccd camera . connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . collect the fluorescence images of the oxygen - sensitive dye flowing in the cell culture chamber . 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 . 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 . estimate the oxygen gradients by analyzing the fluorescence images using the stern - volmer equation according to references 10 and 11 . note : the entire microfluidic device is fabricated using the soft lithography replica molding process13 . fabrication of molds for the pdms layers in the microfluidic device 
 design the microfluidic channel patterns using commercially available illustration or drawing software . submit the file to a company for high - resolution transparency photomask printing14.clean silicon wafers with acetone ( 99.5% ) , isopropyl alcohol ( ipa ; 99.9% ) , and buffered oxide etch ( boe ; 6:1 nh4f.hf ) . rinse with deionized ( di ) water and dehydrate the wafer with a nitrogen gun.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 . note : the spin coating condition should yield su-8 2050 photoresist layers with a thickness of approximately 75 m on the wafers after optical lithography.soft bake the wafers on a 65 c hotplate for 3 min and then at 95 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 / cm . post - exposure bake ( peb ) the wafers at 65 c for 2 min and then at 95 c for 7 min.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.place the wafer and 100 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.take the silanized wafers out of the desiccator and tape them to 15 cm diameter petri dishes for the following soft lithography process : 
  design the microfluidic channel patterns using commercially available illustration or drawing software . clean silicon wafers with acetone ( 99.5% ) , isopropyl alcohol ( ipa ; 99.9% ) , and buffered oxide etch ( boe ; 6:1 nh4f.hf ) . rinse with deionized ( di ) water and dehydrate the wafer with a nitrogen gun . 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 . note : the spin coating condition should yield su-8 2050 photoresist layers with a thickness of approximately 75 m on the wafers after optical lithography . soft bake the wafers on a 65 c hotplate for 3 min and then at 95 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 / cm . post - exposure bake ( peb ) the wafers at 65 c for 2 min and then at 95 c for 7 min . 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 . place the wafer and 100 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 . take the silanized wafers out of the desiccator and tape them to 15 cm diameter petri dishes for the following soft lithography process : 
 fabrication and assembly of pdms layers 
 preparation of the pdms pre - polymer mix pdms monomer ( base ) and the curing agent at a 10:1 ratio ( v / v ) . fabrication of the pc - embedded top layer 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.place the mold overnight in a 60 c oven for pdms curing . make sure that the mold is on a horizontal plane.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.slowly embed a 1 mm thick pc film into the fresh pdms layer as a gas diffusion barrier ; expel any bubbles if needed.put the mold overnight in the 60 c oven , and make sure that it is on a horizontal plane.cool down the cured pdms to room temperature . cut the device with a scalpel to an area of approximately 5.5 x 5 cm , which can cover all channel patterns , and peel off the pdms slab from the mold.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 . fabrication of the bottom pdms layer pour 11 g of the pdms pre - polymer onto the mold for the bottom layer . make sure that it is lying on a horizontal plane.cool down the pdms to room temperature . cut the device to an area of approximately 5.5 x 5 cm , which can cover all channel patterns , and peel it off of the mold . fabrication of the pdms membrane 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 . assembly of the device 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.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 c oven overnight to promote bonding.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 cm , 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.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.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 c oven overnight.take the entire fabricated device out of the oven and cool it down to room temperature . preparation of the pdms pre - polymer mix pdms monomer ( base ) and the curing agent at a 10:1 ratio ( v / v ) . mix pdms monomer ( base ) and the curing agent at a 10:1 ratio ( v / v ) . fabrication of the pc - embedded top layer 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.place the mold overnight in a 60 c oven for pdms curing . make sure that the mold is on a horizontal plane.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.slowly embed a 1 mm thick pc film into the fresh pdms layer as a gas diffusion barrier ; expel any bubbles if needed.put the mold overnight in the 60 c oven , and make sure that it is on a horizontal plane.cool down the cured pdms to room temperature . cut the device with a scalpel to an area of approximately 5.5 x 5 cm , which can cover all channel patterns , and peel off the pdms slab from the mold.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 . 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 . place the mold overnight in a 60 c oven for pdms curing . pour an additional 13 g of pdms pre - polymer onto the mold and degas the pdms in the desiccator setup for 60 min . slowly embed a 1 mm thick pc film into the fresh pdms layer as a gas diffusion barrier ; expel any bubbles if needed . put the mold overnight in the 60 c oven , and make sure that it is on a horizontal plane . cut the device with a scalpel to an area of approximately 5.5 x 5 cm , which can cover all channel patterns , and peel off the pdms slab from the mold . 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 . fabrication of the bottom pdms layer pour 11 g of the pdms pre - polymer onto the mold for the bottom layer . make sure that it is lying on a horizontal plane.cool down the pdms to room temperature . cut the device to an area of approximately 5.5 x 5 cm , 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 . 
  pour 11 g of the pdms pre - polymer onto the mold for the bottom layer . cut the device to an area of approximately 5.5 x 5 cm , 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 . fabrication of the pdms membrane 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 . 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 . assembly of the device 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.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 c oven overnight to promote bonding.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 cm , 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.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.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 c oven overnight.take the entire fabricated device out of the oven and cool it down to room temperature . 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 . 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 c oven overnight to promote bonding . 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 cm , 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 . 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 . place a weight ( about 600 g ) on top of the entire bonded device and put it in a 60 c oven overnight . take the entire fabricated device out of the oven and cool it down to room temperature . 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 ) , as examples . for researchers working on other cells and chemokines , day 0 : cell preparation 
 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.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 10 cells in a t75 flask with 10 ml of complete growth medium . note : all cell culture processes are performed in an incubator with a 37 c and 5% co2 atmosphere . 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 . 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 10 cells in a t75 flask with 10 ml of complete growth medium . note : all cell culture processes are performed in an incubator with a 37 c and 5% co2 atmosphere . day 1 : device preparation 
 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.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 . 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.prepare 1 ml of 50 g / ml fibronectin solution by diluting fibronectin stock with dulbecco 's phosphate - buffered saline ( dpbs).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.incubate the entire device overnight in a conventional cell culture incubator . 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 . 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 . 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 . prepare 1 ml of 50 g / ml fibronectin solution by diluting fibronectin stock with dulbecco 's phosphate - buffered saline ( dpbs ) . 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 . day 2 : cell seeding and microscopy imaging 
 cell seeding 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.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.aspirate the supernatant after centrifuging and add an appropriate amount of the serum - free medium to make the final cell density 1 x 10 cells / ml . take out the microfluidic device prepared on day 1 . count the cells using a hemocytometer or another automatic cell counting instrument.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 l of the cell suspension from the outlet of the chemical gradient channel.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 . preparation of reagents for chemical gradient generation prepare the chemical ( sdf-1 ) 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 l / min for later use . preparation of the reagents for oxygen gradient generation 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 l / min for later use . microfluidic device setup after the 5 hr incubation , take the entire device out and place it on a 15 cm diameter petri dish . transfer the petri dish to a live cell imaging microscope in an incubator.connect the tubing from the syringes for the chemical gradient generation to the inlets of the chemical gradient channel . turn on the syringe pump with the syringes for chemical gradient generation.connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . turn on the syringe pump with the syringes for oxygen gradient generation.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 . cell seeding 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.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.aspirate the supernatant after centrifuging and add an appropriate amount of the serum - free medium to make the final cell density 1 x 10 cells / ml . take out the microfluidic device prepared on day 1 . count the cells using a hemocytometer or another automatic cell counting instrument.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 l of the cell suspension from the outlet of the chemical gradient channel.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 . 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 . 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 . aspirate the supernatant after centrifuging and add an appropriate amount of the serum - free medium to make the final cell density 1 x 10 cells / ml . take out the microfluidic device prepared on day 1 . count the cells using a hemocytometer or another automatic cell counting instrument . 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 l of the cell suspension from the outlet of the chemical gradient channel . 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 . preparation of reagents for chemical gradient generation prepare the chemical ( sdf-1 ) 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 l / min for later use . prepare the chemical ( sdf-1 ) 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 l / min for later use . preparation of the reagents for oxygen gradient generation 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 l / min for later use . 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 l / min for later use . microfluidic device setup 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.connect the tubing from the syringes for the chemical gradient generation to the inlets of the chemical gradient channel . turn on the syringe pump with the syringes for chemical gradient generation.connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . turn on the syringe pump with the syringes for oxygen gradient generation.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 . after the 5 hr incubation , take the entire device out and place it on a 15 cm diameter petri dish . transfer the petri dish to a live cell imaging microscope in an incubator . connect the tubing from the syringes for the chemical gradient generation to the inlets of the chemical gradient channel . connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . 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 . day 3 : data collection and analysis 
 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)1516 . 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)1516 . note : the chemical and oxygen gradients can be characterized before or after the cell experiments . 
 numerical simulation of the chemical gradient 
 estimate the laminar flow nature of microfluidics using computational fluidic dynamics ( cfd ) simulation . estimate the laminar flow nature of microfluidics using computational fluidic dynamics ( cfd ) simulation . experimental characterization of the oxygen gradient 
 prepare an oxygen - sensitive fluorescent dye , tris(2,2-bipyridyl ) ruthenium(iii ) chloride hexahydrate , solution in water at a concentration of 5 mg / ml.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 l / min ( identical to the flow rates used for the chemical gradient generation).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.measure the fluorescence intensity using an inverted fluorescence microscope with the excitation light passing through a 470  20 nm optical filter . collect the emission light through a 515 nm long - pass emission filter using a cool ccd camera.connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . turn on the syringe pump with the syringes for oxygen gradient generation.collect the fluorescence images of the oxygen - sensitive dye flowing in the cell culture chamber.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.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.estimate the oxygen gradients by analyzing the fluorescence images using the stern - volmer equation according to references 10 and 11 . prepare an oxygen - sensitive fluorescent dye , tris(2,2-bipyridyl ) ruthenium(iii ) chloride hexahydrate , solution in water at a concentration of 5 mg / ml . 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 l / min ( identical to the flow rates used for the chemical gradient generation ) . turn on the syringe pump with the syringes for chemical gradient generation . measure the fluorescence intensity using an inverted fluorescence microscope with the excitation light passing through a 470  20 nm optical filter . collect the emission light through a 515 nm long - pass emission filter using a cool ccd camera . connect the high - purity tubing from the syringes containing naoh and pyrogallol to the inlets of the oxygen gradient channel . collect the fluorescence images of the oxygen - sensitive dye flowing in the cell culture chamber . 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 . 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 . estimate the oxygen gradients by analyzing the fluorescence images using the stern - volmer equation according to references 10 and 11 . 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 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 . 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 . 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 ) 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 . 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 . 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 concentration is higher . the results suggest the sdf-1 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 ) . ( 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 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 ) 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 chemokine gradient across the width of the cell culture chamber for cell migration studies . the gradients , from left to right of the chamber , are characterized , and the results show consistent gradient profiles across the width of the chamber . 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 . 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 . ( 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  sd , obtained from three independent experimental sets , and 10 cells were analyzed in each experiment . the statistical significantly different ( unpaired student 's t - test , p < 0.01 ) 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 . 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 can not 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 .
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 ) 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 can not 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 .