垂直化学的酸素勾配の下で細胞移動研究のためのポリジメチルシロキサン - ポリカーボネートマイクロ流体デバイス

The overall goal of this experiment is to establish a polydimethylsiloxane-polycarbonate microfluidic device to mammalian cell migration under various combinations of perpendicular chemical and oxygen gradients. This method can help answer key questions in mammalian cell migration studies including the effects of these gradients on migration of different types of cells or cells with ote genes. The main advantage of this technique is that the entire experimental setup can be conducted in a conventional incubator for optimal cell culture conditions without tedious setup and bulky instrumentation.

The implications of this technique extended towards diagnosis of cancer cell metastasis capability as the cell motility and the various physiologically-related gradients can be characterized using the microfluidic device. This method can provide insights into mammalian cell migration. It can also be applied to other cell studies such as in vitro cell-based drug testing on the combinations of drug concentrations and oxygen tensions.

Transfer two grams of polydimethylsiloxane, or PDMS pre-polymer onto the mold with the top layer fluidic channel patterns to make a thin layer of PDMS. Then, place the mold in a desiccator setup to degas the PDMS for 60 minutes. After degassing, place the mold overnight in a 60-degree Celsius oven for PDMS curing.

Make sure the mold is in a horizontal plane. After cooling the mold to room temperature, pour an additional 13 grams of PDMS pre-polymer onto the mold, then degas the PDMS in the desiccator setup for 60 minutes. Slowly imbed a one-millimeter-thick polycarbonate, or PC film into the fresh PDMS layer as a gas-diffusion barrier.

Expel any bubbles that are present. Then, place the mold in the 60-degree Celsius oven overnight. Make sure that the mold is on a horizontal plane.

After cooling down the cured PDMS to room temperature, cut the device with a scalpel to an area of approximately 5.5 by five square centimeters which can cover all channel patterns. Then, peal the PDMS lab from the mold. Next, punch holes for inlets and outlets using a two-millimeter-diameter biopsy punch.

Store the fabricated top PDMS-PC layer away from ambient dust for later assembly. To assemble the device, first place the fabricated PDMS-PC top layer and the wafer with the spin-coded PDMS membrane in an oxygen-plasma surface treatment machine with the bonding surfaces facing up. Treat the PDMS surfaces with 90 watts of oxygen plasma for 40 seconds.

Bond the top layer onto the PDMS membrane immediately after the oxygen plasma surface treatment. Place a weight on the top of the bonded layers and put them in a 60-degree Celsius oven overnight to promote bonding. Once the bonded layers have cooled to room temperature, cut the membrane bonded to the top layer with a scalpel to an area of approximately 5.5 by five square centimeters.

Peal the bonded structure from the silicon wafer and punch holes at the inlets and outlets of the chemical gradient channel using a two-millimeter-diameter biopsy punch. Then, place the membrane-bonded top layer and the fabricated PDMS bottom layer in the oxygen plasma surface treatment machine with the bonding surfaces facing up. To activate the PDMS surfaces, use the plasma at 90 watts for 40 seconds.

Attach the top and bottom layers together for bonding immediately after the surface treatment. Place the weight on top of the entire bonded device and leave it overnight in a 60-degree Celsius oven. The next day, take the entire fabricated device out of the oven and cool it to room temperature.

Begin the microfluidic cell migration assay by preparation of the cells in the device as described in the text protocol. On day two, aspirate the medium from the cells that were cultured since day zero and wash the cells with five milliliters of DPBS two times. Aspirate the DPBS and add two milliliters of Trypsin-EDTA, then incubate the cells in the incubator for five minutes to detach the cells from the flask surface.

Once the cells are detached and suspended in the solution, add eight milliliters of serum-free medium to the flask. Transfer all the liquids into a 15-milliliter conical tube and centrifuge it at 140 times G for five minutes at room temperature. Following centrifugation, aspirate the supernatant and add an appropriate amount of serum-free medium for a final cell density of one million cells per milliliter.

Then, take out the microfluidic device prepared on day one as described in the text protocol. Use a one-milliliter syringe with a blunt 14-gauge needle to inject the serum-free medium through the outlet of the chemical gradient channel until the medium flows out from both needles at the inlets. Then, inject 200 microliters 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 the cells have been introduced to the microfluidic channel. Then, place the device in a humid container and keep it in a cell culture incubator for five hours to promote the adhesion of the cells onto the device surface. Meanwhile, prepare the chemical at the desired concentration in serum-free medium.

Draw the serum-free medium with and without the chemical into two separate three-milliliter syringes connected to the high-purity tubing. Then, set up the syringes on a syringe pump with a flow rate of one microliter per minute for later use. Next, make 15 milliliters of a one-molar sodium hydroxide solution and 15 milliliters of 200 milligrams per milliliter pyrogallol solution.

Draw the sodium hydroxide solution into a 15 milliliter syringe connected to a high-purity PFTE tubing and draw the pyrogallol solution into a 15-milliliter syringe connected to a high-purity tubing. Set up the syringes on a syringe pump with a flow rate of five microliters per minute for later use. After the five-hour incubation, take the entire device out and place it on a 15-centimeter-diameter Petri dish.

Fix the device in the Petri dish using adhesive putty, then transfer the Petri dish to a live-cell imaging microscope in an incubator. Connect the tubing from the syringes to the inlets of the chemical gradient channel for generation of the chemical gradient. Then, connect the outlet to the tubing that leads to a waste reservoir.

Turn on the syringe pump with the syringes for chemical gradient generation. Next, connect the high-purity tubing from the syringes containing sodium chloride and pyrogallol to the inlets of the oxygen gradient channel. Connect the tubing to the outlet to collect the waste and turn on the syringe pump with the syringes for oxygen gradient generation.

Add about 15 milliliters of double-distilled water to the Petri dish to keep the device moisturized. Then, set up the live-cell imaging microscope for time-lapsed image capture and take an image every 15 minutes. Perform data collection and analysis as well as characterization of gradients as described in the text protocol.

Shown here are representative results of chemical gradients generated within the microfluidic device for cell migration. The numerical simulation result agrees well with the experimental characterization using fluorescein solution as the chemical. The gradients of other chemical such as chemokine SDF-1 alpha can also be estimated beforehand using the simulation.

Oxygen gradients generated within the device can be experimentally characterized using oxygen-sensitive fluorescence dye. Typically, oxygen gradients from 1%at upstream to 16%at downstream can be stably established within the device. Shown here are typical time-lapsed images collected during the experiments using a live-cell imaging microscope.

After image analysis, the migration trajectories of A549 cells and their average movements can be calculated. Under no gradients, cells show random migration and average movement is close to zero. With only an SDF-1 alpha chemokine gradient, cells show chemotactic behavior and migrate towards the high-chemokine-gradient direction as expected.

With only an oxygen gradient, cells move towards the low-oxygen direction showing an aerotactic behavior that has not been well-studied for mammalian cells. Interestingly, with both chemokine and oxygen gradients, the cells move toward the low-oxygen gradient showing that the oxygen gradient has dominant effects. This observation requires further study.

While attempting this procedure, it is important to remember to well-level the device during the fabrication and eliminate air bubbles within the microfluidic device setup during the cell migration experiment. After watching this video, you should have a good understanding of how to prepare a PDMS-PC microfluidic device and setup the experiments to study mammalian cell migration under combinations of chemical and oxygen gradients.