The overall goal of this procedure is to pattern cells or substrates by distributing them into complex long and closed microchannels using simple bench top tools available in most biological laboratories. This method can help answer key questions in the field of biology regarding cell-to-cell and cell-to-protein interactions. The main advantage of this technique is that it is easy to perform and it only requires tools that are readily available in most biological laboratories.

Generally, individuals new to this method will struggle because it requires optimization of the incubation conditions for the cells and substrates chosen by the researcher. Visual demonstration of this method is critical, as the proper placement of the PDMS stamp and solution significantly improves the reproducibility of the experiment. Start by drawing out the layout of the microchannel using a CAD drawing tool.

Then, print the drawing onto white paper. Next, use isopropanol to clean a glass slide that is large enough to accommodate the design. And then dry the slide with an air gun.

Attach adhesive tape to the cleaned glass slide, taking care not to trap any air bubbles. Then, tape the sides of the glass slide onto the paper with the design, so that the tape side is facing up. Use a scalpel to cut the tape on the glass slide, using the paper design as a reference.

Once cut, peel off the tape from the unwanted areas of the glass slide. Next, rinse the glass slide with isopropanol to remove any residual adhesive. And then use an air gun to dry the surface.

Once patterned and cleaned, bake the glass slide in an oven at 65 degrees Celsius for 30 minutes. Then, gently roll over the tape with a rubber roller, to remove air bubbles and allow the slide to cool to room temperature in a petri dish. Begin by mixing 10 parts of a PDMS elastomer with 1 part of its curing agent and stir the mixture vigorously.

Then, place the mixture into a vacuum chamber to de-gas the mixture until no air bubbles remain. Pour a 2mm thick layer of the mixture onto the adhesive tape mold, and de-gas the mold until all air bubbles disappear. Once cooled, use a scalpel to cut the PDMS layer at least 5mm away from the features.

And then, peel the cured PDMS off the mold using a pair of tweezers. Punch a single inlet hole with a 1mm biopsy punch, preferably at the end of a network of microchannels. Then, clean the PDMS cast with adhesive tape, to remove dust particles from the device.

Sterilize the PDMS microchannel device by rinsing it with a solution of 70%ehtanol, followed by sterile deionized water, and expose it to UV light for 30 minutes. Use tweezers to place the sterile PDMS cast onto a sterile glass cover slip, and apply gentle pressure using the tip of the tweezers. Next, place a droplet of poly d lysine in sodium tetraborate buffer on the inlet of the microchannels.

Place the petri dish under vacuum for 10 minutes. During this time, observe air coming out of the channel in the form of bubbles. Release the vacuum and observe the substrate solution flowing into the microchannel Incubate the dish at 37 degrees Celsius for one hour.

Then, carefully peel off the PDMS stamp using sterile tweezers, and wash the pattern three times with deionized water. Add a 1%BSA solution to the glass cover slip, to reduce non-specific adhesion, and incubate the sample overnight at 37 degrees Celsius. The next day, aspirate the BSA solution.

To start the indirect patterning of cells, prepare a cell suspension in serum free culture medium as described in the accompanying text protocol. Add the cell suspension to the patterned petri dish, completely submerging the patterned region in cell suspension. Then, incubate the petri dish at 37 degrees Celsius in an incubator for 15 minutes to allow cells to attach to the patterned substrate.

Following incubation, aspirate the excess cell suspension from the petri dish and wash the patterned cells three times with PBS, while gently shaking for ten seconds to remove the unattached cells. Then, add the appropriate culture medium to the patterned cell cultures, and incubate the patterned cells at 37 degrees Celsius in a carbon dioxide buffered incubator. For the direct patterning of cells, first, sterilize the microfluidic device by rinsing it with a solution of 70%ethanol followed by deionized water.

Then, soak the device overnight in a solution of 1%BSA to prevent non-specific cell adhesion to the PDMS. Dry the device at room temperature, and then attach it to the bottom of the tissue culture treated petri dish. Place 4 to 8 microliters of the cell suspension, enough to fill the microchannels, on the inlet, completely covering the hole.

Then, put the petri dish in a vacuum chamber for 10 minutes. Release the vacuum and observe the cell suspension flowing into the microchannel. Incubate the petri dish at 37 degrees Celsius in a carbon dioxide buffered incubator, for up to one hour.

Then, remove the dish from the incubator and use a pair of tweezers to carefully peel the PDMS from the dish. Wash the pattern with PBS, and then add cell culture media to the petri dish. When finished, place the cells back into the incubator.

Shown here are embryonic rat cortical neurons patterned on poly d lysine and laminin surfaces. The location of the cells demonstrate general adherence to the patterned areas and growth along the PDL and laminin stripes. Shown here, at a higher magnification, are C2C12 myoblasts.

This cell type also adheres to the patterned areas and demonstrates fusion into multinucleated myotubes. This technique has also been adapted for direct cell patterning, shown here with fibroblasts. This direct cell patterning method has no adverse effect on cell survival.

Following this procedure, other methods like fluorescent microscopy can be performed in order to answer additional questions involving protein localization or cell-substrate interactions. While attempting this procedure, it is important to remember that proper placement of the droplet of cells or substrate solution to cover the entire inlay is critical for success. After its development, this technique paved the way for researchers in the field of neuroscience to explore photoreceptor axon guidance in retinal biology.

After watching this video, you should have a good understanding of how to pattern cells and substrate with gas permeable PDMS microfluidic devices using negative pressure. Thanks for watching and good luck with your experiment.