単一細胞レベルでのキャビテーション気泡（複数可） - 細胞相互作用し、得られた生体効果を調査するための表面パターニングとマイクロ流体システム

The overall goal of this experimental procedure is to investigate cavitation induced bioeffects in microfluidic confinement using surface patterning to precisely control the generation of tandem bubbles and the location and shape of the individual target cells. This microfluidic system enables experimentation of this transit cavitation bubbles and cells that is relevant to therapeutic and sound publication and sound operation. The main advantage of this technique comes from the improve the precision from surface patterning.

It allows us to study the bioeffects of individual cells and is a high stream loading from reliable bubble-bubble interaction. Visual demonstration of the procedures is critical as the surface patterning and cell preparation in the chip steps are complex involving various techniques and tips. Perform all the microfabrication procedures in a clean room while wearing a clean room suit.

Design the area of each gold dot within 25 to 30 square microns, so that it is large enough to absorb laser energy for bubble generation, but small enough to avoid individual cells adhering to it. Clean the glass slide in a chemical hood according to the text protocol, then proceed to the spin coating hood. Program the spin coder to accelerate to 1000 RPM over two seconds and to maintain that speed for five seconds then have it ramp to 3000 RPM over three seconds and maintain that speed for 30 seconds.

Next, secure the glass slide to the spin coder using the vacuum. Then, cover the slide with P20 and start the spin cycle. Next, apply NFR negative photo resist using the same cycle.

Now, bake the slide on a hot plate at 95 degrees celsius for 60 seconds. Once cooled to room temperature perform the photolithography. Mount the chrome mask onto the mask aligner and make sure that the pattern side is facing down towards the slide.

Then, set the photolithography recipe to hard exposure mode with nine seconds of UV exposure and align the glass substrate with the mask. After the UV exposure, bake the slide at 95 degrees celsius for one minute, and then let it cool as before. To develop the pattern on the slide, place it in developer solution for 60 seconds, then wash the slide with distilled water and dry it with nitrogen gas.

After confirming the pattern by microscopic inspection bake the slide at 120 degrees celsius for five minutes and let it cool to room temperature. Now, clean the slide with plasma in the reactive ion etching machine for 90 seconds at 500 tor and 100 watts. Using an E-beam evaporator, tape the sample to the plate holder.

Program the machine to deposit a 5 nanometer layer of titanium, followed by a 15 nanometer layer of gold. Once that position is complete, ventilate the machine. Next, soak the slide overnight in a beaker of photoresist remover solvent to remove the gold resting on top of the NFR resist.

The next day, wash the slide with acetone, followed by IPA and then dry it with nitrogen. Rinse the slide with DI water and dry it again with nitrogen. Now, heat the slide at 115 degrees celsius for five minutes.

Then, clean the gold dot pattern slide using an oxygen plasma asher at 100 watts for 90 seconds. Set the area of each fibronectin encoded island to be within 700 to 900 square microns to facilitate adequate hela cell spreading in a square region while minimizing the chances of multiple cells aggregating on the island. Fabrication is much like that of the gold pattern.

Spin code the slide as before using S18 positive photoresist and bake on the coding at 115 degrees celsius. Then, perform photolithography, aligning the marks on the mask with those on the substrate. Check the pattern on the central portion to confirm the correct angle and adjust the standoff distance from the gold dots to the H pattern.

Run the UV exposure for nine seconds with no post bake. The next difference is that the development step only takes 45 seconds. For the reactive ion etching, set the parimeters to 500 tor 100 watts, and 90 seconds.

Then, apply a drop of PLLG peg passivating solution onto a piece of paraffin film and sandwich the solution to the pattern side of the slide. Avoid trapping bubbles. After 45 minutes, remove the slide from the film.

Rinse the slide with DI water and then dry it with nitrogen. Next, soak the slide consecutively in photoresist remover 1165. Then 50 percent 1165 in DI water.

Then just DI water. During each bath, agitate the solution in an ultrasound bath for 90 seconds. Now, dry the slide on a hot plate, then seal it in a desiccator and store it at 4 degrees celsius.

Assemble the slides into a microchannel chip as described in the text protocol. Pay careful attention to shield the patterned area of the glass substrate with the small PDMS slab before using reactive ion etching to remove the PLLG peg from the peripheral area. Retrieve the patterned glass from the RIE machine and remove the small PDMS slab.

After treating the microchannel PDMS with a reduced dose of oxygen plasma, align it to the patterned glass substrate under a stereoscope. Bring the micro channel PDMS and the patterned glass substrate in conformal contact. Now, proceed to use the chip.

First, prime the chip by flowing PBS down the microchannel for 30 minutes at one microliter per minute. Then infuse the chip with fibronectin solution for 45 minutes at the same flow rate. While waiting, prepare the cells at five million cells per milliliter.

Healthy status and high confluencey is critical to this procedure. Later, replace the solution flowing through the chip with PBS and increase the flow rate to ten microliters per minute. Let the PBS flow for five minutes.

Next, inject the prepared cells into the chip with a syringe. When one drop has flowed out of the tubing stop the injection and clamp the outlet. Then, place the chip in an incubator for 30 minutes.

Later, release the outlet and flush the chip with cell culture medium at ten microliters per minute for five minutes. After five minutes, drop the flow to 0.75 microliters per minute and transfer the chip and pump to an incubator for two hours. After two hours, proceed with tandem bubble generation viewing the chip under an inverted microscope with two pulsed NDI lasers on timing controls directed at the gold dot pattern and with a high speed camera ready to capture the results.

For 50 micron bubbles, set the delay between the two lasers to 2.5 microseconds and set their power to ten microjoules. Then, synchronize the high speed camera recording with the lasers and make records at two million frames per second with 200 nanosecond exposures. Thus, the dynamics of bubble expansion, collapse bubble-bubble interaction, and jet formation are recorded.

Bubble-bubble interactions and a variety of cavitation induced bioeffects were studied at the single cell level using the described technique, such as the transient interactions of tandem bubbles with the jet formation the visualization of the resultant flow field and the calculation of the jet speed. Directional jetting flow around the tandem bubble is around ten meters per second, is about ten microns wide and is thus capable of producing an impulsive and localized sheer stress and stress gradient onto nearby target cells. Tandem bubble induced cell membrane deformation was also studied.

Membrane deformation and recovery are highlighted by the displacement of functionalized beads attached to the leading edge of the cell membrane. From the coordinates of a triad of adjacent beads, the local area strain was calculated. This schematic shows the maximum area change at different locations on the cell surface.

The leading edge is primarily stretched, while the trailing edge or lateral sides of the cell are compressed indicating heterogeneity and cell deformation produced by the tandem bubble induced jetting flow. The temporal variation of the area strain at the cell leading edge is comprised of a few rapid oscillations followed by a large and sustained stretch for about 100 microseconds, and a subsequent gradual recovery on a timescale of several milliseconds. After watching this video, you should have a good understanding of how to make a controllable bubble-bubble interaction to study the bioeffects of single cells with controlled shape and location from surface patterning.

Following this procedure, other measures like cytoskeleton thinning and the conjugal imaging can be further performed to study cell cytoskeleton rearrangement of the tandem bubble treatment. After its development, this technique will pave the way for researchers in the field of subpretic ultrasound to explore captation induced bioeffects on the single cell level. While attempting this procedure, it is important to remember to maintain a good cell confluency and condition for the success during the cell patterning.

Do not forget that working with clean room chemicals and lasers can be extremely hazardous and precautions such as wearing gloves and laser goggles should always be taken while performing this procedure.