Mechanostimulation of Multicellular Organisms Through a High-Throughput Microfluidic Compression System

This protocol explains how to fabricate and use microfluidics technologies to leverage the experimental benefits in sample throughput, automated handling, and the ability to precisely apply mechanical stress while simultaneously visualizing samples. This technique minimizes the manual handling of multicellular biological organisms, such as embryos and organoids, for their immobilization, alignment, and live imaging. It also enables the application of mechanical compression to them for mechanobiology studies.

Fabrication of molds with high aspect ratio features for this microfluidic chip can be challenging with conventional microfabrication techniques. The tips explained in this video article can overcome these challenges. To begin, clean the silicon wafer first with acetone, and then with isopropyl alcohol.

Place the silicon wafer on a 250-degree Celsius hot plate for 30 minutes. for dehydration bake. Coat the silicon wafer with hexamethyldisiloxane in a vapor prime oven.

Place a bottle of SU-8 2100 Photoresist in a 60-degrees Celsius oven for 15 minutes to reduce its viscosity. Pour one milliliter of the heated Photoresist for each inch of the silicon wafer placed on the hot plate until the Photoresist covers most of the surface. Apply pre-spin first at 250 rotations per minute for 30 seconds, and then at 350 rotations per minute for another 30 seconds, both with 100 RPM per second acceleration.

Next, apply a spin first at 500 rotations per minute for 15 seconds with 100 RPM per second acceleration, and then at 1, 450 rotations per minute for 30 seconds with 300 RPM per second acceleration. Remove the edge bead with a clean room swab, and spray acetone on the wafer to remove imperfections and promote uniform coating. Expose the silicone wafer to 350 millijoule per centimeter square UV light through the photo mask.

Using the contact mask aligner, apply post-exposure bake to the silicon wafer, and let it cool down to room temperature. Place the beaker inside another larger beaker, and fill the larger beaker with a fresh developer solution. Place a metal mesh on the beaker.

Place the silicon wafer on the metal mesh upside down. Leave the silicon wafer submerged in the developer for 30 minutes with the stirrer turned on. Transfer the silicon wafer into an ultrasonic bath sonicator filled with the fresh developer for one hour at 40 kilohertz.

Then take the wafer out from the beaker, and wash it with a fresh developer solution. Prepare the pre-cured PDMS solution by mixing the PDMS base with the curing agent at a 10-to-one ratio. Degas the mixture by placing it into a centrifuge for 500 g for five minutes at room temperature.

Pour the pre-cured PDMS on a silicon wafer, and degas it again. Finally, place the uncured PDMS into 60-degrees Celsius oven for curing. Use a scalpel to cut the borders of the cured PDMS region corresponding to the microfluidic chip geometry.

Punch the inlet and outlet holes on PDMS using a biopsy punch, or a needle with a blunt tip. Place the PDMS on the glass slide with its patterned surface facing toward the glass slide after plasma treatment to seal the microchannels via covalent bonding. Allow Oregon-R adult flies to lay eggs on apple juice agar plates, and collect the plates at the desired developmental time after egg laying for the given experiment.

Flood the agar with embryo egg wash, and gently agitate the embryos with a paintbrush to dislodge them from the agar. Transfer the embryos to a 50%bleach solution for 90 seconds, stirring occasionally. Strain the embryos through a tissue sieve, and thoroughly wash away the bleach solution with water.

Transfer the embryos to a 90-millimeter glass Petri dish with enough embryo egg wash to fully cover the embryos. Examine the embryos with transillumination on a dissecting microscope, and select embryos of the desired developmental stage for loading into the microfluidic device. Prime all seven embryo microchannels by filling them with 0.4 micrometers of filtered IPA through the main embryo inlet port.

Replace the IPA with 0.4 micrometers of filtered deionized water. Then replace the DI water with embryo egg wash solution. Collect approximately 100 pre-selected embryos from the glass Petri dish using a glass pipette.

Next, pipette the embryos into the embryo inlet port, and apply approximately three PSI negative pressure to the gas inlet using a portable vacuum pump to open up the embryo microchannels. Then tilt the microfluidic chip downward for the embryos to automatically align and settle into the embryo microchannels. If the embryo microchannel inlets get clogged by multiple embryos entering simultaneously, tilt the microfluidic chip upward, and then down again to clear the clogging.

Based on the required throughput, introduce as many as 300 embryos into the embryo microchannels. Once the embryo loading is completed, remove the vacuum to immobilize the embryos. Then tilt the micro fluidic chip back to the horizontal position.

Connect a portable positive pressure source with a pressure gauge to the gas inlet to apply three PSI compression. If live imaging experiments will be conducted on the mechanically stimulated embryos, place the microfluidic chip on a standard microscope stage glass slide holder with the gas inlet connected to the pressure source. Examine the embryos under a fluorescent microscope inside the microfluidic channels.

Once the compression experiment is completed, the embryos can be collected for downstream analysis. To do this, first, apply the vacuum to the gas inlet to free the embryos. Then tilt the microfluidic chip upward for the embryos to move downward toward the embryo introduction port.

Collect the embryos from the microfluidic chip using a glass pipette. The functionality of the microfluidic device was experimentally determined by loading Drosophila embryos into the compression channels and applying positive pressure to the gas channels. The embryos do not experience significant compression under vacuum, or in neutral pressure states.

They are compressed when positive pressure is applied. Measurements of the decreasing width of the embryos under a microscope demonstrate how gas pressure can be used to obtain a target compression level. Microfluidic devices made in this way allow for chemical stimulation of immobilized samples.

The stimulation allows for high spatiotemporal imaging. A fundamental question in developmental biology is how do mechanical forces regulate protein and gene expression during development? This technology allows us to apply mechanical stimulation to a large number of embryos at once, so that we can isolate sufficient amounts of protein and RNA to perform comparative proteomics, or transcriptomics experiments.

This will enable researchers to learn more about the proteins and genes that are sensitive to mechanical forces.