Image: microfluidic assay I created during microfabrication and prototyping workshop.
Weekly seminars are available for CEMS summer interns, and I attended many incredible courses this week. In addition to an instructional course on how to properly read scientific articles, I attended an incredible microfabrication and prototyping workshop. The course centered around the production of microfluidic assays, which are the foundation for technologies like organs-on-a-chip and cell-on-a-chip drug screening. Microfluidic assays are chips with patterned microchannels that are linked to wells, into which fluids are injected. The flow of fluids through the channels generates concentration gradients of the injected liquids, generating automated, high-throughput systems. The small scale of the chips enables the use of lesser sample volumes and shortens the time of the experiments.
We were guided through each step of the prototyping process, allowed to both make our own assays and then use them to run a test food dye gradient experiment. The first step of the process is using SolidWorks to generate the 3D mold design and then transferring the design to Preform for printing. Once the mold is printed, it must go through extensive post-processing; we wash it in isopropyl alcohol (IPA) for 60-90 minutes, cure it for 60-90 minutes to full solidify the resin, cut the 3D-printed supports, and use finishing tools for surface fine-tuning. Then we prepare the polydimethylsiloxane (PDMS) by mixing a 1:10 ratio of elastomer and curing agent, creating a porous material that allows gas diffusion. We use a vacuum to remove gas from the PDMS and then pour it into the mold, curing it at 48°C. Once the PDMS is cured, we remove the cured resin from the mold and punch holes in the chip for channel tubing. We then insert the chip into an oxygen plasma cleaner to bond the surface of the PDMS to the base in order to create a tight seal on the channels. After curing again at 65°C, we returned to complete the final experiment. We first prepared the blue and yellow dyes and connected tubing between the syringes and the channels. Then we placed the syringes into the syringe pump machine so that the liquid is released at a constant rate. Attached above is the picture of our final product; although most microfluidic experiments don’t use these colored liquids, the color mixing introduced us to how a concentration gradient is generated for such experiments.
Further, on Thursday we received much more patient blood than usual, so in addition to our typical resolvin experiments (explained in my week 1 reflections post), we were able to run viability assays, Propidium iodide (PI) vs. Annexin assays, and test for CD85 and CD163 monocyte markers. The viability assays are critical steps in our mitochondrial revival project (also discussed in my week 1 reflection). In order to determine the impact of mitochondria on cell viability, we must measure the viability before and after treatment. We accomplish this by using an assay that measures the enzyme in charge of cellular metabolism. Since metabolism levels are correlated with cell count, this gives us an accurate estimation of viability. We measure enzyme levels through measuring absorbance of the treated samples– the greater the viability, the greater the absorbance measured. The PI vs annexin assay is critical in determining the cause of death of cells; PI is an indicator of necrosis (passive, accidental cell death caused by environmental changes), while annexin is a protein that binds to a lipid indicator of apoptosis (active, programmed cell death).
When performing monocyte experiments, it’s important to track monocyte differentiation in order to determine monocyte function. Monocytes begin in the M0 stage, at which point they are unstimulated and undifferentiated. In our experiments, however, we’re exposing them to lipopolysaccharide (LPS), which is a microbe-associated molecular pattern that triggers cytokine secretion to alert immune cells to destroy invading pathogens. It also triggers differentiation into M1 and M2 phases, M1 being macrophages that release pro-inflammatory cytokines and M2 releasing anti-inflammatory cytokines. We detect the stage of the monocytes by determining the markers on the outside of the cell using the CD85 and CD163 markers mentioned above, since CD85 is an M1 marker and CD163 is an M2 marker.
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