Microfluidic Approach to Resolve Simultaneous and Sequential Cytokine Secretion of Individual Polyfunctional Cells

In this project, we focus on developing and applying novel analytical strategies which allow for the direct, quantitative, and deep analysis of cellular functionalities at single cell resolution. And so by going beyond pure one data point measurement, we aim to better understand and exploit this multi-functionality which is coming from complex responses in the host and in tissue. To obtain a comprehensive understanding of these functionalities is complex, difficult, and requires interdisciplinary approaches.

And so one challenge in this context is to ensure that the dynamic range of the assay developed is appropriate to capture the quantity and dynamics of a secreted protein, which can vary greatly across samples, preparations, and tissues. Disruption in the regulation of the immune response can induce various disorder, leading to mild fever, to potentially life-threatening complication. Moving beyond traditional endpoint measurements, we propose to directly measure the dysregulation and activation of immune cells dynamically on a single cell level to gain additional insight.

So cytokine are usually detected at a specific time point as a concentration in the supernatant, a percentage of activated cells. Bulk measurement to quantify secretion of each individual cells, masking cellular heterogeneity with endpoint measurements that do not distinguish between simultaneous and secondary secretion overlooking the dynamic aspect of the response. Cellular polyfunctionality refers to an immune cell's ability to secrete multiple cytokines simultaneously.

This allows them to fine tune their response against threats, for instance, and to measure this polyfunctionality dynamically, meaning over time, can give an over insights into the different nuances of an immune response. To begin, place the glass slide with holes on a clean surface with the plasma activated side facing upwards. Carefully remove the protective layer from one side of the double-sided adhesive tape in the direction of cutting.

Align the tape with the edges of the glass slide and the drilled holes. Place the tape on the glass slide starting from the short edge and make sure of good adherence. After removing the second protective layer from the tape, place the second glass slide with the activated surface facing downwards.

Using a flat board, press the two glass slides together while applying the upper body strength for 10 seconds. Flip the chamber so that the holes face the user. Apply a small amount of UV curable glue in the ring below the nano port.

Slide the nano port down the hole in the glass slide using a microtubing and add a ring of UV curable glue around the port. Load one milliliter of 1%solution prepared in fluorinated oil into a syringe. Inject the solution through a PTFE syringe filter and a needle attached to PTFE microtubing into the observation chamber.

After a minute of incubation, use nitrogen pressure to flush out the coating solution under a fume hood. Using a different syringe assembly, rinse the chamber solely with fluorinated oil. To begin, add streptavidin functionalized nanoparticles to tubes for TNF alpha, interleukin one beta, and interleukin six detection.

Dilute the streptavidin plus nanoparticles in equal volumes of PBS. Then add the biotinylated capture antibody solutions to tubes and incubate for 30 minutes at room temperature. Next, add 0.01%of one millimolar D-biotin solution to each tube and incubate for five minutes.

Then hold a neodymium magnet close to the tube to collect the particles. Discard the clear supernatant and immediately resuspend the nanoparticles in a mixture of pluronic F-127 and PBS and incubate for 30 minutes. After incubating the particles for 30 minutes in storage buffer, stir them to ensure a uniform mixture.

Immediately before encapsulation, combine the conjugated nanoparticles for interleukin six, TNF alpha, and interleukin one beta. Now wash the particles with complete media using the magnet as demonstrated earlier, and resuspend the beads in complete media. Afterward, add detection antibodies for interleukin six, TNF alpha, and interleukin one beta to achieve the final concentration of 10 nanomolar.

To begin, fill a one milliliter syringe with 500 microliters of continuous phase. Attach the PTFE microtubing to a 27 gauge 0.75 inch needle. Mount the assembly onto the syringe and subsequently onto the syringe pump.

Then, punch a hole using a 0.75 millimeter biopsy punch in the middle of a PDMS cutout. Pull approximately three centimeters of PTFE tubing through the hole in the cutout and push the assembly into the top of a 200 microliter pipette tip. Seal the connector with UV curable glue on top of the pipette, and attach the other side of the tube to a 23 gauge 1.25 inch needle.

Next, fill two one milliliter syringes with 500 microliters of light mineral oil. Attach two 23 gauge needles with the custom made attachments and mount them onto the syringe pump. Using the syringe pump control software, aspirate functionalized nanoparticle and cell solution into the pipette tips of the aqueous phases.

After cleaning the prepared observation chamber, clamp the chamber into the printed chamber holder equipped with two neodymium magnets at 30 degrees. Now, open both ports and plug a paper towel in the upper port to absorb the excess outer phase during filling. Connect the continuous phase to the top inlet of the microfluidic chip.

Then, flush the chip with the continuous phase for approximately 30 seconds at a flow rate of 1800 microliters per hour. Attach the pipette tips of the aqueous solutions to the two middle inlets of the chip. Start the flow of the aqueous solution at 200 microliters per hour for each solution.

Once the liquid appears at the outlet, begin the fluorinated oil phase flow at 800 microliters per hour. Wait until stable droplet production is established, indicated by a homogeneous gray shiny solution at the outlet. Then, connect the PTFE microtubing to the outlet port to collect the droplets.

Using the ferrule module of a finger tight one piece fitting, direct them into an observation chamber. Once the chamber is filled, stop the flow and close off the ports with port plugs using finger tight pressure. After droplet production, flush the chip with fluorinated oil, followed by nitrogen to blow out any remaining liquid.

Mount the chamber holder on the microscope with a well plate format stage. Then, using the 10x objective, switch the microscope to the bright-field channel. Focus on the immobilized droplets in the bright-field and move to the center of the chamber.

Then, activate the automated focusing system. Adjust the measurement plane on the bright-field channel so that droplet edges appear as sharp black circles easily distinguishable from the oil phase and background. Go through each of the fluorescence channels, setting the optimal measurement plane for them.

For relocation measurements, focus on the nanoparticle aggregate in the FITC, TRITC, and cyanine five channels.