Integrating this methodology with droplet-based microfluidics helps to answer key questions related to the field of systems immunology to code cellular heterogeneity and communication between single cells or pathogens. The main advantage of this technique that we developed is, that this tip loading procedure allows the encapsulation of rare cells in droplets closely matching the predicted percent distribution. Visual demonstration of this method is critical for comprehending how to use pipette tips to load cells to gendered droplets and to retrieve cells from the droplets.
For tip loading for aqueous droplet generation, first add three milliliters of surfactant to two milliliters of flourinated oil and draw the oil phase mixture into a 2.5 milliliter syringe. Remove any air bubbles from the syringe and connect the syringe to a piece of Teflon tubing of an appropriate length. Load two sample syringes with an appropriate bi-compatible mineral oil, and connect the syringe to a second piece of Teflon tubing of an appropriate length.
Next, punch a five millimeter diameter Polydimethylsiloxane or PDMS plug from a cured EDMS slab, and punch a one millimeter hole through the center of the plug. Insert the plug tightly into top end of a 200 microliter pipette-tip and insert the tubing attached to the bi-compatible mineral oil syringe into the plug. Depress the syringe plunger slowly to fill the pipette-tip with mineral oil pushing out all the residual air.
Carefully place both sample syringes and the flourinated oil syringe, onto a syringe pump. When all of the air has been removed, lower each pipette-tip into the sample solution and aspirate about 100 microliters of cell sample into the tips. Next, insert both of the sample-containing pipette-tips into the two inner inlets of the PDMS chip and insert the tube containing the oil phase mixture into the outer inlet.
Set the flow rates on the syringe pump as indicated And enter and set the dimensions of each syringe. Start the pump to flush the sample solution through the inner channels of the device, and the oil phase through the outer channel of the device. Then plug a piece of tubing of an appropriate length into the outlet to begin collecting the droplets when the droplet formation is stable, collecting the droplets in a lock tube.
When all of the droplets have been collected, add 200 microliters of RPMI medium, without serum, on top of the droplets and incubate the sample with the hole on the lid of the lock tube for the appropriate experimental time period. For emulsion breaking, first add two milliliters of PFO to eight milliliters of flouinated oil and use a syringe to remove the excess oil from the bottom of the droplet collection tube. Then add 100 microliters of 20%PFO solution to the emulsion to release the encapsulated cells into the aqueous phase, and tap the tube briefly to mix.
Do not forget that PFO can be harmful to the cells. And therefore, keep the contact with PFO to a minimum. After one to two minutes, spin the solution at the lowest possible relative centrifugal force for 30 seconds and immediately transfer 550 microliters of the aqueous fraction into a new locked tube containing 500 microliters of cold PBS, supplemented with 2%Fetal Calf Serum, or FCS.
Let any residual oil sink to the bottom of the new lock tube and carefully transfer 950 microliters of the cell containing aqueous phase to a new lock tube. Make sure that the oil is not transferred along with the aqueous phase into the new lock tube. Then collect the cells by centrifugation and re-suspend the pellet in 300 microliters of fresh cold PBS supplemented with 2%FCS.
Using the tip loading approach as just demonstrated, Jurkat T cell encapsulation rates coincident with the statistically predicted values can be obtained. Remarkably, even with adherent cells like tumor cells, which tend to clump, or rare cells such as plasmacytoid dendritic cells, an improved encapsulation efficiency is observed. During this representative encapsulation, the droplets regenerated using ultra-low gelling temperature agarose and gelled after production to form agarose hydrogel beads that allowed subsequent downstream analysis via microscopy and flow cytometry.
Microscopic analysis revealed that cell pairing was achieved at different combinations indicative high throughput cell pairing and analysis of the same population of hydrogel beads by flow cytometry, revealed that beads without cells could be separated from the beads with cells based on their distinct forward and sideward scatter patterns. Indeed, gating on the population of beads without cells confirmed a lack of cell encapsulation by the absence of fluorescent signals while gating on the bead population with cells revealed the existence of multiple sub-populations indicative of the encapsulation of differently labeled Jurkat T cells. While working with this method, it is important to remember that cell concentration is a limiting factor, and must be optimized to insure efficient cell encapsulation and cell pairing.
Following this procedure, not only can one or two cells be encapsulated in droplets with high efficiency, but multiple cells, or cell types, can be encapsulated for a more accurate replication of cellular micro environment. This technique will pave the way for researchers in the fields of immunology and related disciplines for optimal utilization of scar cell populations and their efficient encapsulation in droplets, to study cellular behavior in a noise-free environment.
