Droplet-based Cytotoxicity Assay to Assess Chimeric Antigen Receptor T cells at the Single-cell Level

Our research focuses on assessing the functional diversity of CAR T-cells by encapsulating single effector CAR T-cells with target cells to better understand their cytotoxic potential at the single cell level. Current cytotoxicity assays are limited because they're often performed in bulk. This overlooks differences between individual CAR T-cells and fails to identify distinct subpopulations with varying responses to targets.

Our technology enables multiplex cell killing assays in a single cell format using standard flow cytometers and cell orders. This gives more precise results than bulk protocols, since there's no interference from neighboring cells. We hope that this type of research will allow us and other scientists to better examine the finer details of our CARs and how they function at the single cell level.

To begin, dilute stock solutions of two fluorescent dyes in a 1:5, 000 ratio in Dulbecco's PBS. Transfer the cells into separate 15 milliliter tubes. Centrifuge the tubes at 300g for five minutes.

Remove the supernatant and resuspend the effector cell pellets in the working solution of the violet fluorescent dye. Resuspend the target cell pellets in the working solution of the far red fluorescent dye. Incubate the stained cells for 20 minutes in a 37 degrees Celsius humidified carbon dioxide incubator.

After incubation, centrifuge the cells at 300g for five minutes to pellet them. Resuspend the cell pellets in 15 milliliters of complete RPMI 1640 medium to wash them. Wash and centrifuge again.

Then remove the supernatant and resuspend the effector cell pellets in 225 microliters of complete RPMI 1640 medium and the target cell pellets in 450 microliters of the same medium. Add 30 microliters of a gradient medium to the effector cell suspensions and 60 microliters to the target cell suspensions. Mix the gradient medium stock solution well before pipetting.

Next, pre dilute the Granzyme B substrate stock with complete RPMI 1640 medium. Pipette peridium iodide stock solution and diluted Granzyme B substrate to the effector cell suspensions and target cell suspensions. Then pipette to mix the solutions well.

To encapsulate the cells, preheat the encapsulation cartridge and stabilizing solution to room temperature. Add complete RPMI 1640 medium 33%stabilizing solution for cells and 10%gradient medium. And mix them to prepare a stock of outer medium.

For each encapsulation sample, mix 65 microliters of prepared effector cell suspension with 65 microliters of prepared target cell suspension in a 1.5 milliliter tube. Resuspend the cells well with a pipette. Load the indicated wells of the encapsulation cartridge with reagents in the given order.

Reese suspends cells well with a pipette right before loading. Seal the gasket carefully onto the cartridge. Transfer the cartridge to the microfluidics device, then start encapsulation as per the user manual.

After the run is complete, remove the cartridge and harvest the droplets from well D by resuspending them in the overlaying medium and transferring to a two milliliter DNA low binding tube with a lid. Wash well D with the remaining outer medium from well A to collect the remaining droplets. When the droplets have sedimented in the collection tubes, confirm two clearly distinct phases in the tubes before microscopic examination.

Fill approximately one third of a 10 microliter pipette tip with droplets from the surface of the droplet layer. Then fill up the remaining approximately two thirds of the pipette tip with the overlaying media. Immediately load the sample onto an eight chamber glass slide.

Examine the droplets microscopically at 4x and 20x magnification to confirm droplet loading with cells. For incubation, use a 21 gauge syringe needle to carefully puncture the lid of the required number of two milliliter DNA low binding tubes. Add one milliliter of outer medium to each incubation tube.

Resuspend the droplets generated in the overlaying media and split each production into three prepared incubation tubes. Place the tubes upright in the incubator for two, four or six hours of incubation after droplet generation. After incubation, transfer a small number of droplets to a microscope slide.

Analyze the droplets by bright-field and fluorescence microscopy using a standard fluorescence microscope with appropriate laser and filter configuration. Resuspend each droplet sample in the overlaying medium. Transfer the resuspended samples to five milliliter FAC tubes.

Analyze the droplets with a standard flow cytometer. Record intensity height signals for forward scatter, side scatter and the examined fluorophores. Wash the flow cytometer through the sample injection port After each set of droplet acquisitions using standard clean and rinse solutions.

CAR T-cells were successfully enriched to a purity of over 98%for both donors using anti NGFR magnetic beads and their CD4-CD8 ratio was quantified at 0.73 with a naive like memory phenotype observed. Non transduced T cells were used as controls in the experiment. Fluorescence microscopy after four hours of incubation demonstrated Granzyme B secretion in droplets with JeKo-1 cells coencapsulated with CAR T-cells whereas no secretion was detected with non-transduced T-cells.

Flow cytometry identified distinct droplet populations based on cell types encapsulated, confirming that pronounced Granzyme B secretion and cytotoxic activity occurred in droplets containing CAR T-cells and JeKo-1 target cells. Granzyme B secretion and target cell killing by CAR T-cells exhibited a time dependent increase with more than 30%CAR T-cells secreting Granzyme B after six hours and more than 20%of CAR T-cells killing target cells as indicated by propidium iodide positivity.