Droplet‐Based Combinatorial Assay for Cell Cytotoxicity and Cytokine Release Evaluation

Abstract

Balancing the maximal efficacy of interferon gamma (IFN-ɣ)-based therapies with its side effects is a great challenge for future cytokine treatments. To achieve this, the development of single-cell technologies that study IFN-ɣ release in correlation with antitumor activity would represent a huge step forward. To this end, droplet-based microfluidics is employed to quantitatively investigate IFN-ɣ secretion from single natural killer (NK) cells in correlation with their cytotoxic activity against a specific target. The method relies on co-encapsulation of NK-92 cells, target cancer cells, polystyrene beads conjugated with specific IFN-ɣ capture antibodies, and fluorescently labeled detection antibodies inside water-in-oil compartments. The secreted cytokines are captured and detected by localized fluorescence at the periphery of the beads. NK-92's cytotoxicity is evaluated simultaneously by means of a fluorescent DNA intercalating agent, which penetrates the membranes of dead target cells. To deepen the understanding of the role of the cytokine in antitumor immunomodulation, the impact of different doses of human recombinant IFN-ɣ on the cytolytic activity of NK-92 cells shows a trend that the higher the dose the lower the cytolytic activity of NK cells. The developed method represents a simple quantitative approach to unravel the complex heterogeneity of NK cells toward IFN-ɣ secretion and cytolytic activity.

Single cell profiling reveals functional heterogeneity and serial killing in human peripheral and ex vivo-generated CD34+ progenitor derived Natural Killer cells

“…To this end we identified 74% of total lytic events in droplets that showed positive secretory function as well. In this way we demonstrated a correlation between cytotoxic and secretory function of NK cells, which others were not able to find(Antona et al, 2020b; Yamanaka et al, 2012b). We believe that this discrepancy is explained by a short experimental protocol of 4 hours leading to a missed cytolytic fraction that also test positive for IFNγ.…”

“…To the best of our knowledge, we report for the first time on a droplet-based platform that allows monitoring both functions in single primary NK cells simultaneously in real time. A similar platform was developed by Antona et al that also studied these functions in droplets, however their platform was designed for end-point based analysis and thus cannot address the temporal dynamics of cellular interactions(Antona et al, 2020b). We adapted the “In-drop sandwich immunoassay”, as described by Eyer.et al(Bounab et al, 2020; Eyer et al, 2017) to allow the combinatorial investigation of IFNγ release and NK cell cytotoxicity in a time dependent manner.…”

Cell Culture in Microfluidic Droplets

“…In all these cases, chemical species (nutrient, stimulus) must be transported to the cells. In other experiments, proteins secreted by encapsulated cells are captured on a sensor; this is for the example case when detecting the secretions of individual cells or of a spheroid coencapsulated in a microfluidic droplet with a bead functionalized for protein capture . Knowing the time scale for transport to and from the cell is essential for the efficient design of microfluidic experiments.…”

“…In the study of cellular interactions, two different cell types may need to be coencapsulated in droplets . The number of cells of each type per droplet then follows a Poisson distribution.…”

“…This has been achieved by coencapsulating the cells and a capture antibody immobilized on beads in droplets. Specific soluble proteins were then captured on the beads and were quantified by imaging through the addition of a fluorescently labeled secondary antibody ,,,, (Figure a,b). Alternatively, cancer cells have been coencapsulated with FRET-based cleavable peptide substrate to measure their capacity for ECM proteolysis. , Such measurements have shed light on the heterogeneity of protein secretion at the cell and at spheroid level (e.g., VEGF secreted by spheroids of MSCs), which would not have been possible using bulk characterization methods.…”