Abstract
Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Here, we present a fully automated droplet-based microfluidic platform that allowed the real-time monitoring of effector-target cell interactions and killing, allowing the screening of over 60,000 droplets identifying 2000 individual cellular interactions monitored over 10 h. During the course of incubation, we observed that the dynamics of cytotoxicity within the Natural Killer (NK) cell population varies significantly over the time. Around 20% of the total NK cells in droplets showed positive cytotoxicity against paired K562 cells, most of which was exhibited within first 4 h of cellular interaction. Using our single cell analysis platform, we demonstrated that the population of NK cells is composed of individual cells with different strength in their effector functions, a behavior masked in conventional studies. Moreover, the versatility of our platform will allow the dynamic and resolved study of interactions between immune cell types and the finding and characterization of functional sub-populations, opening novel ways towards both fundamental and translational research.
Highlights
Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells
Since a well-functioning immune response is the result of combined efforts of multiple, and diverse individual cells, understanding heterogeneity within immune cell populations is crucial for a better understanding of the overall immune response and the effector functions in a cell population
Primary Natural Killer (NK) cells were obtained from buffy coats of healthy donors (Sanquin) after written informed consent according to the Declaration of Helsinki and all experimental protocols concur to institutional guidelines
Summary
Cytotoxicity is a vital effector mechanism used by immune cells to combat pathogens and cancer cells. While conventional cytotoxicity assays rely on averaged end-point measures, crucial insights on the dynamics and heterogeneity of effector and target cell interactions cannot be extracted, emphasizing the need for dynamic single-cell analysis. Several other alternative approaches have been developed, e.g. fluorescent probes to detect cell v iability[6], metabolic MTT assay[7], enzyme-based LDH assays[8], and luciferase transduced cell-based bioluminescence assays[9] Even though these assays have provided insight into fundamental cellular behaviour, these studies are performed in bulk and generate averaged r esponses[10]. Bulk-based studies fail to address the functional heterogeneity underlying a given cell population by masking the phenotype, gene expression, and the mechanism of cellular communication in between individual immune cells[11,12]. Despite the numerous attempts to overcome this challenge, a novel approach that meets the requirements is a necessity
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
