Future technologies for biologics drug discovery
We introduce a new approach to collect and quantify single-cell secretions without crosstalk in monodisperse droplets formed by precisely structured microparticles, enabling high-throughput screening based on this critical cell function. The ability to analyze and sort cells based on secretions (antibodies, cytokines, proteases, or other enzymes) has implications in understanding cellular heterogeneity fundamental to biology and creating new biotechnology products, such as biologics and cell therapies. Recently, droplet microfluidics has emerged as a powerful approach to perform single-cell secretion screening in high-throughput, using compartmentalization in a small volume to accumulate secreted factors to high levels for accurate detection. Despite this utility, the necessity of specialized equipment and expertise on the end user hinders its widespread adoption. A platform that is fully compatible with standard lab equipment (e.g. pipettes, flow cytometers) has the potential to dramatically extend the reach of single-cell screening technology.
Our particle-templated droplet, i.e. “Dropicle”, approach is unique in that pre-fabricated particles are used to form monodisperse emulsions that encapsulate single cells, requiring only standard lab equipment for the end user. Cavity-containing microparticles are loaded into wellplates and due to their morphology settle upright with their cavities exposed. Cells are loaded into the microparticle cavities and adhere via integrin binding sites. Biocompatible oil and surfactant are added and the suspension is agitated by pipetting to create incrementally smaller water-in-oil droplets. These resulting dropicles are monodisperse, maintaining a size defined by the particle geometry (CV< 6%), while excess fluid is partitioned into surrounding smaller satellite droplets. Secretions from encapsulated cells are captured on the associated particles via protein A binding sites. Particles and associated cells and secretions are transferred back to aqueous phase enabling downstream labeling and screening with standard flow cytometers.
It was observed that seeded cells filled the cavities of the particles according to single-poisson statistics (in contrast to typical double-poisson statistics for single-cell, single-particle pairs in drops). After dropicle formation cells maintained high viability over 24 hours ( >80%). Initial tests with anti-IL-8 producing CHO cells demonstrate the ability to capture and label secretions on particles containing cells without crosstalk to neighboring particles. Further we demonstrate the ability to isolate cells associated with high anti-IL-8 signal in high-throughput using commercial flow cytomtery systems ( >100 sorts/s).
Using this dropicle platform researchers can perform droplet-based assays using standard lab equipment without sacrificing the precision of droplet microfluidics. Since dropicles are formed simultaneously, compartmentalization is rapid ( >400k in 30s) and can be easily scaled to accomodate large population screens. Further, the associated particle enables additional functionality such as physicochemical cues or cell specific capture antibodies to select out specific cell types. Our results demonstrate new capabilities for lab-on-a-particle technologies that can accelerate automation of single-cell assays.