Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS

Abstract

In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5–25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.

Droplet flow cytometry for single-cell analysis

“…[45][46][47] By selectively controlling the wettability of microchannels, microuidic devices enabled a hierarchical generation of monodisperse W/O/W DEs encapsulated with individual cells. [47][48][49][50] There are two main approaches to generate W/O/W DEs: the two-step batch method and the one-step continuous ow method using microuidic devices. 51 In the rst approach, water phase and oil phase are mixed by stirring to form W/O droplets, which can then be encapsulated by another water phase via constantly adding more water under the stirring condition.…”

“…The usage of uorinated oil (e.g., HFE-7500 and FC-40) acting as a selective barrier can reduce hydrophilic mass transfer and communication between DEs. 49,55,56 The aqueous external phase allows the suspension of DEs in nutrient medium for long-term cell culture and the compatibility with commercial cytometers for high-throughput assays 18,49,57,58 (with the sorting speed >10 7 events per hour 31,32,56 ).…”

“…Moreover, the isolated cells from heterogeneous populations allow downstream bioinformatics analysis (e.g., tandem genomic, epigenomic and transcriptomic analyses) of the same target, revealing the biological functions of massive genetic pathways. [48][49][50]…”

“…Droplet microuidics 16,17 has emerged as an incredible tool in this effort, which can compartmentalise individual cells in pico-litre microdroplets containing a highly reproducible volume of a reaction mixture. Unlike conventional water-in-oil (W/O) droplets, two main architectures of droplets suspended in aqueous solutions are compatible with FC: water-in-oil-inwater (W/O/W) 18 double emulsion (DE) droplets and hydrogel droplets. 19 The pairing of FC and droplets allows a complete characterisation of individual cells beyond the conventional surface and intracellular biomarkers, featuring high throughput, sensitivity and dynamic range, and low cost.…”

Counting of Enzymatically Amplified Affinity Reactions in Hydrogel Particle-Templated Drops

“…These cytometers are often equipped with more sensitive detectors like photomultiplier tubes that will also help further distinguish positive and negative signals. While others have analyzed double emulsions in flow cytometers 48,49 since dropicles are supported by a hydrogel particle core they may be more likely to sustain fluid shear stresses in a flow cytometer that can operate with an oil continuous phase, for example the On-Chip Sort 50 . Ultimately, we envision the hydrogel particles can be produced at Please do not adjust margins Please do not adjust margins centralized sites and conveniently distributed to enable highly sensitive immunoassays for various biomarkers.…”