Biology subjects

Brower, K. K.

Publications and source records attributed to Brower, K. K..

4 recordsLinked to original sources

bioRxiv2022

Alleviating cell lysate-induced inhibition to enable RT-PCR from single cells in picoliter-volume double emulsion droplets

Microfluidic droplet assays enable single-cell PCR and sequencing assays at unprecedented scale, with most methods encapsulating cells within nanoliter sized single emulsion droplets (water-in-oil). Encapsulating cells within picoliter double emulsion (DE) (water-in-oil-in-water) droplets allows sorting on commercially available FACS machines, making it possible to isolate single cells based on phenotypes of interest for downstream sequencing and PCR analyses. However, DE droplets must be within the range of large cells (<20 pl) for sorting on standard cytometers, posing challenges for molecular biology as prior reports suggest that reverse transcription (RT) and PCR amplification cannot proceed efficiently at volumes below 1 nL due to cell lysate-induced inhibition. To overcome this limitation, we used a plate-based RT-PCR assay designed to mimic reactions in picoliter droplets to systematically quantify and ameliorate the inhibition. We find that RT-PCR is blocked by lysate-induced cleavage of nucleic acid probes and primers, but that this cleavage can be efficiently alleviated through heat lysis that simultaneously inactivates inhibitory lysate components. We further show that the magnitude of RT-PCR inhibition depends strongly on cell type, but that RT-PCR can proceed in low-picoscale reaction volumes for most cell lines tested. Finally, we demonstrate one-step RT-PCR from single cells in 20 pL double emulsion droplets with fluorescence detectable via FACS. These results open up exciting new avenues for improving picoscale droplet RT-PCR reactions and expanding microfluidic droplet based single-cell analysis technologies.

bioengineering↗

bioRxiv2022

Systematic Characterization of Double Emulsion Droplets for Biological Applications

Double emulsion droplets (DEs) are water/oil/water droplets that can be sorted via Fluorescence-Activated Cell Sorting (FACS), allowing for new opportunities in high-throughput cellular analysis, enzymatic screening, and synthetic biology. These applications require stable, uniform droplets with predictable microreactor volumes. However, predicting DE droplet size, shell thickness, and stability as a function of flow rate has remained challenging for monodisperse single core droplets and those containing biologically-relevant buffers, which influence bulk and interfacial properties. As a result, developing novel DE-based bioassays has typically required extensive initial optimization of flow rates to find conditions that produce stable droplets of the desired size and shell thickness. To address this challenge, we conducted systematic size parameterization quantifying how differences in flow rates and buffer properties (viscosity and interfacial tension at water/oil interfaces) alter droplet size and stability, across 6 inner aqueous buffers used across applications such as cellular lysis, microbial growth, and drug delivery, quantifying the size and shell thickness of >22,000 droplets overall. We restricted our study to stable single core droplets generated in a 2-step dripping-dripping formation regime in a straightforward PDMS device. Using data from 138 unique conditions (flow rates and buffer composition), we also demonstrated that a recent physically-derived size law of Wang et al1 can accurately predict double emulsion shell thickness for >95% of observations. Finally, we validated the utility of this size law by using it to accurately predict droplet sizes for a novel bioassay that requires encapsulating growth media for bacteria in droplets. This work has the potential to enable new screening-based biological applications by simplifying novel DE bioassay development.

bioengineering↗

bioRxiv2020

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

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 phenotyping via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying size and morphology as well as a heterogeneous cell mixture of a whole dissociated flatworm (5 - 25 m in diameter) within highly monodisperse double emulsions (35 m 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 phenotyping 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 screening. ABSTRACT FIGURE O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/139311v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@9d2028org.highwire.dtl.DTLVardef@1aa55b3org.highwire.dtl.DTLVardef@1e05eb3org.highwire.dtl.DTLVardef@1695a88_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Optimized double emulsion flow cytometry with high-throughput single droplet isolation

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 m nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two industry-standard FACS instruments. We report the first quantitative plate statistics for double emulsion droplet isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, an advance in droplet sorting comparable with the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics.

bioengineering↗