Microfluidics in Single-Cell Virology: Technologies and Applications

Abstract

Microfluidics has proven to be a powerful tool for probing biology at the single-cell level. However, it is only in the past 5 years that single-cell microfluidics has been used in the field of virology. An array of strategies based on microwells, microvalves, and droplets is now available for tracking viral infection dynamics, identifying cell subpopulations with particular phenotypes, as well as high-throughput screening. The insights into the virus-host interactions gained at the single-cell level are unprecedented and usually inaccessible by population-based experiments. Therefore, single-cell microfluidics, which opens new avenues for mechanism elucidation and development of antiviral therapeutics, would be a valuable tool for the study of viral pathogenesis.

Keywords: heterogeneity; microfluidics; single-cell analysis; transcriptomic analysis; viral infection dynamics; virology.

Figure 1, Key Figure.

Single-cell microfluidics technologies and their typical applications in virology. Microwell-, valve-, and droplet-based strategies are applied in virology studies. Features and typical application opportunities of these methods are presented.

Figure 2.

Microfluidic platforms for kinetic analysis of viral infection dynamics in single cells. (A) One of ten bull’s-eyes on a PDMS microwell device, each containing 2,500 sub-nanoliter volume wells. VSV infected PC3 cells were loaded into the microwell array. They were engineered so that RFP and GFP signals indicated the production of infectious virus particles and the activation of cellular innate immunity, respectively. A model was used to process the fluorescent trajectories from each single cell and derive kinetic parameters. Delay-time: the first data point above the limit of detection (LOD); maximum signal: the highest intensity reached; rise-time: the period between the delay-time and the time when 85% of the maximum signal was reached; production-rate: speed of the production, obtained by fitting the first four data-points above the LOD with an exponential curve. Adapted with permission from [34]. (B) A device composed of 4 groups of 1,600 microchambers for single-cell cultivation, which could be sealed with pneumatic valves. A complete time course of infection by a virus expressing a fluorescent reporter could be recorded. Adapted with permission from [35]. (C) Single-cell analysis of viral infection dynamics could inform mechanism of actions of antiviral drugs [39].

Figure 3.

Transcriptome analysis of virus-host interactions at the single-cell level. (A) ScRNA-seq reveals distinct cell subpopulations. Combined with phenotypical analysis, single-cell transcriptome analysis could be used to investigate viral diversity and identify cell subpopulations with specific phenotypes. (B-D) Characteristics of main scRNA-seq technologies (Fluidigm C1, Drop-seq and 10X) applied in the field of virology.

Figure 4.

An integrated valve-based microfluidic system for multi-parameter measurements. A device containing 144 assay chambers and the schematic of one unit are shown. Inset shows a single cell is trapped in chamber II. By switching the valves, on-chip cell loading, lysis, proximity ligation and product retrieval could be accomplished stepwisely. Droplet digital PCR (ddPCR) was then used for quantification of proteins or mRNA. Adapted from [75], under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).