Author Correction: High throughput single cell counting in droplet-based microfluidics.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-throughput multiplexed fluorescence-activated droplet sorting.

Fluorescence-activated droplet sorting (FADS) is one of the most important features provided by droplet-based microfluidics. However, to date, it does not allow to compete with the high-throughput multiplexed sorting capabilities offered by flow cytometery. Here, we demonstrate the use of a dielectrophoretic-based FADS, allowing to sort up to five different droplet populations simultaneously. Our system provides means to select droplets of different phenotypes in a single experimental run to separate initially heterogeneous populations. Our experimental results are rationalized with the help of a numerical model of the actuation of droplets in electric fields providing guidelines for the prediction of sorting designs for upscaled or downscaled microsystems.

Microfluidics as a Strategic Player to Decipher Single-Cell Omics?

Most cell studies are performed at a population level, relying on the assumption of a normal distribution of the function and fate of a cell among a population. However, technologies allowing single-cell analysis (SCA) have recently arisen and have led to increasing evidence of cell population heterogeneity and its importance. Tremendous amounts of new data could now be uncovered to redefine our understanding of cell omics. Microfluidics has emerged as a major technological player in this new era and is gradually increasing in use among biology laboratories, mainly due to the single-cell high-throughput handling solutions it offers. In this review, we assess its use and relevance for omics analysis at the single-cell level, with a specific focus on compartment-based microfluidic approaches.

High throughput single cell counting in droplet-based microfluidics.

Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

Multiplex Detection of Rare Mutations by Picoliter Droplet Based Digital PCR: Sensitivity and Specificity Considerations.

In cancer research, the accuracy of the technology used for biomarkers detection is remarkably important. In this context, digital PCR represents a highly sensitive and reproducible method that could serve as an appropriate tool for tumor mutational status analysis. In particular, droplet-based digital PCR approaches have been developed for detection of tumor-specific mutated alleles within plasmatic circulating DNA. Such an approach calls for the development and validation of a very significant quantity of assays, which can be extremely costly and time consuming. Herein, we evaluated assays for the detection and quantification of various mutations occurring in three genes often misregulated in cancers: the epidermal growth factor receptor (EGFR), the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and the Tumoral Protein p53 (TP53) genes. In particular, commercial competitive allele-specific TaqMan® PCR (castPCR™) technology, as well as TaqMan® and ZEN™ assays, have been evaluated for EGFR p.L858R, p.T790M, p.L861Q point mutations and in-frame deletions Del19. Specificity and sensitivity have been determined on cell lines DNA, plasmatic circulating DNA of lung cancer patients or Horizon Diagnostics Reference Standards. To show the multiplexing capabilities of this technology, several multiplex panels for EGFR (several three- and four-plexes) have been developed, offering new "ready-to-use" tests for lung cancer patients.

Parallelized ultra-high throughput microfluidic emulsifier for multiplex kinetic assays.

Droplet-based microfluidic technologies are powerful tools for applications requiring high-throughput, for example, in biochemistry or material sciences. Several systems have been proposed for the high-throughput production of monodisperse emulsions by parallelizing multiple droplet makers. However, these systems have two main limitations: (1) they allow the use of only a single disperse phase; (2) they are based on multiple layer microfabrication techniques. We present here a pipette-and-play solution offering the possibility of manipulating simultaneously 10 different disperse phases on a single layer device. This system allows high-throughput emulsion production using aqueous flow rates of up to 26 ml/h (>110 000 drops/s) leading to emulsions with user-defined complex chemical composition. We demonstrate the multiplex capabilities of our system by measuring the kinetics of ß-galactosidase in droplets using nine different concentrations of a fluorogenic substrate.

[Digital PCR compartmentalization II. Contribution for the quantitative detection of circulating tumor DNA]. / PCR digitale en micro-compartiments - II. Apport pour la détection quantitative d'ADN tumoral circulant.

Genetic markers are now widely used in the clinics, particularly in cancer patient management. Indeed, these tumor markers can help in the diagnosis and prognosis of the disease, and provide valuable information for treatment orientation in the context of personalized medicine. The presence of circulating cell-free tumor DNA (cftDNA) and thus of tumor markers in the blood can be considered to partly avoid the use of solid biopsies. The use of blood samples, as liquid biopsies, is less invasive and described as more representative of tumor heterogeneity. However, cftDNA can be found in blood in low proportion that can vary according to the nature and the progression of the tumor. For these reasons, the use of highly sensitive, specific and ideally quantitative methods for its detection are required. These requirements constituted until recently a technological limit, which now can be overcome thanks to digital PCR. This technology could now become a very efficient and non-invasive tool in oncology, complementary to conventional diagnostic techniques.

Mechanical control of cell flow in multicellular spheroids.

Collective cell motion is observed in a wide range of biological processes. In tumors, physiological gradients of nutrients, growth factors, or even oxygen give rise to gradients of proliferation. We show using fluorescently labeled particles that these gradients drive a velocity field resulting in a cellular flow in multicellular spheroids. Under mechanical stress, the cellular flow is drastically reduced. We describe the results with a hydrodynamic model that considers only convection of the particles by the cellular flow.