iSort enables automated complex microfluidic droplet sorting in an effort to democratize technology.

Fluorescence-activated droplet sorting (FADS) is a widely used microfluidic technique for high-throughput screening. However, it requires highly trained specialists to determine optimal sorting parameters, and this results in a large combinatorial space that is challenging to optimize systematically. Additionally, it is currently challenging to track every single droplet within a screen, leading to compromised sorting and "hidden" false-positive events. To overcome these limitations, we have developed a setup in which the droplet frequency, spacing, and trajectory at the sorting junction are monitored in real time using impedance analysis. The resulting data are used to continuously optimize all parameters automatically and to counteract perturbations, resulting in higher throughput, higher reproducibility, increased robustness, and a beginner-friendly character. We believe this provides a missing piece for the spreading of phenotypic single-cell analysis methods, similar to what we have seen for single-cell genomics platforms.

A microfluidic Braille valve platform for on-demand production, combinatorial screening and sorting of chemically distinct droplets.

Droplet microfluidics is a powerful tool for a variety of biological applications including single-cell genetics, antibody discovery and directed evolution. All these applications make use of genetic libraries, illustrating the difficulty of generating chemically distinct droplets for screening applications. This protocol describes our Braille Display valving platform for on-demand generation of droplets with different chemical contents (16 different reagents and combinations thereof), as well as sorting droplets with different chemical properties, on the basis of fluorescence signals. The Braille Display platform is compact, versatile and cost efficient (only ~US$1,000 on top of a standard droplet microfluidics setup). The procedure includes manufacturing of microfluidic chips, assembly of custom hardware, co-encapsulation of cells and drugs into droplets, fluorescence detection of readout signals and data analysis using shared, freely available LabVIEW and Python packages. As a first application, we demonstrate the complete workflow for screening cancer cell drug sensitivities toward 74 conditions. Furthermore, we describe here an assay enabling the normalization of the observed drug sensitivity to the number of cancer cells per droplet, which additionally increases the robustness of the system. As a second application, we also demonstrate the sorting of droplets according to enzymatic activity. The drug screening application can be completed within 2 d; droplet sorting takes ~1 d; and all preparatory steps for manufacturing molds, chips and setting up the Braille controller can be accomplished within 1 week.

Microfluidics as an Enabling Technology for Personalized Cancer Therapy.

Tailoring patient-specific treatments for cancer is necessary in order to achieve optimal results but requires new diagnostic approaches at affordable prices. Microfluidics has immense potential to provide solutions for this, as it enables the processing of samples that are not available in large quantities (e.g., cells from patient biopsies), is cost efficient, provides a high level of automation, and allows the set-up of complex models for cancer studies. In this review, individual solutions in the fields of genetics, circulating tumor cell monitoring, biomarker analysis, phenotypic drug sensitivity tests, and systems providing controlled environments for disease modeling are discussed. An overview on how these early stage achievements can be combined or developed further is showcased, and the required translational steps before microfluidics becomes a routine tool for clinical applications are critically discussed.

A microfluidics platform for combinatorial drug screening on cancer biopsies.

Screening drugs on patient biopsies from solid tumours has immense potential, but is challenging due to the small amount of available material. To address this, we present here a plug-based microfluidics platform for functional screening of drug combinations. Integrated Braille valves allow changing the plug composition on demand and enable collecting >1200 data points (56 different conditions with at least 20 replicates each) per biopsy. After deriving and validating efficient and specific drug combinations for two genetically different pancreatic cancer cell lines and xenograft mouse models, we additionally screen live cells from human solid tumours with no need for ex vivo culturing steps, and obtain highly specific sensitivity profiles. The entire workflow can be completed within 48 h at assay costs of less than US$ 150 per patient. We believe this can pave the way for rapid determination of optimal personalized cancer therapies.

A Versatile, Low-Cost, Multiway Microfluidic Sorter for Droplets, Cells, and Embryos.

Partitioning and sorting particles, including molecules, cells and organisms, is an essential prerequisite for a diverse range of applications. Here, we describe a very economical microfluidic platform (built from parts costing about U.S. $6800 for a stand-alone system or U.S. $3700, when mounted on an existing fluorescence microscope connected to a computer) to sort droplets, cells and embryos, based on imaging data. Valves operated by a Braille display are used to open and close microfluidic channels, enabling sorting at rates of >2 Hz. Furthermore, we show microfluidic 8-way sorting for the first time, facilitating the simultaneous separation and collection of objects with diverse characteristics/phenotypes. Due to the high flexibility in the size of objects that can be sorted, the low cost, and the many possibilities enabled by imaging technology, we believe that our approach nicely complements existing FACS and µFACS technology.

Droplet-based microfluidics in drug discovery, transcriptomics and high-throughput molecular genetics.

Droplet-based microfluidics enables assays to be carried out at very high throughput (up to thousands of samples per second) and enables researchers to work with very limited material, such as primary cells, patient's biopsies or expensive reagents. An additional strength of the technology is the possibility to perform large-scale genotypic or phenotypic screens at the single-cell level. Here we critically review the latest developments in antibody screening, drug discovery and highly multiplexed genomic applications such as targeted genetic workflows, single-cell RNAseq and single-cell ChIPseq. Starting with a comprehensive introduction for non-experts, we pinpoint current limitations, analyze how they might be overcome and give an outlook on exciting future applications.

Functional single-cell hybridoma screening using droplet-based microfluidics.

Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1:10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.