Efficient full-length IgG secretion and sorting from single yeast clones in droplet picoreactors.

The search for new antibodies is a major field of pharmaceutical research that remains lengthy and costly due to the need for successive library screenings. Existing in vitro and in vivo antibody discovery processes require that libraries are repeatedly subcloned to switch the antibody format or the secretory host, a resource-intensive process. There is an urgent need for an antibody identification platform capable of screening large antibody libraries in their final soluble format. Previous attempts to develop such a platform have struggled to combine large antibody libraries with screening of high specificity, while retaining sufficient library diversity coverage (ability to detect rare events). Here, we describe a new antibody screening platform based on the encapsulation of antibody secreting yeast cells into picoreactor droplets. We developed and optimized a Yarrowia lipolytica yeast strain capable of growing and secreting full-length human IgGs in picoreactors, and applied a microfluidics-based high-throughput screening approach to sort and recover target-specific antibody-secreting yeasts. Critically, the direct recovery of secretory yeasts allows for downstream screening and antibody characterization, without the need to reformat or subclone the coding sequences. We successfully increased the diversity coverage of sorting the antibody library without compromising sorting specificity by developing a new fluorescence signal processing methodology. By combining this drastically enhanced sorting efficiency with the high-throughput capability of droplet microfluidics, and the rapid growth of Y. lipolytica, our new platform is capable of screening millions of antibodies per day and enriching for target-specific ones in 4 days. This platform will enable the efficient screening of antibody libraries in a variety of contexts, including primary screening of synthetic libraries, affinity maturation, and identification of multi-specific or cross-reactive antibodies.

DNA-Encoded Immunoassay in Picoliter Drops: A Minimal Cell-Free Approach.

Immunoassays have emerged as indispensable bioanalytical tools but necessitate long preliminary steps for the selection, production, and purification of the antibody(ies) to be used. Here is explored the paradigm shift of creating a rapid and purification-free assay in picoliter drops where the antibody is expressed from coding DNA and its binding to antigens concomitantly characterized in situ. Efficient synthesis in bulk of various functional variable domains of heavy-chain only antibodies (VHH) using reconstituted cell-free expression media, including an anti-green fluorescent protein VHH, is shown first. A microfluidic device is then used to generate monodisperse drops (30 pL) containing all the assay components, including a capture scaffold, onto which the accumulation of VHH:antigen produces a specific fluorescent signal. This allows to assess, in parallel or sequentially at high throughput (500 Hz), the VHH-antigen binding and its specificity in less than 3 h, directly from a VHH-coding DNA, for multiple VHH sequences, various antigens and down to DNA concentrations as low as 12 plasmids per drop. It is anticipated that the ultraminiaturized format, robustness, and programmability of this novel cell-free immunoassay concept will constitute valuable assets in fields as diverse as antibody discovery, point-of-care diagnostics, synthetic biology, and/or bioanalytical assays.

Bidirectional hybrid erythritol-inducible promoter for synthetic biology in Yarrowia lipolytica.

BACKGROUND: The oleaginous yeast Yarrowia lipolytica is increasingly used as a chassis strain for generating bioproducts. Several hybrid promoters with different strengths have been developed by combining multiple copies of an upstream activating sequence (UAS) associated with a TATA box and a core promoter. These promoters display either constitutive, phase-dependent, or inducible strong expression. However, there remains a lack of bidirectional inducible promoters for co-expressing genes in Y. lipolytica. RESULTS: This study built on our previous work isolating and characterizing the UAS of the erythritol-induced genes EYK1 and EYD1 (UAS-eyk1). We found an erythritol-inducible bidirectional promoter (BDP) located in the EYK1-EYL1 intergenic region. We used the BDP to co-produce YFP and RedStarII fluorescent proteins and demonstrated that the promoter's strength was 2.7 to 3.5-fold stronger in the EYL1 orientation compared to the EYK1 orientation. We developed a hybrid erythritol-inducible bidirectional promoter (HBDP) containing five copies of UAS-eyk1 in both orientations. It led to expression levels 8.6 to 19.2-fold higher than the native bidirectional promoter. While the BDP had a twofold-lower expression level than the strong constitutive TEF promoter, the HBDP had a 5.0-fold higher expression level when oriented toward EYL1 and a 2.4-fold higher expression level when oriented toward EYK1. We identified the optimal media for BDP usage by exploring yeast growth under microbioreactor conditions. Additionally, we constructed novel Golden Gate biobricks and a destination vector for general use. CONCLUSIONS: In this research, we developed novel bidirectional and hybrid bidirectional promoters of which expression can be fine-tuned, responding to the need for versatile promoters in the yeast Y. lipolytica. This study provides effective tools that can be employed to smoothly adjust the erythritol-inducible co-expression of two target genes in biotechnology applications. BDPs developed in this study have potential applications in the fields of heterologous protein production, metabolic engineering, and synthetic biology.

High-affinity autoreactive plasma cells disseminate through multiple organs in patients with immune thrombocytopenic purpura.

The major therapeutic goal for immune thrombocytopenic purpura (ITP) is to restore normal platelet counts using drugs to promote platelet production or by interfering with mechanisms responsible for platelet destruction. Eighty percent of patients with ITP possess anti-integrin αIIbß3 IgG autoantibodies that cause platelet opsonization and phagocytosis. The spleen is considered the primary site of autoantibody production by autoreactive B cells and platelet destruction. The immediate failure in approximately 50% of patients to recover a normal platelet count after anti-CD20 rituximab-mediated B cell depletion and splenectomy suggests that autoreactive, rituximab-resistant, IgG-secreting B cells (IgG-SCs) reside in other anatomical compartments. We analyzed more than 3,300 single IgG-SCs from spleen, bone marrow, and/or blood of 27 patients with ITP, revealing high interindividual variability in affinity for αIIbß3, with variations over 3 logs. IgG-SC dissemination and range of affinities were, however, similar for each patient. Longitudinal analysis of autoreactive IgG-SCs upon treatment with the anti-CD38 mAb daratumumab demonstrated variable outcomes, from complete remission to failure with persistence of high-affinity anti-αIIbß3 IgG-SCs in the bone marrow. This study demonstrates the existence and dissemination of high-affinity autoreactive plasma cells in multiple anatomical compartments of patients with ITP that may cause the failure of current therapies.

Author Correction: High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

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

High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.

Mining the antibody repertoire of plasma cells and plasmablasts could enable the discovery of useful antibodies for therapeutic or research purposes1. We present a method for high-throughput, single-cell screening of IgG-secreting primary cells to characterize antibody binding to soluble and membrane-bound antigens. CelliGO is a droplet microfluidics system that combines high-throughput screening for IgG activity, using fluorescence-based in-droplet single-cell bioassays2, with sequencing of paired antibody V genes, using in-droplet single-cell barcoded reverse transcription. We analyzed IgG repertoire diversity, clonal expansion and somatic hypermutation in cells from mice immunized with a vaccine target, a multifunctional enzyme or a membrane-bound cancer target. Immunization with these antigens yielded 100-1,000 IgG sequences per mouse. We generated 77 recombinant antibodies from the identified sequences and found that 93% recognized the soluble antigen and 14% the membrane antigen. The platform also allowed recovery of ~450-900 IgG sequences from ~2,200 IgG-secreting activated human memory B cells, activated ex vivo, demonstrating its versatility.

Single-cell deep phenotyping of IgG-secreting cells for high-resolution immune monitoring.

Studies of the dynamics of the antibody-mediated immune response have been hampered by the absence of quantitative, high-throughput systems to analyze individual antibody-secreting cells. Here we describe a simple microfluidic system, DropMap, in which single cells are compartmentalized in tens of thousands of 40-pL droplets and analyzed in two-dimensional droplet arrays using a fluorescence relocation-based immunoassay. Using DropMap, we characterized antibody-secreting cells in mice immunized with tetanus toxoid (TT) over a 7-week protocol, simultaneously analyzing the secretion rate and affinity of IgG from over 0.5 million individual cells enriched from spleen and bone marrow. Immunization resulted in dramatic increases in the range of both single-cell secretion rates and affinities, which spanned at maximum 3 and 4 logs, respectively. We observed differences over time in dynamics of secretion rate and affinity within and between anatomical compartments. This system will not only enable immune monitoring and optimization of immunization and vaccination protocols but also potentiate antibody screening.

Chromatin immunoprecipitation in microfluidic droplets: towards fast and cheap analyses.

Genetic organization is governed by the interaction of DNA with histone proteins, and differential modifications of these proteins is a fundamental mechanism of gene regulation. Histone modifications are primarily studied through chromatin immunoprecipitation (ChIP) assays, however conventional ChIP procedures are time consuming, laborious and require a large number of cells. Here we report for the first time the development of ChIP in droplets based on a microfluidic platform combining nanoliter droplets, magnetic beads (MB) and magnetic tweezers (MT). The droplet approach enabled compartmentalization and improved mixing, while reducing the consumption of samples and reagents in an integrated workflow. Anti-histone antibodies grafted to MB were used as a solid support to capture and transfer the target chromatin from droplets to droplets in order to perform chromatin immunoprecipitation, washing, elution and purification of DNA. We designed a new ChIP protocol to investigate four different types of modified histones with known roles in gene activation or repression. We evaluated the performances of this new ChIP in droplet assay in comparison with conventional methods. The proposed technology dramatically reduces analytical time from a few days to 7 hours, simplifies the ChIP protocol and decreases the number of cells required by 100 fold while maintaining a high degree of sensitivity and specificity. Therefore this droplet-based ChIP assay represents a new, highly advantageous and convenient approach to epigenetic analyses.

FISH-in-CHIPS: A Microfluidic Platform for Molecular Typing of Cancer Cells.

Microfluidics offer powerful tools for the control, manipulation, and analysis of cells, in particular for the assessment of cell malignancy or the study of cell subpopulations. However, implementing complex biological protocols on chip remains a challenge. Sample preparation is often performed off chip using multiple manually performed steps, and protocols usually include different dehydration and drying steps that are not always compatible with a microfluidic format.Here, we report the implementation of a Fluorescence in situ Hybridization (FISH) protocol for the molecular typing of cancer cells in a simple and low-cost device. The geometry of the chip allows integrating the sample preparation steps to efficiently assess the genomic content of individual cells using a minute amount of sample. The FISH protocol can be fully automated, thus enabling its use in routine clinical practice.

High throughput micropatterning of interspersed cell arrays using capillary assembly.

A novel technology is reported to immobilize different types of particles or cells on a surface at predefined positions with a micrometric precision. The process uses capillary assembly on arrays of crescent-shaped structures with different orientations. Sequential assemblies in different substrate orientations with different types of particles allow for the creation of imbricated and multiplexed arrays. In this work up to four different types of particles were deterministically localized on a surface. Using this process, antibody coated microparticles were assembled on substrates and used as capture patterns for the creation of complex cell networks. This new technology may have numerous applications in biology, e.g. for fast cell imaging, cell-cell interactions studies, or construction of cell arrays.

FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool.

Microfluidic systems bear promise to provide new powerful tools for the molecular characterization of cancer cells, in particular for the routine detection of multiple cancer biomarkers using a minute amount of the sample. However, taking miniaturized cell-based assays into the clinics requires the implementation and validation of complex biological protocols on chip, as well as the development of disposable microdevices produced at a low cost. Based on a recently developed microfluidic chip made of Cyclic Olefin Copolymer for cell immobilization with minimal dead volume and controlled shear stress, we developed a protocol performed entirely in the liquid phase, allowing the immobilization and fixation of cells and their quantitative characterization by fluorescence in situ hybridization. We demonstrated first in cell lines and then in two clinical case studies the potential of this method to perform quantitative copy number measurement and clinical scoring of the amplification of the ERBB2 gene, a decisive biomarker for the prescription of HER2+ related targeted therapies. This validation was performed in a blind protocol in two clinical case studies, in reference to the gold standard and clinically used method based on glass slides. We obtained a comparable reproducibility and a minor difference in apparent amplification, which can be corrected by internal calibration. The method thus reaches the standard of robustness needed for clinical use. The protocol can be fully automated, and its consumption of samples and DNA probes is reduced as compared to glass slide protocols by a factor of at least 10. The total duration of the assay is divided by two.

A three dimensional thermoplastic microfluidic chip for robust cell capture and high resolution imaging.

We present a low cost microfluidic chip integrating 3D micro-chambers for the capture and the analysis of cells. This device has a simple design and a small footprint. It allows the implementation of standard biological protocols in a chip format with low volume consumption. The manufacturing process relies on hot-embossing of cyclo olefin copolymer, allowing the development of a low cost and robust device. A 3D design of microchannels was used to induce high flow velocity contrasts in the device and provide a selective immobilization. In narrow distribution channels, the liquid velocity induces a shear stress that overcomes adhesion forces and prevents cell immobilization or clogging. In large 3D chambers, the liquid velocity drops down below the threshold for cell attachment. The devices can be operated in a large range of input pressures and can even be handled manually using simple syringe or micropipette. Even at high flow injection rates, the 3D structures protect the captured cell from shear stress. To validate the performances of our device, we implemented immuno-fluorescence labeling and Fluorescence in Situ Hybridization (FISH) analysis on cancer cell lines and on a patient pleural effusion sample. FISH is a Food and Drug Administration approved cancer diagnostic technique that provides quantitative information about gene and chromosome aberration at the single cell level. It is usually considered as a long and fastidious test in medical diagnosis. This process can be easily implanted in our platform, and high resolution fluorescence imaging can be performed with reduced time and computer intensiveness. These results demonstrate the potential of this chip as a low cost, robust, and versatile tool adapted to complex and demanding protocols for medical diagnosis.

Mutant Huntingtin alters retrograde transport of TrkB receptors in striatal dendrites.

Huntingtin (HTT), the protein mutated in Huntington's disease (HD), controls transport of the neurotrophin, brain-derived neurotrophic factor (BDNF), within corticostriatal neurons. Transport and delivery of BDNF to the striatum are reduced in disease, which contributes to striatal neuron degeneration. BDNF released by cortical neurons activates TrkB receptors at striatal dendrites to promote striatum survival. However, it remains to be determined whether transport of TrkB, the BDNF receptor, depends on HTT and whether such transport is altered in mutant situation. Here we show that TrkB binds to and colocalizes with HTT and dynein. Silencing HTT reduces vesicular transport of TrkB in striatal neurons. In HD, the polyQ expansion in HTT alters the binding of TrkB-containing vesicles to microtubules and reduces transport. Using a combination of microfluidic devices that isolate dendrites from cell bodies and BDNF coupled to quantum dots, we selectively analyzed TrkB retrograde transport in response to BDNF stimulation at dendrite terminals. We show that the retrograde transport of TrkB vesicles within striatal dendrites and the BDNF/TrkB-induced signaling through ERK phosphorylation and c-fos induction are decreased in neurons from an HD mouse model. Together, our findings demonstrate that HTT is a crucial regulator of TrkB trafficking. Transport defects in HD are not restricted to BDNF transport in cortical neurons but also affect trafficking of its ligand-bound receptor in the striatal neurons. This transport alteration may further impair BDNF-TrkB survival signaling within the corticostriatal connection that is most affected in HD.

High-resolution analyses of cell fusion dynamics in a biochip.

In this study, we analyzed the electrofusion of two cells in a biochip that has been developed to perform the capture by dielectrophoresis and the electrofusion of pairs of cells. The good transparency of the microsystem allowed analyzing the details of the fusion events. By staining one of the cells, the mixing of the two cytosols could be observed during the electrofusion experiment. We show for the first time the rapidity of the mixing of the two cytosols: less than 5 s under our experimental conditions. By comparing these experimental results to a numerical simulation, we found that the rate of this phenomenon is compatible with a diffusion-only mechanism, showing that during the fusion, the two cell membranes in contact are affected by very rapid structural changes and do not limit the exchange of the cytosols between the two cells. A point of interest is the use of dielectric structures to concentrate the electric field and of positive dielectrophoresis to capture cells in the area where the electric field is more intense. This technique allows the increase of the cell-to-cell contact and limits cell cytosol leakages during the fusion process.

Fabrication of thermoplastics chips through lamination based techniques.

In this work, we propose a novel strategy for the fabrication of flexible thermoplastic microdevices entirely based on lamination processes. The same low-cost laminator apparatus can be used from master fabrication to microchannel sealing. This process is appropriate for rapid prototyping at laboratory scale, but it can also be easily upscaled to industrial manufacturing. For demonstration, we used here Cycloolefin Copolymer (COC), a thermoplastic polymer that is extensively used for microfluidic applications. COC is a thermoplastic polymer with good chemical resistance to common chemicals used in microfluidics such as acids, bases and most polar solvents. Its optical quality and mechanical resistance make this material suitable for a large range of applications in chemistry or biology. As an example, the electrokinetic separation of pollutants is proposed in the present study.