Abstract Cytotoxic CD8⁺ T cells kill target cells through brief cell–cell encounters, but pooled genetic screens cannot readily link perturbations in individual T cells to the fate of the target cells they engage. We developed droplet single-cell CRISPR screening to pair individual primary human CD8⁺ T cells with cancer cells, measure rapid target-cell death, and recover sgRNAs from phenotype-defined droplets. Applied across primary T cells from multiple donors, the platform recovered regulators of T cell receptor signaling, synapse formation, granule exocytosis and cytotoxic differentiation, and identified negative regulators of killing, including established inhibitory nodes such as PTEN , RASA2 and FOXO1 , together with AFAP1L2 and components of the mTORC1 pathway. Validation across bispecific engager and TCR-engineered settings showed that selected hits modulate target-cell killing across recognition modalities and tumor models. Unexpectedly, perturbation of RPTOR or RHEB enhanced cytotoxic execution while reducing mTORC1 output, increasing AKT phosphorylation, and attenuating anabolic programs. Transient pharmacologic mTORC1 inhibition reproduced this rapid-killing state and improved antitumor activity after adoptive transfer. These results establish an interaction-resolved pooled genetic strategy for mapping cytotoxicity regulators and reveal that transient modulation of mTORC1 can shift T cells from anabolic growth toward rapid cytotoxic execution to enhance antitumor activity.

Cell-cell interactions are essential for the proper functioning of multicellular organisms. For example, T cells interact with antigen-presenting cells (APCs) through specific T-cell receptor (TCR)-antigen interactions during an immune response. Fluorescence-activated droplet sorting (FADS) is a high-throughput technique for efficiently screening cellular interaction events. Unfortunately, current droplet sorting instruments have significant limitations, most notably related to analytical throughput and complex operation. In contrast, commercial fluorescence-activated cell sorters offer superior speed, sensitivity, and multiplexing capabilities, although their use as droplet sorters is poorly defined and underutilized. Herein, we present a universally applicable and simple-to-implement workflow for generating double emulsions and performing multicolor cell sorting using a commercial FACS instrument. This workflow achieves a double emulsion detection rate exceeding 90%, enabling multicellular encapsulation and high-throughput immune cell activation sorting for the first time. We anticipate that the presented droplet sorting strategy will benefit cell biology laboratories by providing access to an advanced microfluidic toolbox with minimal effort and cost investment.

We present a portable imaging flow cytometer comprising a smartphone, a small-footprint optical framework, and a PDMS-based microfluidic device. Flow cytometric analysis is performed in a sheathless manner via elasto-inertial focusing with a custom-written Android program, integrating a graphical user interface (GUI) that provides a high degree of user control over image acquisition. The proposed system offers two different operational modes. First, "post-processing" mode enables particle/cell sizing at throughputs of up to 67 000 particles/s. Alternatively, "real-time" mode allows for integrated cell/particle classification with machine learning at throughputs of 100 particles/s. To showcase the efficacy of our platform, polystyrene particles are accurately enumerated within heterogeneous populations using the post-processing mode. In real-time mode, an open-source machine learning algorithm is deployed within a custom-developed Android application to classify samples containing cells of similar size but with different morphologies. The flow cytometer can extract high-resolution bright-field images with a spatial resolution <700 nm using the developed machine learning-based algorithm, achieving classification accuracies of 97% and 93% for Jurkat and EL4 cells, respectively. Our results confirm that the smartphone imaging flow cytometer (sIFC) is capable of both enumerating single particles in flow and identifying morphological features with high resolution and minimal hardware.

Nowadays, the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes. The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes. While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems, the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts. Only a few commercial microfluidic systems for enzyme analysis are currently available, and the transformation of numerous published prototypes into commercial platforms is still to be streamlined. This review presents the state-of-the-art, recent trends, and perspectives in applying microfluidic tools in the functional and structural analysis of biocatalysts. We discuss the advantages and disadvantages of available technologies, their reproducibility and robustness, and readiness for routine laboratory use. We also highlight the unexplored potential of microfluidics to leverage the power of machine learning for biocatalyst development.

ADVERTISEMENT RETURN TO ISSUEPREVReviewNEXTRecent Advances in Droplet MicrofluidicsYun DingYun DingInstitute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zürich, SwitzerlandMore by Yun DingView Biographyhttp://orcid.org/0000-0002-7784-7758, Philip D. HowesPhilip D. HowesInstitute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zürich, SwitzerlandMore by Philip D. HowesView Biographyhttp://orcid.org/0000-0002-1862-8395, and Andrew J. deMello*Andrew J. deMelloInstitute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zürich, Switzerland* E-mail: [email protected]More by Andrew J. deMelloView Biographyhttp://orcid.org/0000-0003-1943-1356Cite this: Anal. Chem. 2020, 92, 1, 132–149Publication Date (Web):November 26, 2019Publication History Published online26 November 2019Published inissue 7 January 2020https://pubs.acs.org/doi/10.1021/acs.analchem.9b05047https://doi.org/10.1021/acs.analchem.9b05047review-articleACS PublicationsCopyright © 2019 American Chemical SocietyRequest reuse permissionsArticle Views10094Altmetric-Citations194LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose SUBJECTS:Biotechnology,Drug discovery,Fluid dynamics,Genetics,Liquids Get e-Alerts

Droplet-based microfluidic technologies have proved themselves to be of significant utility in the performance of high-throughput chemical and biological experiments. By encapsulating and isolating reagents within femtoliter-nanoliter droplet, millions of (bio) chemical reactions can be processed in a parallel fashion and on ultra-short timescales. Recent applications of such technologies to genetic analysis have suggested significant utility in low-cost, efficient and rapid workflows for DNA amplification, rare mutation detection, antibody screening and next-generation sequencing. To this end, we describe and highlight some of the most interesting recent developments and applications of droplet-based microfluidics in the broad area of nucleic acid analysis. In addition, we also present a cursory description of some of the most essential functional components, which allow the creation of integrated and complex workflows based on flowing streams of droplets.

Herein, we assess the functionality of magnetic helical microswimmers as basic tools for the manipulation of soft materials, including microdroplets and single cells. Their ability to perform a range of unit operations is evaluated and the operational challenges associated with their use are established. In addition, we also report on interactions observed between the head of such helical swimmers and the boundaries of droplets and cells and discuss the possibilities of assembling an artificial swimming microorganism or a motorized cell.

We present the use of microfluidic "V-junctions" as a droplet generation strategy that incorporates enhanced performance characteristics when compared to more traditional "T-junction" formats. This includes the ability to generate target-sized droplets from the very first one, efficient switching between multiple input samples, the production of a wide range of droplet sizes (and size gradients) and the facile generation of droplets with residence time gradients. Additionally, the use of V-junction droplet generators enables the suspension and subsequent resumption of droplet flows at times defined by the user. The high degree of operational flexibility allows a wide range of droplet sizes, payloads, spacings and generation frequencies to be obtained, which in turn provides for an enhanced design space for droplet-based experimentation. We show that the V-junction retains the simplicity of operation associated with T-junction formats, whilst offering functionalities normally associated with droplet-on-demand technologies.

SmallVolume 10, Issue 10 p. 1953-1957 Communication Artificial Bacterial Flagella for Remote-Controlled Targeted Single-Cell Drug Delivery Rami Mhanna, Corresponding Author Rami Mhanna Institute of Robotics and Intelligent Systems, ETH Zurich, Zurich, CH-8092 SwitzerlandE-mail: rami.mhanna@dep.uminho.ptSearch for more papers by this authorFamin Qiu, Famin Qiu Institute of Robotics and Intelligent Systems, ETH Zurich, Zurich, CH-8092 SwitzerlandSearch for more papers by this authorLi Zhang, Li Zhang Department of Mechanical and Automation Engineering, The Chinese University of Hong Kong, Hong Kong SAR, ChinaSearch for more papers by this authorYun Ding, Yun Ding Institute of Robotics and Intelligent Systems, ETH Zurich, Zurich, CH-8092 SwitzerlandSearch for more papers by this authorKaori Sugihara, Kaori Sugihara Laboratory of Biosensors and Bioelectronics, ETH Zurich, Zurich, CH-8092 SwitzerlandSearch for more papers by this authorMarcy Zenobi-Wong, Marcy Zenobi-Wong Cartilage Engineering + Regeneration, ETH Zurich, Zurich, CH-8093 SwitzerlandSearch for more papers by this authorBradley J. Nelson, Bradley J. Nelson Institute of Robotics and Intelligent Systems, ETH Zurich, Zurich, CH-8092 SwitzerlandSearch for more papers by this author Rami Mhanna, Corresponding Author Rami Mhanna Institute of Robotics and Intelligent Systems, ETH Zurich, Zurich, CH-8092 SwitzerlandE-mail: rami.mhanna@dep.uminho.ptSearch for more papers by this authorFamin Qiu, Famin Qiu Institute of Robotics and Intelligent Systems, ETH Zurich, Zurich, CH-8092 SwitzerlandSearch for more papers by this authorLi Zhang, Li Zhang Department of Mechanical and Automation Engineering, The Chinese University of Hong Kong, Hong Kong SAR, ChinaSearch for more papers by this authorYun Ding, Yun Ding Institute of Robotics and Intelligent Systems, ETH Zurich, Zurich, CH-8092 SwitzerlandSearch for more papers by this authorKaori Sugihara, Kaori Sugihara Laboratory of Biosensors and Bioelectronics, ETH Zurich, Zurich, CH-8092 SwitzerlandSearch for more papers by this authorMarcy Zenobi-Wong, Marcy Zenobi-Wong Cartilage Engineering + Regeneration, ETH Zurich, Zurich, CH-8093 SwitzerlandSearch for more papers by this authorBradley J. Nelson, Bradley J. Nelson Institute of Robotics and Intelligent Systems, ETH Zurich, Zurich, CH-8092 SwitzerlandSearch for more papers by this author First published: 10 March 2014 https://doi.org/10.1002/smll.201303538Citations: 155Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract An approach for batch preparation of liposome-functionalized microdevices is demonstrated for remotely controlled single-cell drug delivery. The liposome functionalized artificial bacterial flagella exhibit corkscrew swimming in 3D with micrometer positioning precision by applying an external rotating magnetic field. The devices are also capable of delivering water-soluble drugs to single cells in vitro. Citing Literature Supporting Information As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Filename Description smll201303538-sup-0001-S1.pdf1.5 MB Supplementary smll201303538-sup-0002-S2.wmv278.5 KB Supplementary Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume10, Issue10May 28, 2014Pages 1953-1957 RelatedInformation

Inspired by flagellar propulsion of bacteria such as E. coli, artificial bacterial flagella (ABFs) are magnetic swimming microrobots with helical shapes. ABFs are capable of performing precise three-dimensional (3D) navigation in fluids under low-strength rotating magnetic fields making them attractive tools for targeted drug delivery. Further biomedical functionalization of these swimming microrobots is essential to enhance their biological and medical performances. We report the successful functionalization of titanium-coated ABFs with temperature-sensitive dipalmitoylphosphatidylcholine (DPPC)-based liposomes, known as "smart" drug carriers. Liposome coating on the surface of ABFs was confirmed using quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescent probes. The functionalized ABFs (f-ABFs) showed the ability to incorporate both hydrophilic and hydrophobic drugs. Finally, thermally triggered release of calcein (a common drug analog) from f-ABFs was demonstrated. These f-ABFs have the potential to be used in targeted and triggered drug delivery, microfluidic devices and biosensing. (C) 2014 Elsevier B.V. All rights reserved.