Techniques to analyze and sort single cells based on functional outputs, such as secreted products, have the potential to transform our understanding of cellular biology, as well as accelerate the development of next generation cell and antibody therapies. However, secreted molecules rapidly diffuse away from cells, and analysis of these products requires specialized equipment and expertise to compartmentalize individual cells and capture their secretions. Herein we demonstrate the use of suspendable microcontainers to sort single viable cells based on their secreted products at high-throughput using only commonly accessible laboratory infrastructure. Our microparticles act as solid supports which facilitate cell attachment, partition uniform aqueous compartments, and capture secreted proteins. Using this platform, we demonstrate highthroughput screening of stably-and transiently-transfected producer cells based on relative IgG production as well as screening of B lymphocytes and hybridomas based on antigen-specific antibody production using commercially available flow sorters. Leveraging the high-speed sorting capabilities of standard sorters, we sorted >1,000,000 events in less than an hour. The reported microparticles can be easily stored, and distributed as a consumable reagent amongst researchers, democratizing access to high-throughput functional cell screening.

A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.

Techniques to analyze and sort single cells based on secreted products have the potential to transform our understanding of cellular biology as well as accelerate the development of next generation cell and antibody therapies. However, secretions are rapidly transported away from cells, such that specialized equipment and expertise has been required to compartmentalize cells and capture their secretions. Herein we demonstrate the use of cavity-containing hydrogel microparticles to perform functional single-cell secretion analysis and sorting using only commonly accessible lab infrastructure. These microparticles act as a solid support which facilitates cell attachment, templates formation of uniform aqueous compartments which prevent cross-talk between cells, and captures secreted proteins. Using this platform we demonstrate high-throughput analysis and sorting of Chinese Hamster Ovary cells based on their relative production of human IgG using commercially available flow sorters.Microparticles are easily distributed and used, democratizing access to high-throughput functional cell screening.

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The majority of clinically approved therapeutics target membrane proteins (MPs), highlighting the need for tools to study this important category of proteins. To overcome limitations with recombinant MP expression, whole cell screening techniques have been developed that present MPs in their native conformations. Whereas many such platforms utilize adherent cells, here we introduce a novel suspension cell-based platform termed "biofloating" that enables quantitative analysis of interactions between proteins displayed on yeast and MPs expressed on mammalian cells, without need for genetic fusions. We characterize and optimize biofloating and illustrate its sensitivity advantage compared to an adherent cell-based platform (biopanning). We further demonstrate the utility of suspension cell-based approaches by iterating rounds of magnetic-activated cell sorting selections against MP-expressing mammalian cells to enrich for a specific binder within a yeast-displayed antibody library. Overall, biofloating represents a promising new technology that can be readily integrated into protein discovery and development workflows.