Cell-cell interactions are important to numerous biological systems, including tissue microenvironments, the immune system, and cancer. However, current methods for studying cell combinations and interactions are limited in scalability, allowing just hundreds to thousands of multicell assays per experiment; this limited throughput makes it difficult to characterize interactions at biologically relevant scales. Here, we describe a paradigm in cell interaction profiling that allows accurate grouping of cells and characterization of their interactions for tens to hundreds of thousands of combinations. Our approach leverages high-throughput droplet microfluidics to construct multicellular combinations in a deterministic process that allows inclusion of programmed reagent mixtures and beads. The combination droplets are compatible with common manipulation and measurement techniques, including imaging, barcode-based genomics, and sorting. We demonstrate the approach by using it to enrich for chimeric antigen receptor (CAR)-T cells that activate upon incubation with target cells, a bottleneck in the therapeutic T cell engineering pipeline. The speed and control of our approach should enable valuable cell interaction studies.

Droplet libraries consisting of many reagents encapsulated in separate droplets are necessary for applications of microfluidics, including combinatorial chemical synthesis, DNA-encoded libraries, and massively multiplexed PCR. However, existing approaches for generating them are laborious and impractical. Here, we describe an automated approach using a commercial array spotter. The approach can controllably emulsify hundreds of different reagents in a fraction of the time of manual operation of a microfluidic device, and without any user intervention. We demonstrate that the droplets produced by the spotter are similarly uniform to those produced by microfluidics and automate the generation of a ~ 2 mL emulsion containing 192 different reagents in ~ 4 h. The ease with which it can generate high diversity droplet libraries should make combinatorial applications more feasible in droplet microfluidics. Moreover, the instrument serves as an automated droplet generator, allowing execution of droplet reactions without microfluidic expertise.

Single-cell RNA sequencing has emerged as a powerful tool for characterizing cells, but not all phenotypes of interest can be observed through changes in gene expression. Linking sequencing with optical analysis has provided insight into the molecular basis of cellular function, but current approaches have limited throughput. Here, we present a high-throughput platform for linked optical and gene expression profiling of single cells. We demonstrate accurate fluorescence and gene expression measurements on thousands of cells in a single experiment. We use the platform to characterize DNA and RNA changes through the cell cycle and correlate antibody fluorescence with gene expression. The platform's ability to isolate rare cell subsets and perform multiple measurements, including fluorescence and sequencing-based analysis, holds potential for scalable multi-modal single-cell analysis.

Cytokines are produced by leukocytes in blood and may be used as indicators of malignancies or infections. The objective of this study was to develop a strategy for immunosensing cytokines in whole, unprocessed human blood. Microfluidic droplet generation was employed to fabricate ∼400 μm diameter microcapsules with a hydrogel shell and an aqueous core containing sensing microbeads. The hydrogel shell was composed of poly(ethylene glycol) forming a thin (∼10 μm) immunoisolation layer protecting antibody-modified microbeads inside the capsule from immune cells on the outside. The microbeads were functionalized with antibodies against cytokines of interest: interferon (IFN)-γ and tumor necrosis factor (TNF)-α. While nonfouling, a hydrogel shell was permeable to cytokine molecules; these molecules were captured on microbeads and were detected with fluorescently labeled secondary antibodies. Calibration of encapsulated immunoassays with known concentrations of cytokines revealed a limit of detection of 14.8 and 14.4 pM for IFN-γ and TNF-α, respectively. We also demonstrated the concept of multi-cytokine detection by fabricating distinct populations of capsules carrying either anti-IFN-γ or anti-TNF-α microbeads and dispensing these capsules into a solution containing both cytokine types. Importantly, when placed into whole blood for 16 h, microcapsules were free of leukocytes, effectively protecting sensing beads from the blood components. To further demonstrate utility of this strategy, encapsulated microbeads were used for detection of IFN-γ in blood of patients with latent tuberculosis infection (LTBI) and unexposed healthy controls. When compared to gold standard technology (interferon gamma release assay or IGRA), our encapsulated immunoassay accurately predicted LTBI diagnosis in 11 out of 14 patients. Overall, encapsulation of immunoassays represents a promising strategy for keeping sensing elements operational in a highly fouling complex environment such as blood.

Droplet microfluidics enables massively-parallel analysis of single cells, biomolecules, and chemicals, making it valuable for high-throughput screens. However, many hydrophobic analytes are soluble in carrier oils, preventing their quantitative analysis with the method. We apply Printed Droplet Microfluidics to construct defined reactions with chemicals and cells incubated under air on an open array. The method interfaces with most bioanalytical tools and retains hydrophobic compounds in compartmentalized reactors, allowing their quantitation.

Although the elementary unit of biology is the cell, high-throughput methods for the microscale manipulation of cells and reagents are limited. The existing options either are slow, lack single-cell specificity, or use fluid volumes out of scale with those of cells. Here we present printed droplet microfluidics, a technology to dispense picoliter droplets and cells with deterministic control. The core technology is a fluorescence-activated droplet sorter coupled to a specialized substrate that together act as a picoliter droplet and single-cell printer, enabling high-throughput generation of intricate arrays of droplets, cells, and microparticles. Printed droplet microfluidics provides a programmable and robust technology to construct arrays of defined cell and reagent combinations and to integrate multiple measurement modalities together in a single assay.

3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro, and typically require 3D culture systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) gel shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures.Statement of SignificanceOur paper combines an interesting new way for making capsules with cultivation of difficult-to-maintain primary epithelial cells (hepatocytes). The microcapsules described here will enable high density suspension culture of hepatocytes or other cells and may be used as building blocks for engineering tissues. (C) 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Conducting polymer hydrogel is fabricated atop gold or ITO electrodes and is functionalized with monoclonal antibodies. Binding of interferon-γ molecules causes redox properties of conductive hydrogel to change in a concentration-dependent fashion without the need for washing or sample handling steps. This conductive hydrogel remains functional in a fouling media such as whole blood.

A major challenge in tissue engineering is to develop robust protocols for differentiating ES and iPS cells to functional adult tissues at a clinically relevant scale. The goal of this study is to develop a high throughput platform for generating bioactive, stem cell-laden microgels to direct differentiation in a well-defined microenvironment. We describe a droplet microfluidics system for fabricating microgels composed of polyethylene glycol and heparin, with tunable geometric, mechanical, and chemical properties, at kHz rates. Heparin-containing hydrogel particles sequestered growth factors Nodal and FGF-2, which are implicated in specifying pluripotent cells to definitive endoderm. Mouse ESCs were encapsulated into heparin microgels with a single dose of Nodal and FGF-2, and expressed high levels of endoderm markers Sox17 and FoxA2 after 5 days. These results highlight the use of microencapsulation for tailoring the stem cell microenvironment to promote directed differentiation, and may provide a straightforward path to large scale bioprocessing in the future. STATEMENT OF SIGNIFICANCE:Multicellular spheroids and microtissues are valuable for tissue engineering, but fabrication approaches typically sacrifice either precision or throughput. Microfluidic encapsulation in polymeric biomaterials is a promising technique for rapidly generating cell aggregates with excellent control of microenvironmental parameters. Here we describe the microfluidic fabrication of bioactive, heparin-based microgels, and demonstrate the adsorption of heparin-binding growth factors for enhancing directed differentiation of embryonic stem cells toward endoderm. This approach also facilitated a ∼90-fold decrease in consumption of exogenous growth factors compared to conventional differentiation protocols.

Monitoring activity of single cells has high significance for basic science and diagnostic applications.

Lentivectors are widely used for gene delivery and have been increasingly tested in clinical trials. However, achieving safe, localized, and sufficient gene expression remain key challenges for effective lentivectoral therapy. Localized and efficient gene expression can be promoted by developing material systems to deliver lentivectors. Here, we address the utility of microgel encapsulation as a strategy for the controlled release of lentivectors. Three distinct routes for ionotropic gelation of alginate were incorporated into microfluidic templating to create lentivector-loaded microgels. Comparisons of the three microgels revealed marked differences in mechanical properties, crosslinking environment, and ultimately lentivector release and functional gene expression in vitro. Gelation with chelated calcium demonstrated low utility for gene delivery due to a loss of lentivector function with acidic gelation conditions. Both calcium carbonate gelation, and calcium chloride gelation, preserved lentivector function with a more sustained transduction and gene expression over 4 days observed with calcium chloride gelated microgels. The validation of these two strategies for lentivector microencapsulation may provide a platform for controlled gene delivery.

Targeted cancer therapies are designed to deactivate signaling pathways used by cancer cells for survival. However, cancer cells are often able to adapt by activating alternative survival pathways, thereby acquiring drug resistance. An emerging theory is that autocrine or paracrine growth factor signaling in the cancer microenvironment represent an important mechanism of drug resistance. In the present study we wanted to examine whether paracrine interactions between groups of melanoma cells result in resistance to vemurafenib - an FDA approved drug targeting the BRAF mutation in metastatic melanoma. We used a vemurafenib-resistant melanoma model which secretes fibroblast growth factor (FGF)-2 to test our hypothesis that this is a key paracrine mediator of resistance to vemurafenib. Sensitive cells treated with media conditioned by resistant cells did not protect from the effects of vemurafenib. To query paracrine interactions further we fabricated a microfluidic co-culture device with two parallel compartments, separated by a 100 μm wide hydrogel barrier. The gel barrier prevented resorting/contact of cells while permitting paracrine cross-talk. In this microfluidic system, sensitive cells did become refractive to the effects of vemurafenib when cultured adjacent to resistant cells. Importantly, incorporation of FGF-2 capture probes into the gel barrier separating the two cell types prevented onset of resistance to vemurafenib. Microfluidic tools described here allow for more sensitive analysis of paracrine signals, may help better understand signaling in the cancer microenvironment and may enable development of more effective cancer therapies.

Long-term in vitro maintenance of primary hepatocytes has been a challenge in hepatic tissue engineering, since these cells dedifferentiate and lose their phenotype in standard culture conditions. Some of the most successful culture systems employ collagen or Matrigel matrices. As an alternative matrix, our laboratory has been exploring the use of heparin-containing hydrogels as scaffolds for cultivation of functional hepatocytes. The present study characterized a heparin gel sandwich culture system for maintenance of primary rat hepatocytes. The heparin gel layers were fabricated via UV light induced thiol-ene coupling reaction of thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEGDA) precursors. Analysis of hepatic function revealed that cells sustained albumin secretion for at least three weeks at similarly high levels for collagen and heparin gel sandwich culture systems. Furthermore, cytochrome P450 activity of hepatocytes was similar to 1.6 times higher in heparin gels compared to collagen gels. Another distinct advantage of heparin is its ability to bind and release growth factors in a controlled manner. We observed that after 24 days hepatocytes cultured in heparin gel sandwich containing hepatocyte growth factor (HGF) produced similar to 1.8 times higher amounts of albumin compared to cells in collagen sandwiches. Overall, we demonstrate heparin gel sandwich to be an excellent system for maintaining differentiated, polarized primary hepatocytes for over three weeks. The biomaterial used in this study, heparin-PEG hydrogel, offers significant flexibility in terms of tuning mechanical properties, adjusting heparin content or introducing photolabile chemistries to promote gel degradation. We see multiple applications for this novel system in hepatocyte maintenance and stem cell differentiation studies. (C) 2014 Elsevier Ltd. All rights reserved.

The development of a novel photodegradable heparin‐based hydrogel for cultivation and retrieval of embryonic stem cells is described. Mouse embryonic stem cells cultured atop the gel with encapsulated growth factors (GFs) express higher levels of differentiation markers compared to a standard protocol employing soluble GFs. Beyond improving differentiation of stem cells, the novel hydrogels can be used to release specific stem cell colonies without disturbing neighboring cells. This way, stem cell colonies can be retrieved at different time points and from different locations of the culture surface for polymerase chain reaction (PCR) analysis without the loss of the microenvironment context. The ability to retrieve some stem cell colonies without disturbing neighboring colonies will open possibilities for characterizing in‐dish heterogeneity of stem cell phenotype and will also allow to conserve cells/reagents. Overall, the bioactive photodegradable hydrogel developed in this study may offer new possibilities for cultivation and analysis of stem cells as well as other cell types.

Inhibitory antibodies to two specific human and mouse Notch ligands, Jagged1 and Jagged2, are generated and shown to have beneficial effects in a goblet cell metaplasia asthma model; systemic Jagged1 inhibition transdifferentiates secretory cells into ciliated cells in the mouse, demonstrating that Jagged1 from ciliated cells normally holds back secretory cells to adopt the ciliated fate.

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.

Sorted out: Soft-lithography micropatterning of a photodegradable poly(ethylene glycol) hydrogel was utilized to sort cell-bound microbeads. Biofunctionalized microbeads were patterned, screened for cell binding activity, and isolated from arrays by UV irradiation. The technique is a simple approach for sorting and downstream processing of cultured cells. 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. 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.

Biosensors are of great significance because of their capability to resolve a potentially large number of analytical problems and challenges in very diverse areas such as defense, homeland security, agriculture and food safety, environmental monitoring, medicine, pharmacology, industry, etc. The expanding role of biosensing in society and a real-world environment has led to an exponential growth of the R&D efforts around the world. The world market for biosensor devices, according to Global Industry Analysts, Inc., is expected to reach $12 billion by 2015. Such expedient growth is driven by several factors including medical and health problems, such as a growing population with a high risk of diabetes and obesity, and the rising incidence of chronic diseases such as heart disease, stroke, cancer, chronic respiratory diseases, tuberculosis, etc.; significant problems with environmental monitoring; and of course serious challenges in security and military applications and agriculture/food safety. A review paper in the biosensor technology area may be structured based on (i) the principles of detection, such as the type of transducer platform, bioanalytical principles (affinity or kinetic), and biorecognition elements origin/properties (i.e. antibodies, enzymes, cells, aptamers, etc.), and (ii) the application area. This review follows the latter strategy and focuses on the applications. This allows discussion on how different sensing strategies are brought to bear on the same problem and highlights advantages/disadvantages of these sensing strategies. Given the broad range of biosensor related applications, several particularly relevant areas of application were selected for review: biological threat agents, chemical threat agents, and medicine.