To develop biomimetic three-dimensional (3D) tissue constructs for drug screening and biological studies, engineered blood vessels should be integrated into the constructs to mimic the drug administration process . The development of perfusable vascularized 3D tissue constructs for studying the drug administration process through an engineered endothelial layer remains an area of intensive research. Here, we report the development of a simple 3D vascularized liver tissue model to study drug toxicity through the incorporation of an engineered endothelial layer. Using a sacrificial bioprinting technique, a hollow microchannel was successfully fabricated in the 3D liver tissue construct created with HepG2/C3A cells encapsulated in a gelatin methacryloyl hydrogel. After seeding human umbilical vein endothelial cells (HUVECs) into the microchannel, we obtained a vascularized tissue construct containing a uniformly coated HUVEC layer within the hollow microchannel. The inclusion of the HUVEC layer into the scaffold resulted in delayed permeability of biomolecules into the 3D liver construct. In addition, the vascularized construct containing the HUVEC layer showed an increased viability of the HepG2/C3A cells within the 3D scaffold compared to that of the 3D liver constructs without the HUVEC layer, demonstrating a protective role of the introduced endothelial cell layer. The 3D vascularized liver model presented in this study is anticipated to provide a better and more accurate liver model system for future drug toxicity testing.

To create life‐like movements, living muscle actuator technologies have borrowed inspiration from biomimetic concepts in developing bioinspired robots. Here, the development of a bioinspired soft robotics system, with integrated self‐actuating cardiac muscles on a hierarchically structured scaffold with flexible gold microelectrodes is reported. Inspired by the movement of living organisms, a batoid‐fish‐shaped substrate is designed and reported, which is composed of two micropatterned hydrogel layers. The first layer is a poly(ethylene glycol) hydrogel substrate, which provides a mechanically stable structure for the robot, followed by a layer of gelatin methacryloyl embedded with carbon nanotubes, which serves as a cell culture substrate, to create the actuation component for the soft body robot. In addition, flexible Au microelectrodes are embedded into the biomimetic scaffold, which not only enhance the mechanical integrity of the device, but also increase its electrical conductivity. After culturing and maturation of cardiomyocytes on the biomimetic scaffold, they show excellent myofiber organization and provide self‐actuating motions aligned with the direction of the contractile force of the cells. The Au microelectrodes placed below the cell layer further provide localized electrical stimulation and control of the beating behavior of the bioinspired soft robot.

Activation of cardiac fibroblasts (CF) into myofibroblasts is considered to play an essential role in cardiac remodeling and fibrosis. A limiting factor in studying this process is the spontaneous activation of CFs when cultured on two-dimensional (2D) culture plates. Here, a simplified 3D hydrogel platform of contractile cardiac tissue, stimulated by transforming growth factor-β1 (TGF-β1), is presented to recapitulate a fibrogenic micro-environment. It was hypothesized that the quiescent state of CFs can be maintained by mimicking the mechanical stiffness of native heart tissue. To test this hypothesis, a 3D cell culture model consisting of cardiomyocytes and CFs encapsulated within mechanically engineered gelatin methacryloyl (GelMA) hydrogel, was developed. The study shows that CFs maintain their quiescent phenotype in mechanically tuned hydrogels. Additionally, treatment with a beta-adrenergic agonist increased beating frequency, demonstrating physiologic-like behavior of the heart constructs. Subsequently, quiescent CFs within the constructs were activated by the exogenous addition of TGF-β1. The expression of fibrotic protein markers (and the functional changes in mechanical stiffness) in the fibrotic-like tissues were analyzed to validate the model. Overall, this 3D engineered culture model of contractile cardiac tissue enabled controlled activation of CFs, demonstrating the usability of this platform to study fibrotic remodeling.

No abstract

Droplet-based microfluidics has emerged as a powerful tool in synthetic biology. For many applications, chemical functionalization of the droplets is a key process. Therefore, a straightforward and broadly applicable approach is developed to functionalize the inner periphery of microfluidic droplets with diverse reactive groups and components. Instead of covalent modification of the droplet-stabilizing surfactants, this method relies on cholesteroltagged DNA that self-assembles at the droplet periphery. The cholesteroltagged DNA serves as an attachment handle for the recruitment of complementary DNA. The complementary DNA can carry diverse functional groups. We exemplify our method by demonstrating the attachment of amine groups, DNA nanostructures, microspheres, a minimal actin cortex, and leukemia cells to the droplet periphery. It is further shown that the DNAmediated attachment to the droplet periphery is temperature-responsive and reversible. It is envisioned that droplet functionalization via DNA handles will help to tailor droplet interfaces for diverse applications-featuring programmable assembly, unique addressability, and stimuli-responsiveness.The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adfm.201808647. not universally applicable-it depends on the success of the chemical synthesis and can interfere with the stability and the physical properties of the droplets. Moreover, the binding of the functional group to the droplet periphery is irreversible.DNA nanotechnology, [12] on the other hand can attain the programmable assembly of arbitrary nanoscale architectures like DNA-based lattices, [13] nanopores, [14][15][16] or lid-containing boxes. [17,18] DNA has also been used as a scaffold or linker to assemble secondary components including proteins, [19] gold nanoparticles [20] and liposomes. [21] In addition, networks of emulsion droplets [22,23] or colloid-coated droplets [24] have been created using DNA linkers. Yet in all cases, the linkage was based on biotinylated DNA, which requires additional efforts to graft streptavidin onto the droplet surface. Furthermore, it has never been demonstrated that it is possible to functionalize the interior of block-copolymer surfactantstabilized droplets with DNA.Here, we present a broadly applicable method for functionalizing microfluidic droplets utilizing the hydrophobic interaction of cholesterol-tagged DNA with the droplet-stabilizing surfactant. Notably, the interaction of cholesterol with perfluorinated chains has never been described or exploited before. We show that DNA handles can serve as reversible anchoring points for various components including reactive groups, DNA nanostructures, beads, proteins or even cells. The use of off-the-shelf available DNA holds considerable advantages compared to standard methods for droplet functionalization, including: the broad scope of options for site-directed chemical functionalization, the addressability and programmability due to specific base pairin...

Balancing the maximal efficacy of interferon gamma (IFN-ɣ)-based therapies with its side effects is a great challenge for future cytokine treatments. To achieve this, the development of single-cell technologies that study IFN-ɣ release in correlation with antitumor activity would represent a huge step forward. To this end, droplet-based microfluidics is employed to quantitatively investigate IFN-ɣ secretion from single natural killer (NK) cells in correlation with their cytotoxic activity against a specific target. The method relies on co-encapsulation of NK-92 cells, target cancer cells, polystyrene beads conjugated with specific IFN-ɣ capture antibodies, and fluorescently labeled detection antibodies inside water-in-oil compartments. The secreted cytokines are captured and detected by localized fluorescence at the periphery of the beads. NK-92's cytotoxicity is evaluated simultaneously by means of a fluorescent DNA intercalating agent, which penetrates the membranes of dead target cells. To deepen the understanding of the role of the cytokine in antitumor immunomodulation, the impact of different doses of human recombinant IFN-ɣ on the cytolytic activity of NK-92 cells shows a trend that the higher the dose the lower the cytolytic activity of NK cells. The developed method represents a simple quantitative approach to unravel the complex heterogeneity of NK cells toward IFN-ɣ secretion and cytolytic activity.

Natural killer (NK) cells are key players of the innate immune system. Due to their rapid cytotoxicity against infectious pathogens, hematologic malignancies, and solid tumors, NK cells represent solid candidates for cell-based immunotherapy. Despite the progress made in recent years, the heterogeneity in their cytotoxic behavior represents a drawback. With the goal of screening the intrinsic diversity of NK cells, droplet-based microfluidic technology is exploited to develop a single-cell time-efficient cytotoxicity assay. Toward this end, NK-92 cells are coencapsulated with hematological tumor cell lines in water-in-oil droplets of different sizes and their cytotoxic activity is evaluated. The effect of droplet-based confinement on NK cytotoxicity is investigated by controlling the droplet volume. The successful optimization of the droplet size allows for time efficiency compared to cytotoxicity assays based on flow cytometry. Additionally, the ability of individual NK-92 cells to kill multiple target cells in series is explored, expanding the knowledge about the serial killing process dynamics. The developed droplet-based microfluidic assay does not require the labeling of NK cells and represents a step toward developing of a forthcoming process for the selection of NK cells with the highest cytotoxicity against specific targets.

Fluorescence correlation spectroscopy (FCS) is a sensitive technique commonly applied for studying the dynamics of nanoscale-labeled objects in solution. Current analysis of FCS data is largely based on the assumption that the labeled objects are stochastically displaced due to Brownian motion. However, this assumption is often invalid for microscale objects, since the motion of these objects is dominated by Stokes drag and settling or rising effects, rather than stochastic Brownian motion. To utilize the power of FCS for systems with nonstochastic displacements of objects, the collection and analysis of FCS data have to be reconceptualized. Here, we extended the applicability of FCS for the detection and analysis of periodically passing objects. Toward this end, we implemented droplet-based microfluidics, in which monodispersed droplets containing fluorescent marker are flowing equally spaced within microchannels. We show by simulations and experiments that FCS can sensitively quantify the flow-rates, variability, and content of rapidly passing droplets. This information can be derived at high temporal resolution, based on the intensity fluctuations generated by only 5–10 passing droplets. Moreover, by utilizing the periodicity of the flowing droplets for noise reduction by averaging, FCS can monitor accurately the droplets flow even if their fluorescence intensity is negligible. Hence, extending FCS for periodically passing objects converts it into a powerful analytical tool for high-throughput droplet-based microfluidics. Moreover, based on the principles described here, FCS can be straightforwardly applied for a variety of systems in which the passing of objects is periodic rather than stochastic.