Microfluidic production and characterization of biofunctionalized giant unilamellar vesicles for targeted intracellular cargo delivery.

Lipid-based vesicles have found widespread applications in the life sciences, allowing for fundamental insights into membrane-based processes in cell biology and as carrier systems for drug delivery purposes. So far, mostly small unilamellar vesicles (SUVs) with diameters of ~100 nm have been applied as carrier systems for biomedical applications. Despite this progress, several systematic limitations have arisen due to SUV dimensions, e.g., the size and total amount of applicable cargo is limited. Giant unilamellar vesicles (GUVs) might offer a pragmatic alternative for efficient cargo delivery. However, due to the lack of reliable high-throughput production technologies for GUV-carrier systems, only little is known about their interaction with cells. Here we present a microfluidic-based mechanical droplet-splitting pipeline for the production of carrier-GUVs with diameters of ~2 µm. The technology developed allows for highly efficient cargo loading and unprecedented control over the biological and physicochemical properties of GUV membranes. By generating differently charged (between -31 and + 28 mV), bioligand-conjugated (e.g. with E-cadherin, NrCam and antibodies) and PEG-conjugated GUVs, we performed a detailed investigation of attractive and repulsive GUV-cell interactions. Fine-tuning of these interactions allowed for targeted cellular GUV delivery. Moreover, we evaluated strategies for intracellular GUV cargo release by lysosomal escape mediated by the pH sensitive lipid DOBAQ, enabling cytoplasmic transmission. The presented GUV delivery technology and the systematic characterization of associated GUV-cell interactions could provide a means for more efficient drug administration and will pave the way for hitherto impossible approaches towards a targeted delivery of advanced cargo such as microparticles, viruses or macromolecular DNA-robots.

Droplet-Based Cytotoxicity Assay: Implementation of Time-Efficient Screening of Antitumor Activity of Natural Killer Cells.

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.

Electrically Driven Microengineered Bioinspired Soft Robots.

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.

Reconceptualizing Fluorescence Correlation Spectroscopy for Monitoring and Analyzing Periodically Passing Objects.

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.

Bioprinted 3D vascularized tissue model for drug toxicity analysis.

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 in vivo. 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 in vitro liver model system for future drug toxicity testing.

Engineered 3D Cardiac Fibrotic Tissue to Study Fibrotic Remodeling.

Activation of cardiac fibroblasts 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 cardiac fibroblasts when cultured on two-dimensional (2D) culture plates. In this study, a simplified three-dimensional (3D) hydrogel platform of contractile cardiac tissue, stimulated by transforming growth factor-ß1 (TGF-ß1), is presented to recapitulate a fibrogenic microenvironment. It is hypothesized that the quiescent state of cardiac fibroblasts 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 cardiac fibroblasts encapsulated within a mechanically engineered gelatin methacryloyl hydrogel, is developed. The study shows that cardiac fibroblasts maintain their quiescent phenotype in mechanically tuned hydrogels. Additionally, treatment with a beta-adrenergic agonist increases beating frequency, demonstrating physiologic-like behavior of the heart constructs. Subsequently, quiescent cardiac fibroblasts within the constructs are activated by the exogenous addition of TGF-ß1. The expression of fibrotic protein markers (and the functional changes in mechanical stiffness) in the fibrotic-like tissues are analyzed to validate the model. Overall, this 3D engineered culture model of contractile cardiac tissue enables controlled activation of cardiac fibroblasts, demonstrating the usability of this platform to study fibrotic remodeling.