Evaluation of Acoustophoretic and Dielectrophoretic Forces for Droplet Injection in Droplet-Based Microfluidic Devices.

Acoustophoretic forces have been successfully implemented into droplet-based microfluidic devices to manipulate droplets. These acoustophoretic forces in droplet microfluidic devices are typically generated as in acoustofluidic devices through transducer actuation of a piezoelectric substrate such as lithium niobate (LiNbO3), which is inherently accompanied by the emergence of electrical fields. Understanding acoustophoretic versus dielectrophoretic forces produced by electrodes and transducers within active microfluidic devices is important for the optimization of device performance during design iterations. In this case study, we design microfluidic devices with a droplet injection module and report an experimental strategy to deduce the respective contribution of the acoustophoretic versus dielectrophoretic forces for the observed droplet injection. Our PDMS-based devices comprise a standard oil-in-water droplet-generating module connected to a T-junction injection module featuring actuating electrodes. We use two different electrode geometries produced within the same PDMS slab as the droplet production/injection channels by filling low-melting-point metal alloy into channels that template the electrode geometries. When these electrodes are constructed on LiNbO3 as the substrate, they have a dual function as a piezoelectric transducer, which we call embedded liquid metal interdigitated transducers (elmIDTs). To decipher the contribution of acoustophoretic versus dielectrophoretic forces, we build the same devices on either piezoelectric LiNbO3 or nonpiezo active glass substrates with different combinations of physical device characteristics (i.e., elmIDT geometry and alignment) and operate in a range of phase spaces (i.e., frequency, voltage, and transducer polarity). We characterize devices using techniques such as laser Doppler vibrometry (LDV) and infrared imaging, along with evaluating droplet injection for our series of device designs, constructions, and operating parameters. Although we find that LiNbO3 device designs generate acoustic fields, we demonstrate that droplet injection occurs only due to dielectrophoretic forces. We deduce that droplet injection is caused by the coupled dielectrophoretic forces arising from the operation of elmIDTs rather than by acoustophoretic forces for this specific device design. We arrive at this conclusion because equivalent droplet injection occurs without the presence of an acoustic field using the same electrode designs on nonpiezo active glass substrate devices. This work establishes a methodology to pinpoint the major contributing force of droplet manipulation in droplet-based acoustomicrofluidics.

An Efficient Method for the Production of High-Purity Bioinspired Large Unilamellar Vesicles.

In order to recapitulate complex eukaryotic compartmentalization, synthetic biology aims to recreate cellular membrane-lined compartments from the bottom-up. Many important cellular organelles and cell-produced extracellular vesicles are in the size range of several hundreds of nanometers. Although attaining a fundamental characterization and mimicry of their cellular functions is a compelling goal, the lack of methods for controlled vesicle formation in this size range has hindered full understanding. Here, we show the optimization of a simple and efficient protocol for the production of large unilamellar vesicles (LUVs) with a median diameter in the range of 450-550 nm with high purity. Importantly, we rely on commercial reagents and common laboratory equipment. We thoroughly characterize the influence of different experimental parameters on the concentration and size of the resulting vesicles and assess changes in their lipid composition and surface charge. We provide guidance for researchers to optimize LUV production further to suit specific applications.

Vascularized 3D Human Skin Models in the Forefront of Dermatological Research.

In vitro engineered skin models are emerging as an alternative platform to reduce and replace animal testing in dermatological research. Despite the progress made in recent years, considerable challenges still exist for the inclusion of diverse cell types within skin models. Blood vessels, in particular, are essential in maintaining tissue homeostasis and are one of many primary contributors to skin disease inception and progression. Substantial efforts in the past have allowed the successful fabrication of vascularized skin models that are currently utilized for disease modeling and drugs/cosmetics testing. This review first discusses the need for vascularization within tissue-engineered skin models, highlighting their role in skin grafting and disease pathophysiology. Second, the review spotlights the milestones and recent progress in the fabrication and utilization of vascularized skin models. Additionally, advances including the use of bioreactors, organ-on-a-chip devices, and organoid systems are briefly explored. Finally, the challenges and future outlook for vascularized skin models are addressed.

Bottom-up Assembled Synthetic SARS-CoV-2 Miniviruses Reveal Lipid Membrane Affinity of Omicron Variant Spike Glycoprotein.

The ongoing COVID-19 pandemic has been brought on by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike glycoprotein (S), which decorates the viral envelope forming a corona, is responsible for the binding to the angiotensin-converting enzyme 2 (ACE2) receptor and initiating the infection. In comparison to previous variants, Omicron S presents additional binding sites as well as a more positive surface charge. These changes hint at additional molecular targets for interactions between virus and cell, such as the cell membrane or proteoglycans on the cell surface. Herein, bottom-up assembled synthetic SARS-CoV-2 miniviruses (MiniVs), with a lipid composition similar to that of infectious particles, are implemented to study and compare the binding properties of Omicron and Alpha variants. Toward this end, a systematic functional screening is performed to study the binding ability of Omicron and Alpha S proteins to ACE2-functionalized and nonfunctionalized planar supported lipid bilayers. Moreover, giant unilamellar vesicles are used as a cell membrane model to perform competitive interaction assays of the two variants. Finally, two cell lines with and without presentation of the ACE2 receptor are used to confirm the binding properties of the Omicron and Alpha MiniVs to the cellular membrane. Altogether, the results reveal a significantly higher affinity of Omicron S toward both the lipid membrane and ACE2 receptor. The research presented here highlights the advantages of creating and using bottom-up assembled SARS-CoV-2 viruses to understand the impact of changes in the affinity of S for ACE2 in infection studies.

Bottom-Up Assembly of Bioinspired, Fully Synthetic Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid membrane-enclosed compartments released by cells for intercellular communication in homeostasis and disease. Studies have shown great therapeutic potential of EVs, including but not limited to regenerative and immunomodulatory therapies. Additionally, EVs are promising next-generation drug delivery systems due to their biocompatibility, low immunogenicity, and inherent target specificity. However, clinical application of EVs is so far limited due to challenges in scaling up production, high heterogeneity, batch-to-batch variation, and limited control over composition. Although attaining a fundamental characterization of EVs' functions is a compelling goal, these limitations have hindered a full understanding. Therefore, there is rising interest in exploiting the beneficial properties of EVs while gaining better control over their production and composition. Herein, we describe a method for the bottom-up assembly of bioinspired, fully synthetic vesicles that mimic the most important biophysical and biochemical properties of natural EVs.

Compact holographic sound fields enable rapid one-step assembly of matter in 3D.

Acoustic waves exert forces when they interact with matter. Shaping ultrasound fields precisely in 3D thus allows control over the force landscape and should permit particulates to fall into place to potentially form whole 3D objects in "one shot." This is promising for rapid prototyping, most notably biofabrication, since conventional methods are typically slow and apply mechanical or chemical stress on biological cells. Here, we realize the generation of compact holographic ultrasound fields and demonstrate the one-step assembly of matter using acoustic forces. We combine multiple holographic fields that drive the contactless assembly of solid microparticles, hydrogel beads, and biological cells inside standard labware. The structures can be fixed via gelation of the surrounding medium. In contrast to previous work, this approach handles matter with positive acoustic contrast and does not require opposing waves, supporting surfaces or scaffolds. We envision promising applications of 3D holographic ultrasound fields in tissue engineering and additive manufacturing.

Design and Development of Extracellular Matrix Protein-Based Microcapsules as Tools for Bacteria Investigation.

The extracellular matrix (ECM) plays an immense role in the homeostasis of tissues and organs, can function as a barrier for infectious agents, but is also exploited by pathogens during infection. Therefore, the development of well-defined 3D ECM models in the form of microcapsules to elucidate the interactions between ECM components and pathogens in confinement and study disease infectivity is important, albeit challenging. Current limitations are mainly attributed to the lack of biocompatible methods for the production of protein-based microcapsules. Herein, hollow ECM-based microcapsules from laminin-111 or laminin-111/collagen IV are generated to investigate the behavior of organisms within confined 3D extracellular matrices. Microcapsules are created using water-in-oil emulsion droplets stabilized by block copolymer surfactants as templates for the charge-mediated attraction of laminin or laminin-collagen proteins to the droplets' inner periphery, allowing for the formation of modular ECM-based microcapsules with tunable biophysical and biochemical properties and organism encapsulation. The release of E. coli-laden ECM-based protein microcapsules into a physiological environment revealed differences in the dynamic behavior of E. coli depending on the constitution of the surrounding ECM protein matrix. The developed ECM-based protein microcapsules have the potential to be implemented in several biomedical applications, including the design of in vitro infection models.

Artificial Cytoskeleton Assembly for Synthetic Cell Motility.

Imitation of cellular processes in cell-like compartments is a current research focus in synthetic biology. Here, a method is introduced for assembling an artificial cytoskeleton in a synthetic cell model system based on a poly(N-isopropyl acrylamide) (PNIPAM) composite material. Toward this end, a PNIPAM-based composite material inside water-in-oil droplets that are stabilized with PNIPAM-functionalized and commercial fluorosurfactants is introduced. The temperature-mediated contraction/release behavior of the PNIPAM-based cytoskeleton is investigated. The reversibility of the PNIPAM transition is further examined in bulk and in droplets and it could be shown that hydrogel induced deformation could be used to controllably manipulate droplet-based synthetic cell motility upon temperature changes. It is envisioned that a combination of the presented artificial cytoskeleton with naturally occurring components might expand the bandwidth of the bottom-up synthetic biology.

Bottom-up assembly of viral replication cycles.

Bottom-up synthetic biology provides new means to understand living matter by constructing minimal life-like systems. This principle can also be applied to study infectious diseases. Here we summarize approaches and ethical considerations for the bottom-up assembly of viral replication cycles.

Bottom-up assembly of target-specific cytotoxic synthetic cells.

Immune vigilance ensures body integrity by eliminating malignant cells through the complex but coordinated cooperation of highly diversified lymphocytes populations. The sheer complexity of the immune system has slowed development of immunotherapies based on top-down genetic engineering of lymphocytes. In contrast, bottom-up assembly of synthetic cell compartments has contributed novel engineering strategies to reverse engineer and understand cellular phenomena as molecularly defined systems. Towards reducing the complexity of immunological systems, herein, a bottom-up approach for controlled assembly of fully-synthetic immune-inspired cells from predefined molecular components based on giant unilamellar vesicles is described. For construction of target-specific cytotoxic immune cells, the Fas-ligand-based apoptosis-inducing immune cell module is combined with an antibody-mediated cellular cytotoxicity-inspired system. The designed immune cells identify leukemia cells by specific surface antigens. Subsequently, they form stable attachments sites and eliminate their targets by induction of apoptosis. A structural and functional characterization of the synthetic immune cells by means of microfluidics, live cell, confocal and electron microscopy, dynamic light scattering as well as flow cytometry is presented. This study demonstrates the bioinspired construction of effector immune cells from defined molecular building blocks, enabling learning-by-building approaches in synthetic immunology.

Synthetic cells with self-activating optogenetic proteins communicate with natural cells.

Development of regulated cellular processes and signaling methods in synthetic cells is essential for their integration with living materials. Light is an attractive tool to achieve this, but the limited penetration depth into tissue of visible light restricts its usability for in-vivo applications. Here, we describe the design and implementation of bioluminescent intercellular and intracellular signaling mechanisms in synthetic cells, dismissing the need for an external light source. First, we engineer light generating SCs with an optimized lipid membrane and internal composition, to maximize luciferase expression levels and enable high-intensity emission. Next, we show these cells' capacity to trigger bioprocesses in natural cells by initiating asexual sporulation of dark-grown mycelial cells of the fungus Trichoderma atroviride. Finally, we demonstrate regulated transcription and membrane recruitment in synthetic cells using bioluminescent intracellular signaling with self-activating fusion proteins. These functionalities pave the way for deploying synthetic cells as embeddable microscale light sources that are capable of controlling engineered processes inside tissues.

Vesicle Induced Receptor Sequestration: Mechanisms behind Extracellular Vesicle-Based Protein Signaling.

Extracellular vesicles (EVs) are fundamental for proper physiological functioning of multicellular organisms. By shuttling nucleic acids and proteins between cells, EVs regulate a plethora of cellular processes, especially those involved in immune signalling. However, the mechanistic understanding concerning the biophysical principles underlying EV-based communication is still incomplete. Towards holistic understanding, particular mechanisms explaining why and when cells apply EV-based communication and how protein-based signalling is promoted by EV surfaces are sought. Here, the authors study vesicle-induced receptor sequestration (VIRS) as a universal mechanism augmenting the signalling potency of proteins presented on EV-membranes. By bottom-up reconstitution of synthetic EVs, the authors show that immobilization of the receptor ligands FasL and RANK on EV-like vesicles, increases their signalling potential by more than 100-fold compared to their soluble forms. Moreover, the authors perform diffusion simulations within immunological synapses to compare receptor activation between soluble and EV-presented proteins. By this the authors propose vesicle-triggered local clustering of membrane receptors as the principle structural mechanism underlying EV-based protein presentation. The authors conclude that EVs act as extracellular templates promoting the local aggregation of membrane receptors at the EV contact site, thereby fostering inter-protein interactions. The results uncover a potentially universal mechanism explaining the unique structural profit of EV-based intercellular signalling.

Synthetic virions reveal fatty acid-coupled adaptive immunogenicity of SARS-CoV-2 spike glycoprotein.

SARS-CoV-2 infection is a major global public health concern with incompletely understood pathogenesis. The SARS-CoV-2 spike (S) glycoprotein comprises a highly conserved free fatty acid binding pocket (FABP) with unknown function and evolutionary selection advantage1,2. Deciphering FABP impact on COVID-19 progression is challenged by the heterogenous nature and large molecular variability of live virus. Here we create synthetic minimal virions (MiniVs) of wild-type and mutant SARS-CoV-2 with precise molecular composition and programmable complexity by bottom-up assembly. MiniV-based systematic assessment of S free fatty acid (FFA) binding reveals that FABP functions as an allosteric regulatory site enabling adaptation of SARS-CoV-2 immunogenicity to inflammation states via binding of pro-inflammatory FFAs. This is achieved by regulation of the S open-to-close equilibrium and the exposure of both, the receptor binding domain (RBD) and the SARS-CoV-2 RGD motif that is responsible for integrin co-receptor engagement. We find that the FDA-approved drugs vitamin K and dexamethasone modulate S-based cell binding in an FABP-like manner. In inflammatory FFA environments, neutralizing immunoglobulins from human convalescent COVID-19 donors lose neutralization activity. Empowered by our MiniV technology, we suggest a conserved mechanism by which SARS-CoV-2 dynamically couples its immunogenicity to the host immune response.

pH-Triggered Assembly of Endomembrane Multicompartments in Synthetic Cells.

By using electrostatic interactions as driving force to assemble vesicles, the droplet-stabilized method was recently applied to reconstitute and encapsulate proteins, or compartments, inside giant unilamellar vesicles (GUVs) to act as minimal synthetic cells. However, the droplet-stabilized approach exhibits low production efficiency associated with the troublesome release of the GUVs from the stabilized droplets, corresponding to a major hurdle for the droplet-stabilized approach. Herein, we report the use of pH as a potential trigger to self-assemble droplet-stabilized GUVs (dsGUVs) by either bulk or droplet-based microfluidics. Moreover, pH enables the generation of compartmentalized GUVs with flexibility and robustness. By co-encapsulating pH-sensitive small unilamellar vesicles (SUVs), negatively charged SUVs, and/or proteins, we show that acidification of the droplets efficiently produces dsGUVs while sequestrating the co-encapsulated material. Most importantly, the pH-mediated assembly of dsGUVs significantly improves the production efficiency of free-standing GUVs (i.e., released from the stabilizing-droplets) compared to its previous implementation.

Bottom-up assembly of biomedical relevant fully synthetic extracellular vesicles.

Extracellular vesicles (EVs) are fundamental for intercellular communication and influence nearly every process in cell physiology. However, because of their intricate molecular complexity, quantitative knowledge on their signaling mechanisms is missing, particularly impeding their therapeutic application. We used a complementary and quantitative engineering approach based on sequential synthetic bottom-up assembly of fully functional EVs with precisely controlled lipid, protein, and RNA composition. We show that the functionalities of synthetic EVs are analogous to natural EVs and demonstrate their programmable therapeutic administration for wound healing and neovascularization therapy. We apply transcriptome profiling to systematically decode synergistic effects between individual EV constituents, enabling analytical dissection and a fundamental understanding of EV signaling.

Proton gradients from light-harvesting E. coli control DNA assemblies for synthetic cells.

Bottom-up and top-down approaches to synthetic biology each employ distinct methodologies with the common aim to harness living systems. Here, we realize a strategic merger of both approaches to convert light into proton gradients for the actuation of synthetic cellular systems. We genetically engineer E. coli to overexpress the light-driven inward-directed proton pump xenorhodopsin and encapsulate them in artificial cell-sized compartments. Exposing the compartments to light-dark cycles, we reversibly switch the pH by almost one pH unit and employ these pH gradients to trigger the attachment of DNA structures to the compartment periphery. For this purpose, a DNA triplex motif serves as a nanomechanical switch responding to the pH-trigger of the E. coli. When DNA origami plates are modified with the pH-sensitive triplex motif, the proton-pumping E. coli can trigger their attachment to giant unilamellar lipid vesicles (GUVs) upon illumination. A DNA cortex is formed upon DNA origami polymerization, which sculpts and deforms the GUVs. We foresee that the combination of bottom-up and top down approaches is an efficient way to engineer synthetic cells.

Can Bottom-Up Synthetic Biology Generate Advanced Drug-Delivery Systems?

Creating a magic bullet that can selectively kill cancer cells while sparing nearby healthy cells remains one of the most ambitious objectives in pharmacology. Nanomedicine, which relies on the use of nanotechnologies to fight disease, was envisaged to fulfill this coveted goal. Despite substantial progress, the structural complexity of therapeutic vehicles impedes their broad clinical application. Novel modular manufacturing approaches for engineering programmable drug carriers may be able to overcome some fundamental limitations of nanomedicine. We discuss how bottom-up synthetic biology principles, empowered by microfluidics, can palliate current drug carrier assembly limitations, and we demonstrate how such a magic bullet could be engineered from the bottom up to ultimately improve clinical outcomes for patients.

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.

Engineering Light-Responsive Contractile Actomyosin Networks with DNA Nanotechnology.

External control and precise manipulation is key for the bottom-up engineering of complex synthetic cells. Minimal actomyosin networks have been reconstituted into synthetic cells; however, their light-triggered symmetry breaking contraction has not yet been demonstrated. Here, light-activated directional contractility of a minimal synthetic actomyosin network inside microfluidic cell-sized compartments is engineered. Actin filaments, heavy-meromyosin-coated beads, and caged ATP are co-encapsulated into water-in-oil droplets. ATP is released upon illumination, leading to a myosin-generated force which results in a motion of the beads along the filaments and hence a contraction of the network. Symmetry breaking is achieved using DNA nanotechnology to establish a link between the network and the compartment periphery. It is demonstrated that the DNA-linked actin filaments contract to one side of the compartment forming actin asters and quantify the dynamics of this process. This work exemplifies that an engineering approach to bottom-up synthetic biology, combining biological and artificial elements, can circumvent challenges related to active multi-component systems and thereby greatly enrich the complexity of synthetic cellular systems.