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.

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.

Author Correction: An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

An optochemical tool for light-induced dissociation of adherens junctions to control mechanical coupling between cells.

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.

Hydrogel micropillars with integrin selective peptidomimetic functionalized nanopatterned tops: a new tool for the measurement of cell traction forces transmitted through αvß3- or α5ß1-integrins.

Poly(ethylene glycol) micropillars with gold nanopatterns on top are functionalized with two integrin selective ligands. This platform is a powerful new tool to determine the specific contribution of traction forces involved in cell adhesion mediated by α5ß1- and αvß3-integrins. Cells adherent via α5ß1-integrins have a tendency to exert higher maximum forces than cells adhering via αvß3-integrins.

Synthesis of nanostructured and biofunctionalized water-in-oil droplets as tools for homing T cells.

Activation, ex vivo expansion of T cells, differentiation into a regulatory subset, and its phenotype-specific high-throughput selection represent major challenges in immunobiology. In part, this is due to the lack of technical means to synthesize suitable 3D extracellular systems to imitate ex vivo the cellular interactions between T cells and antigen-presenting cells (APCs). In this study, we synthesized a new type of gold-linked surfactant and used a drop-based microfluidic device to develop and characterize novel nanostructured and specifically biofunctionalized droplets of water-in-oil emulsions as 3D APC analogues. Combining flexible biofunctionalization with the pliable physical properties of the nanostructured droplets provided this system with superior properties in comparison with previously reported synthetic APC analogues.

Cell adhesion and response to synthetic nanopatterned environments by steering receptor clustering and spatial location.

During adhesion and spreading, cells form micrometer-sized structures comprising transmembrane and intracellular protein clusters, giving rise to the formation of what is known as focal adhesions. Over the past two decades these structures have been extensively studied to elucidate their organization, assembly, and molecular composition, as well as to determine their functional role. Synthetic materials decorated with biological molecules, such as adhesive peptides, are widely used to induce specific cellular responses dependent on cell adhesion. Here, we focus on how surface patterning of such bioactive materials and organization at the nanoscale level has proven to be a useful strategy for mimicking both physical and chemical cues present in the extracellular space controlling cell adhesion and fate. This strategy for designing synthetic cellular environments makes use of the observation that most cell signaling events are initiated through recruitment and clustering of transmembrane receptors by extracellular-presented signaling molecules. These systems allow for studying protein clustering in cells and characterizing the signaling response induced by, e.g., integrin activation. We review the findings about the regulation of cell adhesion and focal adhesion assembly by micro- and nanopatterns and discuss the possible use of substrate stiffness and patterning in mimicking both physical and chemical cues of the extracellular space.

Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands.

Integrin-mediated adhesion is regulated by multiple features of the adhesive surface, including its chemical composition, topography, and physical properties. In this study we investigated integrin lateral clustering, as a mechanism to control integrin functions, by characterizing the effect of nanoscale variations in the spacing between adhesive RGD ligands on cell spreading, migration, and focal adhesion dynamics. For this purpose, we used nanopatterned surfaces, containing RGD-biofunctionalized gold dots, surrounded by passivated gaps. By varying the spacing between the dots, we modulated the clustering of the associated integrins. We show that cell-surface attachment is not sensitive to pattern density, whereas the formation of stable focal adhesions and persistent spreading is. Thus cells plated on a 108-nm-spaced pattern exhibit delayed spreading with repeated protrusion-retraction cycles compared to cells growing on a 58-nm pattern. Cell motility on these surfaces is erratic and nonpersistent, leaving thin membrane tethers bound to the RGD pattern. Dynamic molecular profiling indicated that the adhesion sites formed with the 108-nm pattern undergo rapid turnover and contain reduced levels of zyxin. These findings indicate that a critical RGD density is essential for the establishment of mature and stable integrin adhesions, which, in turn, induce efficient cell spreading and formation of focal adhesions.

Geometric organization of the extracellular matrix in the control of integrin-mediated adhesion and cell function in osteoblasts.

Cell-extracellular matrix (ECM) interactions play a central role in tissue architecture and turnover. Particularly, integrin-mediated cell adhesion participates in biochemical and physical signals. The aim of this study is to investigate the importance of ECM organization for alveolar bone osteoblasts adhesion and to determine the effects on cell functions such as collagen and fibronectin production. By applying new concepts from the nanotechnology to biological systems, we have developed materials decorated with nano-patterns of peptides of the ECM arranged at a distance of 58 or 73 nm. On these surfaces, human osteoblasts from alveolar bone were cultured for 1-96 hr and examined by video and fluorescence microscopy. Protein quantification by western blotting and gene expression by RT-PCR were also performed. Good cell adhesion and spreading was observed on the 58 nm pattern after 30 min, while weak adhesion and increased motility was evident in osteoblasts on the 73 nm pattern, leading to alteration of cell shape and reduction of cell area after 24 hr. Moreover, cells on the 73 nm did not form focal adhesions and failed to organize the cytoskeleton. After 96 hr in culture, osteoblasts on the 73 nm retained intracellular collagen and produced a disorganized fibronectin network. Osteoblast adhesion and intra-and extra-cellular molecules reorganization are regulated not only by the composition but also by the structure of the extracellular environment. Our novel in vitro system makes it possible to elucidate some of the mechanisms necessary for the maintenance of tissue architecture and mechanical strength, as well as for the design of artificial materials for future clinical applications.