Synthetic Cells and Molecules in Cellular Immunotherapy.

Cellular immunotherapy has emerged as an exciting strategy for cancer treatment, as it aims to enhance the body's immune response to tumor cells by engineering immune cells and designing synthetic molecules from scratch. Because of the cytotoxic nature, abundance in peripheral blood, and maturation of genetic engineering techniques, T cells have become the most commonly engineered immune cells to date. Represented by chimeric antigen receptor (CAR)-T therapy, T cell-based immunotherapy has revolutionized the clinical treatment of hematological malignancies. However, serious side effects and limited efficacy in solid tumors have hindered the clinical application of cellular immunotherapy. To address these limitations, various innovative strategies regarding synthetic cells and molecules have been developed. On one hand, some cytotoxic immune cells other than T cells have been engineered to explore the potential of targeted elimination of tumor cells, while some adjuvant cells have also been engineered to enhance the therapeutic effect. On the other hand, diverse synthetic cellular components and molecules are added to engineered immune cells to regulate their functions, promoting cytotoxic activity and restricting side effects. Moreover, novel bioactive materials such as hydrogels facilitating the delivery of therapeutic immune cells have also been applied to improve the efficacy of cellular immunotherapy. This review summarizes the innovative strategies of synthetic cells and molecules currently available in cellular immunotherapies, discusses the limitations, and provides insights into the next generation of cellular immunotherapies.

Functional Integration of Synthetic Cells into 3D Microfluidic Devices for Artificial Organ-On-Chip Technologies.

Microfluidics plays a pivotal role in organ-on-chip technologies and in the study of synthetic cells, especially in the development and analysis of artificial cell models. However, approaches that use synthetic cells as integral functional components for microfluidic systems to shape the microenvironment of natural living cells cultured on-chip are not explored. Here, colloidosome-based synthetic cells are integrated into 3D microfluidic devices, pioneering the concept of synthetic cell-based microenvironments for organs-on-chip. Methods are devised to create dense and stable networks of silica colloidosomes, enveloped by supported lipid bilayers, within microfluidic channels. These networks promote receptor-ligand interactions with on-chip cultured cells. Furthermore, a technique is introduced for the controlled release of growth factors from the synthetic cells into the channels, using a calcium alginate-based hydrogel formation within the colloidosomes. To demonstrate the potential of the technology, a modular plug-and-play lymph-node-on-a-chip prototype that guides the expansion of primary human T cells by stimulating receptor ligands on the T cells and modulating their cytokine environment is presented. This integration of synthetic cells into microfluidic systems offers a new direction for organ-on-chip technologies and suggests further avenues for exploration in potential therapeutic applications.

Optimizing in vitro expression balance of central dogma-related genes using parallel reaction monitoring.

The creation of a self-replicating synthetic cell is an essential to understand life self-replication. One method to create self-replicating artificial cells is to reconstitute the self-replication system of living organisms in vitro. In a living cell, self-replication is achieved via a system called the autonomous central dogma, a system in which central dogma-related factors are autonomously synthesized and genome replication, transcription, and translation are driven by nascent factors. Various studies to reconstitute some processes of the autonomous central dogma in vitro have been conducted. However, in vitro reconstitution of the entire autonomous central dogma system is difficult as it requires balanced expression of several related genes. Therefore, we developed a method to simultaneously quantify and optimize the in vitro expression balance of multiple genes. First, we developed a quantitative mass spectrometry method targeting genome replication-related proteins as a model of central dogma-related factors and acquired in vitro expression profiles of these genes. Additionally, we demonstrated that the in vitro expression balance of these genes can be easily optimized by adjusting the input gene ratio based on the data obtained by the developed method. This study facilitated the easy optimization of the in vitro expression balance of multiple genes. Therefore, extending the scope of this method to other central dogma-related factors will accelerate attempts of self-replicating synthetic cells creation.

Soft Synthetic Cells with Mobile Membrane Ligands for Ex Vivo Expansion of Therapy-Relevant T Cell Phenotypes.

The expansion of T cells ex vivo is crucial for effective immunotherapy but currently limited by a lack of expansion approaches that closely mimic in vivo T cell activation. Taking inspiration from bottom-up synthetic biology, a new synthetic cell technology is introduced based on dispersed liquid-liquid phase-separated droplet-supported lipid bilayers (dsLBs) with tunable biochemical and biophysical characteristics, as artificial antigen presenting cells (aAPCs) for ex vivo T cell expansion. These findings obtained with the dsLB technology reveal three key insights: first, introducing laterally mobile stimulatory ligands on soft aAPCs promotes expansion of IL-4/IL-10 secreting regulatory CD8+ T cells, with a PD-1 negative phenotype, less prone to immune suppression. Second, it is demonstrated that lateral ligand mobility can mask differential T cell activation observed on substrates of varying stiffness. Third, dsLBs are applied to reveal a mechanosensitive component in bispecific Her2/CD3 T cell engager-mediated T cell activation. Based on these three insights, lateral ligand mobility, alongside receptor- and mechanosignaling, is proposed to be considered as a third crucial dimension for the design of ex vivo T cell expansion technologies.

Artificial Receptor in Synthetic Cells Performs Transmembrane Activation of Proteolysis.

The design of artificial, synthetic cells is a fundamentally important and fast-developing field of science. Of the diverse attributes of cellular life, artificial transmembrane signaling across the biomolecular barriers remains a high challenge with only a few documented successes. Herein, the study achieves signaling across lipid bilayers and connects an exofacial enzymatic receptor activation to an intracellular biochemical catalytic response using an artificial receptor. The mechanism of signal transduction for the artificial receptor relies on the triggered decomposition of a self-immolative linker. Receptor activation ensues its head-to-tail decomposition and the release of a secondary messenger molecule into the internal volume of the synthetic cell. Transmembrane signaling is demonstrated in synthetic cells based on liposomes and mammalian cell-sized giant unilamellar vesicles and illustrates receptor performance in cell mimics with a diverse size and composition of the lipid bilayer. In giant unilamellar vesicles, transmembrane signaling connects exofacial receptor activation with intracellular activation of proteolysis. Taken together, the results of this study take a step toward engineering receptor-mediated, responsive behavior in synthetic cells.

Synthetic Vesicles for Sustainable Energy Recycling and Delivery of Building Blocks for Lipid Biosynthesis†.

ATP is a universal energy currency that is essential for life. l-Arginine degradation via deamination is an elegant way to generate ATP in synthetic cells, which is currently limited by a slow l-arginine/l-ornithine exchange. We are now implementing a new antiporter with better kinetics to obtain faster ATP recycling. We use l-arginine-dependent ATP formation for the continuous synthesis and export of glycerol 3-phosphate by including glycerol kinase and the glycerol 3-phosphate/Pi antiporter. Exported glycerol 3-phosphate serves as a precursor for the biosynthesis of phospholipids in a second set of vesicles, which forms the basis for the expansion of the cell membrane. We have therefore developed an out-of-equilibrium metabolic network for ATP recycling, which has been coupled to lipid synthesis. This feeder-utilizer system serves as a proof-of-principle for the systematic buildup of synthetic cells, but the vesicles can also be used to study the individual reaction networks in confinement.

Synthetic Cells from Droplet-Based Microfluidics for Biosensing and Biomedical Applications.

Synthetic cells function as biological mimics of natural cells by mimicking salient features of cells such as metabolism, response to stimuli, gene expression, direct metabolism, and high stability. Droplet-based microfluidic technology presents the opportunity for encapsulating biological functional components in uni-lamellar liposome or polymer droplets. Verified by its success in the fabrication of synthetic cells, microfluidic technology is widely replacing conventional labor-intensive, expensive, and sophisticated techniques justified by its ability to miniaturize and perform batch production operations. In this review, an overview of recent research on the preparation of synthetic cells through droplet-based microfluidics is provided. Different synthetic cells including lipid vesicles (liposome), polymer vesicles (polymersome), coacervate microdroplets, and colloidosomes, are systematically discussed. Efforts are then made to discuss the design of a variety of microfluidic chips for synthetic cell preparation since the combination of microfluidics with bottom-up synthetic biology allows for reproductive and tunable construction of batches of synthetic cell models from simple structures to higher hierarchical structures. The recent advances aimed at exploiting them in biosensors and other biomedical applications are then discussed. Finally, some perspectives on the challenges and future developments of synthetic cell research with microfluidics for biomimetic science and biomedical applications are provided.

Artificial Methylotrophic Cells via Bottom-Up Integration of a Methanol-Utilizing Pathway.

Methanol has gained substantial attention as a substrate for biomanufacturing due to plentiful stocks and nonreliance on agriculture, and it can be sourced renewably. However, due to inevitable complexities in cell metabolism, microbial methanol conversion requires further improvement before industrial applicability. Here, we present a novel, parallel strategy using artificial cells to provide a simplified and well-defined environment for methanol utilization as artificial methylotrophic cells. We compartmentalized a methanol-utilizing enzyme cascade, including NAD-dependent methanol dehydrogenase (Mdh) and pyruvate-dependent aldolase (KHB aldolase), in cell-sized lipid vesicles using the inverted emulsion method. The reduction of cofactor NAD+ to NADH was used to quantify the conversion of methanol within individual artificial methylotrophic cells via flow cytometry. Compartmentalization of the reaction cascade in liposomes led to a 4-fold higher NADH production compared with bulk enzyme experiments, and the incorporation of KHB aldolase facilitated another 2-fold increase above the Mdh-only reaction. This methanol-utilizing platform can serve as an alternative route to speed up methanol biological conversion, eventually shifting sugar-based bioproduction toward a sustainable methanol bioeconomy.

Comprehensive Parameter Space Mapping of Cell Cycle Dynamics under Network Perturbations.

Studies of quantitative systems and synthetic biology have extensively utilized models to interpret data, make predictions, and guide experimental designs. However, models often simplify complex biological systems and lack experimentally validated parameters, making their reliability in perturbed systems unclear. Here, we developed a droplet-based synthetic cell system to continuously tune parameters at the single-cell level in multiple dimensions with full dynamic ranges, providing an experimental framework for global parameter space scans. We systematically perturbed a cell-cycle oscillator centered on cyclin-dependent kinase (Cdk1), enabling comprehensive mapping of period landscapes in response to network perturbations. The data allowed us to challenge existing models and refine a new model that matches the observed response. Our analysis demonstrated that Cdk1 positive feedback inhibition restricts the cell cycle frequency range, confirming model predictions; furthermore, it revealed new cellular responses to the inhibition of the Cdk1-counteracting phosphatase PP2A: monomodal or bimodal distributions across varying inhibition levels, underscoring the complex nature of cell cycle regulation that can be explained by our model. This comprehensive perturbation platform may be generalizable to exploring other complex dynamic systems.

Preparing for the future of precision medicine: synthetic cell drug regulation.

Synthetic cells are a novel class of cell-like bioreactors, offering the potential for unique advancements in synthetic biology and biomedicine. To realize the potential of those technologies, synthetic cell-based drugs need to go through the drug approval pipeline. Here, we discussed several regulatory challenges, both unique to synthetic cells, as well as challenges typical for any new biomedical technology. Overcoming those difficulties could bring transformative therapies to the market and will create a path to the development and approval of cutting-edge synthetic biology therapies. Graphical Abstract.

Dynamics of giant vesicle assembly from thin lipid films.

MOTIVATION: Giant unilamellar vesicles (GUVs), cell-like synthetic micrometer size structures, assemble when thin lipid films are hydrated in aqueous solutions. Quantitative measurements of static yields and distribution of sizes of GUVs obtained from thin film hydration methods were recently reported. Dynamic data such as the time evolution of yields and distribution of sizes, however, is not known. Dynamic data can provide insights into the assembly pathway of GUVs and guidelines for choosing conditions to obtain populations with desired size distributions. APPROACH: We develop the 'stopped-time' technique to characterize the time evolution of the distribution of sizes and molar yields of populations of free-floating GUVs. We additionally capture high resolution time-lapse images of surface-attached GUV buds on the lipid films. We systematically study the dynamics of assembly of GUVs from three widely used thin film hydration methods, PAPYRUS (Paper-Abetted amPhiphile hYdRation in aqUeous Solutions), gentle hydration, and electroformation. FINDINGS: We find that the molar yield versus time curves of GUVs demonstrate a characteristic sigmoidal shape, with an initial yield, a transient, and then a steady state plateau for all three methods. The population of GUVs showed a right-skewed distribution of diameters. The variance of the distributions increased with time. The systems reached steady state within 120 min. We rationalize the dynamics using the thermodynamically motivated budding and merging (BNM) model. These results further the understanding of lipid dynamics and provide for the first-time practical parameters to tailor the production of GUVs of specific sizes for applications.

Cell-Free Expressed Membraneless Organelles Inhibit Translation in Synthetic Cells.

Compartments within living cells create specialized microenvironments, allowing multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation such as P-granules and nucleoli. Synthetic minimal cells are widely used to study many natural processes, including organelle formation. In this work, synthetic cells expressing artificial membrane-less organelles that inhibit translation are described. RGG-GFP-RGG, a phase-separating protein derived from Caenorhabditis elegans P-granules, is expressed by cell-free transcription and translation, forming artificial membraneless organelles that can sequester RNA and reduce protein expression in synthetic cells. The introduction of artificial membrane-less organelles creates complex microenvironments within the synthetic cell cytoplasm and functions as a tool to inhibit protein expression in synthetic cells. The engineering of compartments within synthetic cells furthers the understanding of the evolution and function of natural organelles and facilitates the creation of more complex and multifaceted synthetic lifelike systems.

Controlled exchange of protein and nucleic acid signals from and between synthetic minimal cells.

Synthetic minimal cells are a class of bioreactors that have some, but not all, functions of live cells. Here, we report a critical step toward the development of a bottom-up minimal cell: cellular export of functional protein and RNA products. We used cell-penetrating peptide tags to translocate payloads across a synthetic cell vesicle membrane. We demonstrated efficient transport of active enzymes and transport of nucleic acid payloads by RNA-binding proteins. We investigated influence of a concentration gradient alongside other factors on the efficiency of the translocation, and we show a method to increase product accumulation in one location. We demonstrate the use of this technology to engineer molecular communication between different populations of synthetic cells, to exchange protein and nucleic acid signals. The synthetic minimal cell production and export of proteins or nucleic acids allows experimental designs that approach the complexity and relevancy of natural biological systems. A record of this paper's transparent peer review process is included in the supplemental information.

Synthetic control of living cells by intracellular polymerization.

An emerging cellular engineering method creates synthetic polymer matrices inside cells. By contrast with classical genetic, enzymatic, or radioactive techniques, this materials-based approach introduces non-natural polymers inside cells, thus modifying cellular states and functionalities. Here, we cover various materials and chemistries that have been exploited to create intracellular polymer matrices. In addition, we discuss emergent cellular properties due to the intracellular polymerization, including nonreplicating but active metabolism, maintenance of membrane integrity, and resistance to environmental stressors. We also discuss past work and future opportunities for developing and applying synthetic cells that contain intracellular polymers. The materials-based approach will usher in new applications of synthetic cells for broad biotechnological applications.

Cell Free Expression in Proteinosomes Prepared from Native Protein-PNIPAAm Conjugates.

Towards the goal of building synthetic cells from the bottom-up, the establishment of micrometer-sized compartments that contain and support cell free transcription and translation that couple cellular structure to function is of critical importance. Proteinosomes, formed from crosslinked cationized protein-polymer conjugates offer a promising solution to membrane-bound compartmentalization with an open, semi-permeable membrane. Critically, to date, there has been no demonstration of cell free transcription and translation within water-in-water proteinosomes. Herein, a novel approach to generate proteinosomes that can support cell free transcription and translation is presented. This approach generates proteinosomes directly from native protein-polymer (BSA-PNIPAAm) conjugates. These native proteinosomes offer an excellent alternative as a synthetic cell chassis to other membrane bound compartments. Significantly, the native proteinosomes are stable under high salt conditions that enables the ability to support cell free transcription and translation and offer enhanced protein expression compared to proteinosomes prepared from traditional methodologies. Furthermore, the integration of native proteinosomes into higher order synthetic cellular architectures with membrane free compartments such as liposomes is demonstrated. The integration of bioinspired architectural elements with the central dogma is an essential building block for realizing minimal synthetic cells and is key for exploiting artificial cells in real-world applications.

Chemical Zymogens and Transmembrane Activation of Transcription in Synthetic Cells.

In this work, synthetic cells equipped with an artificial signaling pathway that connects an extracellular trigger event to the activation of intracellular transcription are engineered. Learning from nature, this is done via an engineering of responsive enzymes, such that activation of enzymatic activity can be triggered by an external biochemical stimulus. Reversibly deactivated creatine kinase to achieve triggered production of adenosine triphosphate, and a reversibly deactivated nucleic acid polymerase for on-demand synthesis of RNA are engineered. An extracellular, enzyme-activated production of a diffusible zymogen activator is also designed. The key achievement of this work is that the importance of cellularity is illustrated whereby the separation of biochemical partners is essential to resolve their incompatibility, to enable transcription within the confines of a synthetic cell. The herein designed biochemical pathway and the engineered synthetic cells are arguably primitive compared to their natural counterpart. Nevertheless, the results present a significant step toward the design of synthetic cells with responsive behavior, en route from abiotic to life-like cell mimics.

Dynamic RNA synthetic biology: new principles, practices and potential.

An increased appreciation of the role of RNA dynamics in governing RNA function is ushering in a new wave of dynamic RNA synthetic biology. Here, we review recent advances in engineering dynamic RNA systems across the molecular, circuit and cellular scales for important societal-scale applications in environmental and human health, and bioproduction. For each scale, we introduce the core concepts of dynamic RNA folding and function at that scale, and then discuss technologies incorporating these concepts, covering new approaches to engineering riboswitches, ribozymes, RNA origami, RNA strand displacement circuits, biomaterials, biomolecular condensates, extracellular vesicles and synthetic cells. Considering the dynamic nature of RNA within the engineering design process promises to spark the next wave of innovation that will expand the scope and impact of RNA biotechnologies.

Sequential gentle hydration increases encapsulation in model protocells.

Small, spherical vesicles are a widely used chassis for the formation of model protocells and investigating the beginning of compartmentalized evolution. Various methods exist for their preparation, with one of the most common approaches being gentle hydration, where thin layers of lipids are hydrated with aqueous solutions and gently agitated to form vesicles. An important benefit to gentle hydration is that the method produces vesicles without introducing any organic contaminants, such as mineral oil, into the lipid bilayer. However, compared to other methods of liposome formation, gentle hydration is much less efficient at encapsulating aqueous cargo. Improving the encapsulation efficiency of gentle hydration would be of broad use for medicine, biotechnology, and protocell research. Here, we describe a method of sequentially hydrating lipid thin films to increase encapsulation efficiency. We demonstrate that sequential gentle hydration significantly improves encapsulation of water-soluble cargo compared to the traditional method, and that this improved efficiency is dependent on buffer composition. Similarly, we also demonstrate how this method can be used to increase concentrations of oleic acid, a fatty acid commonly used in origins of life research, to improve the formation of vesicles in aqueous buffer.

pH-Responsive Elastin-Like Polypeptide Designer Condensates.

Biomolecular condensates are macromolecular complexes formed by liquid-liquid phase separation. They regulate key biological functions by reversibly compartmentalizing molecules in cells, in a stimulus-dependent manner. Designing stimuli-responsive synthetic condensates is crucial for engineering compartmentalized synthetic cells that are able to mimic spatiotemporal control over the biochemical reactions. Here, we design and test a family of condensate-forming, pH-responsive elastin-like polypeptides (ELPs) that form condensates above critical pH values ranging between 4 and 7, for temperatures between 20 and at 37 °C. We show that the condensation occurs rapidly, in sharp pH intervals (ΔpH < 0.3). For eventual applications in engineering synthetic cell compartments, we demonstrate that multiple types of pH-responsive ELPs can form mixed condensates inside micron-sized vesicles. When genetically fused with enzymes, receptors, and signaling molecules, these pH-responsive ELPs could be potentially used as pH-switchable functional condensates for spatially controlling biochemistry in engineered synthetic cells.