The fundamental building block of living organisms is the cell, which is the universal biological base of all living entities. This micrometric mass of cytoplasm and the membrane border have fascinated scientists due to the highly complex and multicompartmentalized structure. This specific organization enables numerous metabolic reactions to occur simultaneously and in segregated spaces, without disturbing each other, but with a promotion of inter- and intracellular communication of biomolecules. At present, artificial nano- and microcompartments, whether as single components or self-organized in multicompartment architectures, hold significant value in the study of life development and advanced functional materials and in the fabrication of molecular devices for medical applications. These artificial compartments also possess the properties to encapsulate, protect, and control the release of bio(macro)molecules through selective transport processes, and they are capable of embedding or being connected with other types of compartments. The self-assembly mechanism of specific synthetic compartments and thus the fabrication of a simulated organelle membrane are some of the major aspects to gain insight. Considerable efforts have now been devoted to design various nano- and microcompartments and understand their functionality for precise control over properties. Of particular interest is the use of polymeric vesicles for communication in synthetic cells and colloidal systems to reinitiate chemical and biological communication and thus close the gap toward biological functions. Multicompartment systems can now be effectively created with a high level of hierarchical control. In this way, these structures can not only be explored to deepen our understanding of the functional organization of living cells, but also pave the way for many more exciting developments in the biomedical field.

Synthetic droplets mimicking bio-soft matter droplets formed via liquid-liquid phase separation (LLPS) in living cells have recently been employed in nanobiotechnology for artificial cells, molecular robotics, molecular computing, etc. Temporally controlling the dynamics of synthetic droplets is essential for developing such bio-inspired systems because living systems maintain their functions based on the temporally controlled dynamics of biomolecular reactions and assemblies. This paper reports the temporal control of DNA-based LLPS droplets (DNA droplets). We demonstrate the timing-controlled division of DNA droplets via time-delayed division triggers regulated by chemical reactions. Controlling the release order of multiple division triggers results in order control of the multistep droplet division, i.e., pathway-controlled division in a reaction landscape. Finally, we apply the timing-controlled division into a molecular computing element to compare microRNA concentrations. We believe that temporal control of DNA droplets will promote the design of dynamic artificial cells/molecular robots and sophisticated biomedical applications.

Eukaryotic cells are characterized by multiple chemically distinct compartments, one of the most notable being the nucleus. Within these compartments, there is a continuous exchange of information, chemicals, and signaling molecules, essential for coordinating and regulating cellular activities. One of the main goals of bottom-up synthetic biology is to enhance the complexity of synthetic cells by establishing functional compartmentalization. There is a need to mimic autonomous signaling between compartments, which in living cells, is often regulated at the genetic level within the nucleus. This advancement is key to unlocking the potential of synthetic cells as cell models and as microdevices in biotechnology. However, a technological bottleneck exists preventing the creation of synthetic cells with a defined nucleus-like compartment capable of genetically programmed intercompartment signaling events. Here, we present an approach for creating synthetic cells with distinct nucleus-like compartments that can encapsulate different biochemical mixtures in discrete compartments. Our system enables in situ protein expression of membrane proteins, enabling autonomous chemical communication between nuclear and cytoplasmic compartments, leading to downstream activation of enzymatic pathways within the cell.

Membraneless coacervate microdroplets have long been proposed as model protocells as they can grow, divide, and concentrate RNA by natural partitioning. However, the rapid exchange of RNA between these compartments, along with their rapid fusion, both within minutes, means that individual droplets would be unable to maintain their separate genetic identities. Hence, Darwinian evolution would not be possible, and the population would be vulnerable to collapse due to the rapid spread of parasitic RNAs. In this study, we show that distilled water, mimicking rain/freshwater, leads to the formation of electrostatic crosslinks on the interface of coacervate droplets that not only suppress droplet fusion indefinitely but also allow the spatiotemporal compartmentalization of RNA on a timescale of days depending on the length and structure of RNA. We suggest that these nonfusing membraneless droplets could potentially act as protocells with the capacity to evolve compartmentalized ribozymes in prebiotic environments.

ConspectusCentral to the quest of understanding the emergence of life is to uncover the role of metals, particularly iron, in shaping prebiotic chemistry. Iron, as the most abundant of the accessible transition metals on the prebiotic Earth, played a pivotal role in early biochemical processes and continues to be indispensable to modern biology. Here, we discuss our recent contributions to probing the plausibility of prebiotic complexes with iron, including heme and iron-sulfur clusters, in mediating chemistry beneficial to a protocell. Laboratory experiments and spectroscopic findings suggest plausible pathways, often facilitated by UV light, for the synthesis of heme and iron-sulfur clusters. Once formed, heme displays catalytic, peroxidase-like activity when complexed with amphiphiles. This activity could have been beneficial in two ways. First, heme could have catalytically removed a molecule (H2O2) that could have had degradative effects on a protocell. Second, heme could have helped in the synthesis of the building blocks of life by coupling the reduction of H2O2 with the oxidation of organic substrates. The necessity of amphiphiles to avoid the formation of inactive complexes of heme is telling, as the modern-day electron transport chain possesses heme embedded within a lipid membrane. Conversely, prebiotic iron-sulfur peptides have yet to be reported to partition into lipid membranes, nor have simple iron-sulfur peptides been found to be capable of participating in the synthesis of organic molecules. Instead, iron-sulfur peptides span a wide range of reduction potentials complementary to the reduction potentials of hemes. The reduction potential of iron-sulfur peptides can be tuned by the type of iron-sulfur cluster formed, e.g., [2Fe-2S] versus [4Fe-4S], or by the substitution of ligands to the metal center. Since iron-sulfur clusters easily form upon stochastic encounters between iron ions, hydrosulfide, and small organic molecules possessing a thiolate, including peptides, the likelihood of soluble iron-sulfur clusters seems to be high. What remains challenging to determine is if iron-sulfur peptides participated in early prebiotic chemistry or were recruited later when protocellular membranes evolved that were compatible with the exploitation of electron transfer for the storage of energy as a proton gradient. This problem mirrors in some ways the difficulty in deciphering the origins of metabolism as a whole. Chemistry that resembles some facets of extant metabolism must have transpired on the prebiotic Earth, but there are few clues as to how and when such chemistry was harnessed to support a (proto)cell. Ultimately, unraveling the roles of hemes and iron-sulfur clusters in prebiotic chemistry promises to deepen our understanding of the origins of life on Earth and aids the search for life elsewhere in the universe.

Motile droplets using Marangoni convection are attracting attention for their potential as cell-mimicking small robots. However, the motion of droplets relative to the internal and external environments that generate Marangoni convection has not been quantitatively described. In this study, we used an aqueous two-phase system [poly(ethylene glycol) (PEG) and dextran] in an elongated chamber to generate motile dextran droplets in a constant PEG concentration gradient. We demonstrated that dextran droplets move by Marangoni convection, resulting from the PEG concentration gradient and the active transport of PEG and dextran into and out of the motile dextran droplet. Furthermore, by spontaneously incorporating long DNA into the dextran droplets, we achieved cell-like motility changes controlled by coexisting environment-sensing molecules. The DNA changes its position within the droplet and motile speed in response to external conditions. In the presence of Mg2+, the coil-globule transition of DNA inside the droplet accelerates the motile speed due to the decrease in the droplet\'s dynamic viscosity. Globule DNA condenses at the rear part of the droplet along the convection, while coil DNA moves away from the droplet\'s central axis, separating the dipole convections. These results provide a blueprint for designing autonomous small robots using phase-separated droplets, which change the mobility and molecular distribution within the droplet in reaction with the environment. It will also open unexplored areas of self-assembly mechanisms through phase separation under convections, such as intracellular phase separation.

After tissue injury, inflammatory cells are rapidly recruited to the wound where they clear microbes and other debris, and coordinate the behaviour of other cell lineages at the repair site in both positive and negative ways. In this study, we take advantage of the translucency and genetic tractability of zebrafish to evaluate the feasibility of reprogramming innate immune cells in vivo with cargo-loaded protocells and investigate how this alters the inflammatory response in the context of skin and skeletal repair. Using live imaging, we show that protocells loaded with R848 cargo (which targets TLR7 and TLR8 signalling), are engulfed by macrophages resulting in their switching to a pro-inflammatory phenotype and altering their regulation of angiogenesis, collagen deposition and re-epithelialization during skin wound healing, as well as dampening osteoblast and osteoclast recruitment and bone mineralization during fracture repair. For infected skin wounds, R848-reprogrammed macrophages exhibited enhanced bactericidal activities leading to improved healing. We replicated our zebrafish studies in cultured human macrophages, and showed that R848-loaded protocells similarly reprogramme human cells, indicating how this strategy might be used to modulate wound inflammation in the clinic.

One of the main goals of synthetic biology is to build artificial cells in a bottom-up manner, which not only facilitates the deep understanding of the origin of life and cell function but also plays a critical role in the research fields such as the development of artificial cell chassis, tissue models, engineering drug delivery systems, and drug screening tools. However, achieving this goal is extremely challenging. The complexity of cell structures and the miniaturization and diversity of basic modules pose high requirements for the construction methods. The microfluidic chip, as an advanced microanalysis system, serves as an effective tool for building artificial cells. It can accurately control the structure and local microenvironment of artificial cells, becoming the preferred approach for the current research on synthetic life. This article reviewed the methods of constructing, manipulating, and analyzed artificial cells based on microfluidic chips, emphasized the importance of the microenvironment for life systems and artificial self-sustaining systems. In addition, this article demonstrated the wide applications of artificial cells in multiple critical biomedical fields. Exploring the advantages, disadvantages, and application performance of different microfluidic methods can enrich our knowledge about artificial cell research. Finally, we made an outlook on the development of artificial cell research based on microfluidics, expecting that this field can achieve greater breakthroughs and progress.

The bottom-up construction of artificial cells is beneficial for understanding cell working mechanisms. The glycolysis metabolism mimicry inside artificial cells is challenging. Herein, the glycolytic pathway (Entner-Doudoroff pathway in archaea) is reconstituted inside artificial cells. The glycolytic pathway comprising glucose dehydrogenase (GDH), gluconate dehydratase (GAD), and 2-keto-3-deoxygluconate aldolase (KDGA) converts glucose molecules to pyruvate molecules. Inside artificial cells, pyruvate molecules are further converted into alanine with the help of alanine dehydrogenase (AlaDH) to build a metabolic pathway for synthesizing amino acid. On the other hand, the pyruvate molecules from glycolysis stimulate the living mitochondria to produce ATP inside artificial cells, which further trigger actin monomers to polymerize to form actin filaments. With the addition of methylcellulose inside the artificial cell, the actin filaments form adjacent to the inner lipid bilayer, deforming the artificial cell from a spherical shape to a spindle shape. The spindle-shaped artificial cell reverses to a spherical shape by depolymerizing the actin filament upon laser irradiation. The glycolytic pathway and its further extension to produce amino acids (or ATP) inside artificial cells pave the path to build functional artificial cells with more complicated metabolic pathways.

Bioprinting is an automated bioassembly method that enables the formation of human tissue-like constructs to restore or replace damaged tissues. Regardless of the employed bioprinting method, cells undergo mechanical stress that can impact their survival and function postprinting. In this study, we investigate the use of a synthetic cell-like unit, giant unilamellar vesicles (GUVs), as adjuvants of the cellular function of human cells postprinting, or in future as the complete replacement of human cells. We analyzed the impact of two nozzle-based bioprinting methods (drop-on-demand and extrusion bioprinting) on the structure, stability, and function of GUVs. We showed that over 65% of the GUVs remain intact when printing at 0.5 bar, demonstrating the potential of using GUVs as a synthetic cell source. We further increased the stability of GUVs in a cell culture medium by introducing polyethylene glycol (PEG) into the GUV lipid membrane. The presence of PEG, however, diminished the structural properties of GUVs postprinting, and reduced the interaction of GUVs with human cells. Although the design of PEG-GUVs can still be modified in future studies for better cell-GUV interactions, we demonstrated that GUVs are functional postprinting. Chlorin e6-PEG-GUVs loaded with a fluorescent dye were bioprinted, and they released the dye postprinting only upon illumination. This is a new strategy to deliver carriers, such as growth factors, drugs, nutrients, or gases, inside large bioprinted specimens on a millimeter to centimeter scale. Overall, we showed that printed GUVs can augment the functionality of manufactured human tissues.