Self-assembly of hybrid 3D cultures by integrating living and synthetic cells

BackgroundThe use of quantitative morphological features has been gaining traction as an additional source of information that can be used for cell characterization alongside genomic, transcriptomic, and proteomic data. New...

Advancements in the research on so-called "synthetic (artificial) cells" have been mainly characterized by an important acceleration in all sorts of experimental approaches, providing a growing amount of knowledge and techniques that will shape future successful developments. Synthetic cell technology, indeed, shows potential in driving a revolution in science and technology. On the other hand, theoretical and epistemological investigations related to what synthetic cells "are," how they behave, and what their role is in generating knowledge have not received sufficient attention. Open questions about these less explored subjects range from the analysis of the organizational theories applied to synthetic cells to the study of the "relevance" of synthetic cells as scientific tools to investigate life and cognition; and from the recognition and the cultural reappraisal of cybernetic inheritance in synthetic biology to the need for developing concepts on synthetic cells and to the exploration, in a novel perspective, of information theories, complexity, and artificial intelligence applied in this novel field. In these contributions, we will briefly sketch some crucial aspects related to the aforementioned issues, based on our ongoing studies. An important take-home message will result: together with their impactful experimental results and potential applications, synthetic cells can play a major role in the exploration of theoretical questions as well.

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.

Compartments within living cells create specialized microenvironments, allowing for 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 have been widely used to study many natural processes, including organelle formation. Here we describe a synthetic cell expressing RGG-GFP-RGG, a phase-separating protein derived from LAF-1 RGG domains, to form artificial membraneless organelles that can sequester RNA and reduce protein expression. We create complex microenvironments within synthetic cell cytoplasm and introduce a tool to modulate protein expression in synthetic cells. Engineering of compartments within synthetic cells furthers understanding of evolution and function of natural organelles, as well as it facilitates the creation of more complex and multifaceted synthetic life-like systems.

The bottom-up construction of synthetic cells is one of the most intriguing and interesting research arenas in synthetic biology. Synthetic cells are built by encapsulating biomolecules inside lipid vesicles (liposomes), allowing the synthesis of one or more functional proteins. Thanks to the in situ synthesized proteins, synthetic cells become able to perform several biomolecular functions, which can be exploited for a large variety of applications. This paves the way to several advanced uses of synthetic cells in basic science and biotechnology, thanks to their versatility, modularity, biocompatibility, and programmability. In the previous WIVACE (2012) we presented the state-of-the-art of semi-synthetic minimal cell (SSMC) technology and introduced, for the first time, the idea of chemical communication between synthetic cells and natural cells. The development of a proper synthetic communication protocol should be seen as a tool for the nascent field of bio/chemical-based Information and Communication Technologies (bio-chem-ICTs) and ultimately aimed at building soft-wet-micro-robots. In this contribution (WIVACE, 2013) we present a blueprint for realizing this project, and show some preliminary experimental results. We firstly discuss how our research goal (based on the natural capabilities of biological systems to manipulate chemical signals) finds a proper place in the current scientific and technological contexts. Then, we shortly comment on the experimental approaches from the viewpoints of (i) synthetic cell construction, and (ii) bioengineering of microorganisms, providing up-to-date results from our laboratory. Finally, we shortly discuss how autopoiesis can be used as a theoretical framework for defining synthetic minimal life, minimal cognition, and as bridge between synthetic biology and artificial intelligence.

Advances in artificial/synthetic cells have drawn a new era of nanobiotechnology, which have shown broad prospects in biomedical applications. The rational nanoengineering of synthetic cells that can closely substitute the systematic biological functions of cells is a next grand challenge. Here, a genetically encoded synthetic beta cell, which can sense hyperglycemic conditions to initiate programmed biosynthesis and secretion of insulin is reported. By encapsulating different metal–organic framework‐based artificial organelles with distinctive bifunctionalities, the synthetic cell can undergo programmed, sequential subcellular events, including glucose sensing, initiation of insulin gene transcription and translation, and finally excretion of functional insulin, under hyperglycemic conditions. Glucose uptake assay suggests that the insulin produced by the synthetic cells can successfully promote glucose uptake into mammalian cells. The construction of a higher‐order cell cluster by ligand‐mediated super‐assembly of the synthetic cells is further demonstrated. Such a robust and smart synthetic system that closely mimics the cellular activities of beta cells in response to glucose levels is promising for improving clinical outcomes in diabetes treatment.

Synthetic cells, expressing proteins using cell-free transcription-translation (TXTL), is a technology utilized for a variety of applications, such as investigating natural gene pathways, metabolic engineering, drug development or bioinformatics. For all these purposes, the ability to precisely control gene expression is essential. Various strategies to control gene expression in TXTL have been developed; however, further advancements on gene-specific and straightforward regulation methods are still needed. Here, we present a method of control of gene expression in TXTL using a "silencing oligo": a short oligonucleotide, designed with a particular secondary structure, that binds to the target messenger RNA. We demonstrated that silencing oligo inhibits protein expression in TXTL in a sequence-dependent manner. We showed that silencing oligo activity is associated with RNase H activity in bacterial TXTL. To complete the gene expression control toolbox for synthetic cells, we also engineered a first transfection system. We demonstrated the transfection of various payloads, enabling the introduction of RNA and DNA of different lengths to synthetic cell liposomes. Finally, we combined the silencing oligo and the transfection technologies, demonstrating control of gene expression by transfecting silencing oligo into synthetic minimal cells.

Synthetic minimal cells provide a controllable and engineerable model for biological processes. While much simpler than any live natural cell, synthetic cells offer a chassis for investigating the chemical foundations of key biological processes. Herein, we show a synthetic cell system with host cells, interacting with parasites and undergoing infections of varying severity. We demonstrate how the host can be engineered to resist infection, we investigate the metabolic cost of carrying resistance, and we show an inoculation that immunizes the host against pathogens. Our work expands the synthetic cell engineering toolbox by demonstrating host-pathogen interactions and mechanisms for acquiring immunity. This brings synthetic cell systems one step closer to providing a comprehensive model of complex, natural life.

One of the main questions addressed in the Nationale Wetenschapsagenda (NWA) is our ability to construct a synthetic minimal cell from lifeless, individual components. In biology, this challenge can be compared to bringing a man to the moon. Various international consortia now aim to engineer such synthetic cells, among which the Dutch initiative BaSyC that is supported by a NWO gravity program. The work presented in this thesis served as a pilot study for BaSyC, and focused on the construction and growth of a boundary layer (membrane) that should surround the synthetic cell. In living cells, membranes are composed of various phospholipids that spontaneously orient in a bilayer-like structure that separates the inside from the outside of the cell. Here, we describe the construction of a biosynthetic pathway that yields two phospholipid species critical for life. By combining catabolic and anabolic enzymes purified from various bacteria, a cascade-like biosynthetic pathway could be assembled capable of forming phospholipids from simple water-soluble building blocks that are fed to the system. The synthesis of these phospholipids resulted in the growth of a pre-established membrane that not only served as a barrier, but also supported the function of membrane embedded enzymes, including a system that is responsible for the translocation of proteins across the membrane. Sustainable production of phospholipids and growth of membranes, as well as the functional integration of membrane growth and membrane protein function, presents a first but important step towards the construction of synthetic minimal cells.

Synthetic cells can advance immunotherapy, offering innovative approaches to understanding and enhancing immune responses. This review article delves into the advancements and potential of synthetic cell technologies in immunology, emphasizing their role in understanding and manipulating immune functions. Recent progress in understanding vertebrate immune systems and the challenges posed by diseases highlight the need for innovative research methods, complementing the analysis of multidimensional datasets and genetic engineering. Synthetic immune cell engineering aims to simplify the complexity of immunological systems by reconstructing them in a controlled setting. This approach, alongside high‐throughput strategies, facilitates systematic investigations into immunity and the development of novel treatments. The article reviews synthetic cell technologies, focusing on their alignment with the three laws of immunity: universality, tolerance, and appropriateness. It explores the integration of synthetic cell modules to mimic processes such as controlled T‐cell activation, bacteria engulfment and elimination, or cellular maturation into desirable phenotypes. Together, such advancements expand the toolbox for understanding and manipulating immune functions. Synthetic cell technologies stand at the innovation crossroads in immunology, promising to illuminate fundamental immune system principles and open new avenues for research and therapy.

In the extant lifeforms, the self-sustaining behaviors refer to various well-organized biochemical reactions in spatial confinement, which rely on compartmentalization to integrate and coordinate the molecularly crowded intracellular environment and complicated reaction networks in living/synthetic cells. Therefore, the biological phenomenon of compartmentalization has become an essential theme in the field of synthetic cell engineering. Recent progress in the state-of-the-art of synthetic cells has indicated that multi-compartmentalized synthetic cells should be developed to obtain more advanced structures and functions. Herein, two ways of developing multi-compartmentalized hierarchical systems, namely interior compartmentalization of synthetic cells (organelles) and integration of synthetic cell communities (synthetic tissues), are summarized. Examples are provided for different construction strategies employed in the above-mentioned engineering ways, including spontaneous compartmentalization in vesicles, host-guest nesting, phase separation mediated multiphase, adhesion-mediated assembly, programmed arrays, and 3D printing. Apart from exhibiting advanced structures and functions, synthetic cells are also applied as biomimetic materials. Finally, key challenges and future directions regarding the development of multi-compartmentalized hierarchical systems are summarized; these are expected to lay the foundation for the creation of a "living" synthetic cell as well as provide a larger platform for developing new biomimetic materials in the future.

Tissue functions rely on complex structural, biochemical, and biomechanical cues that guide cellular behavior and organization. Synthetic cells, a promising new class of biomaterials, hold significant potential for mimicking these tissue properties using simplified, nonliving building blocks. Advanced synthetic cell models have already shown utility in biotechnology and immunology, including applications in cancer targeting and antigen presentation. Recent bottom-up approaches have also enabled synthetic cells to assemble into 3D structures with controlled intercellular interactions, creating tissue-like architectures. Despite these advancements, challenges remain in replicating multicellular behaviors and dynamic mechanical environments. Here, we review recent advancements in synthetic cell-based tissue formation and introduce a three-pillar framework to streamline the development of synthetic tissues. This approach, focusing on synthetic extracellular matrix integration, synthetic cell self-organization, and adaptive biomechanics, could enable scalable synthetic tissues engineering for regenerative medicine and drug development.

The recent progresses in bottom-up synthetic biology allow the construction of cell-like systems (also called "synthetic cells") based on the encapsulation of chemicals and biological macromolecules inside lipid vesicles. Synthetic cells are far from being alive, but can be designed in order to imitate biological cells with respect to specific functions. The exchange of chemical signals is one of the most fascinating ones. Experimental papers have shown that synthetic cells can be designed to send and receive molecular signals, inaugurating a new research avenue that can be highly relevant not only for nano-medicine and nano-biotechnology but also for basic understanding of minimal cognitive systems. Here, we shortly present the synthetic cell technology and illustrate how to implement the concept of molecular communication in this field.

Gene-expressing compartments assembled from simple, modular parts, are a versatile platform for creating minimal synthetic cells with life-like functions. By incorporating gene regulatory motifs into their encapsulated DNA templates, in situ gene expression and, thereby, synthetic cell function can be controlled according to specific stimuli. In this work, cell-free protein synthesis within synthetic cells was controlled using light by encoding genes of interest on light-activated DNA templates. Light-activated DNA contained a photocleavable blockade within the T7 promoter region that tightly repressed transcription until the blocking groups were removed with ultraviolet light. In this way, synthetic cells were activated remotely, in a spatiotemporally controlled manner. By applying this strategy to the expression of an acyl homoserine lactone synthase, BjaI, quorum-sensing-based communication between synthetic cells and bacteria was controlled with light. This work provides a framework for the remote-controlled production and delivery of small molecules from nonliving matter to living matter, with applications in biology and medicine.

Bottom-up, chemically formed synthetic cells are usually imaged by optical microscopy, and the cell sizes and shapes are mostly estimated from acquired 2D images. The three-dimensional (3D) structures of a compartmentalised synthetic cell can be analysed by axially stacking 2D images, typically by using a high-resolution imaging systems, such as laser confocal scanning microscopy and light sheet microscopy. However, these techniques require the synthetic cell to be labelled with fluorescent tags, and have performance limits such as being restricted to volumes less than (approximately) 200 μm3. Here, we present the label-free, 3D imaging of soft, free-standing, multicompartment synthetic cell using optical coherence tomography (OCT). The volumes of sub-cellular compartments within individual synthetic cells can be obtained via OCT imaging measurement. The spatial arrangements of the compartments and their contact angle information can be illustrated and measured, respectively. This approach provides a new method to evaluate multiphase soft materials spanning the range of micrometres to millimetres, towards the optimisation of synthetic cell construction for novel biomimetic material development.