Nature Materials

Engineered living materials (ELMs) at the multicelluar level represent an innovation that promises programmable properties for biomedical, environmental, and consumer applications. However, the rational tuning of the mechanical properties of such ELMs from first principles remains a challenge. Here we use synthetic cell-cell adhesins to systematically characterize how rheological and viscoelastic properties of multicellular materials made from living bacteria can be tuned via adhesin strength, cell size and shape, and adhesion logic. We confirmed that the previous results obtained for non-living materials also apply to bacterial ELMs. Additionally, the incorporation of synthetic adhesins, combined with the adaptability of bacterial cells in modifying various cellular parameters, now enables novel and precise control over material properties. Furthermore, we demonstrate that rheology is a powerful tool for actively shaping the microscopic structure of ELMs, enabling control over cell aggregation and particle rearrangement, a key feature for complex material design. These results deepen our understanding of tuning the viscoelastic properties and fine structure of ELMs for applications like bioprinting and microbial consortia design including natural systems.

Smart materials are able to alter one or more of their properties in response to defined stimuli. Our ability to design and create such materials, however, does not match the diversity and specificity of responses seen within the biological domain. We propose that relocation of molecular phenomena from living cells into hydrogels can be used to confer smart functionality to materials. We establish that cell-free protein synthesis can be conducted in agarose hydrogels, that gene expression occurs throughout the material and that co-expression of genes is possible. We demonstrate that gene expression can be controlled transcriptionally (using in gel gene interactions) and translationally in response to small molecule and nucleic acid triggers. We use this system to design and build a genetic device that can alter the structural property of its chassis material in response to exogenous stimuli. Importantly, we establish that a wide range of hydrogels are appropriate chassis for cell-free synthetic biology, meaning a designer may alter both the genetic and hydrogel components according to the requirements of a given application. We probe the relationship between the physical structure of the gel and in gel protein synthesis and reveal that the material itself may act as a macromolecular crowder enhancing protein synthesis. Given the extensive range of genetically encoded information processing networks in the living kingdom and the structural and chemical diversity of hydrogels, this work establishes a model by which cell-free synthetic biology can be used to create autonomic and adaptive materials. Significance statementSmart materials have the ability to change one or more of their properties (e.g. structure, shape or function) in response to specific triggers. They have applications ranging from light-sensitive sunglasses and drug delivery systems to shape-memory alloys and self-healing coatings. The ability to programme such materials, however, is basic compared to the ability of a living organism to observe, understand and respond to its environment. Here we demonstrate the relocation of biological information processing systems from cells to materials. We achieved this by operating small, programmable genetic devices outside the confines of a living cell and inside hydrogel matrices. These results establish a method for developing materials functionally enhanced with molecular machinery from biological systems.

In recent years, matrix viscoelasticity has emerged as a potent regulator of fundamental cellular processes and has been implicated in promoting cancer progression. Alongside viscoelasticity, additional ECM cues have been shown to influence migration decision-making of cancer cells, and spatial confinement is now considered as a potential regulator of metastasis. However, our understanding of these complex processes predominantly relies on purely elastic hydrogels, and the exact relationship between matrix viscoelasticity and spatial confinement in driving epithelial cell mechanotransduction and migration during cancer progression remains unclear. Here, we systematically investigated the interplay between matrix stiffness, viscoelasticity and spatial confinement by engineering soft ([~]0.3 kPa) and stiff ([~]3 kPa) polyacrylamide hydrogels with varying degrees of viscous dissipation, mirroring the mechanical properties of healthy and tumoral conditions in breast tissue. We observed that viscoelasticity modulates cell spreading, focal adhesions and YAP nuclear import in opposite directions on soft and stiff substrates. Strikingly, viscoelasticity enhances migration speed and persistence on soft substrates, while impeding them on stiff substrates via actin retrograde flow regulation. Combining soft micropatterning with viscoelastic hydrogels, we also show that spatial confinement restricts cell migration on soft matrices regardless of matrix viscoelasticity and promotes migration on stiff matrices in a viscoelasticity-dependent fashion. Our findings establish substrate viscoelasticity as a key regulator of epithelial cell functions and unravel the role of the matrix dimensionality in this process. SignificanceWhile matrix elasticity has received significant attention, recent findings underscore the importance of its natural dissipative properties and spatial confinement in regulating cellular processes and tumour invasiveness. However, the intricate interplay between viscoelasticity and spatial confinement in orchestrating epithelial cell behaviour during cancer progression remains elusive. Using micropatterned viscoelastic hydrogels to replicate the mechanical properties encountered during breast tumour progression, we unveil that viscoelasticity modulates cell behaviour and mechanotransduction signals differently on soft and stiff substrates. Increased viscoelasticity enhances migration speed and persistence on soft substrates while impeding them on stiff substrates via actin retrograde flow regulation. Furthermore, spatial confinement restricts cell migration on soft matrices regardless of viscoelasticity, while promoting migration on stiff matrices in a viscoelasticity-dependent manner.

Biologic drug therapies are effective treatments for autoimmune diseases such as rheumatoid arthritis (RA) but may cause significant adverse effects, as they are administered continuously at high doses that can suppress the immune system. Using CRISPR-Cas9 genome editing, we engineered stem cells containing a synthetic gene circuit expressing biologic drugs to antagonize interleukin-1 (IL-1) or tumor necrosis factor (TNF) in an autoregulated, feedback-controlled manner in response to activation of the endogenous chemokine (C-C) motif ligand 2 (Ccl2) promoter. To test this approach in vivo, cells were tissue-engineered into a stable cartilaginous construct and implanted subcutaneously in mice with inflammatory arthritis. Bioengineered anti-cytokine implants mitigated arthritis severity as measured by joint pain, structural damage, and systemic and local inflammation. The coupling of synthetic biology with tissue engineering promises a range of potential applications for treating chronic diseases using custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.Competing Interest StatementThe authors have declared no competing interest.View Full Text

In multicellular organisms, cells and tissues coordinate biochemical signal propagation across length scales spanning microns to meters. Endowing synthetic materials with similar capacities for coordinated signal propagation could allow these systems to adaptively regulate themselves across space and over time. Here we combine ideas from cell signaling and electronic circuitry to design a biochemical waveguide that transmits information in the form of a concentration of a DNA species on a directed path. The waveguide can be seamlessly integrated into a soft material because there is virtually no difference between the chemical or physical properties of the waveguide and the material it is embedded within. We propose the design of DNA strand displacement reactions to construct the system and, using reaction-diffusion models, identify kinetic and diffusive parameters that enable super-diffusive transport of DNA species via autocatalysis. Finally, to support experimental waveguide implementation, we show how a sink reaction could mitigate the spurious amplification of an autocatalyst within the waveguide, allowing for controlled waveguide triggering. Chemical waveguides could facilitate the design of synthetic biomaterials with distributed sensing machinery integrated throughout their structure and enable coordinated self-regulating programs triggered by changing environmental conditions.

Cells in load-bearing tissues experience both solid deformation and interstitial fluid flow during physiological loading, but the mechanisms by which they integrate these biphasic mechanical signals remain poorly understood. Here, we develop a porous, nanoarchitected 3D scaffold that allows simultaneous delivery and control of matrix strain and fluid shear stress. We validated the platform through fatigue loading experiments and simulations of fluid-structure interactions. In static culture, osteoblast-like cells adopted shapes, cytoskeletal architectures, and focal adhesion patterns templated by scaffold geometry. Under cyclic compression, the combined influence of matrix deformation and induced fluid flow disrupted this alignment, producing disordered actin structures and reduced focal adhesion eccentricity. These changes emerged even under low-frequency loading, within the drained poroelastic regime, indicating a high sensitivity of cytoskeletal organization to fluid-solid coupling. Our findings establish a tractable and tunable platform to investigate how cells sense and respond to dynamic biphasic mechanical environments in 3D. Significance StatementCells in tissues such as bone experience mechanical inputs from both matrix deformation and interstitial fluid flow. However, existing in vitro systems often isolate one type of input or lack the ability to control both independently. We engineered a nanoarchitected 3D scaffold that delivers tunable biphasic mechanical inputs by combining structural compression and fluid flow. Without external loads, cells align their cytoskeleton and focal adhesions to the scaffold geometry. When subjected to dynamic loading, they transition to disordered morphologies and less mature focal adhesions, suggesting a transition to migratory states. These results highlight the sensitivity of cells to even subtle biphasic cues and provide a new platform to study how cells integrate multiple mechanical signals in 3D environments.

Every organ surface and body cavity is lined by a confluent collective of epithelial cells. In homeostatic circumstances the epithelial collective remains effectively solid-like and sedentary. But during morphogenesis, remodeling or repair, as well as during malignant invasion or metastasis, the epithelial collective becomes fluid-like and migratory1-4. This conversion from sedentary to migratory behavior has traditionally been understood as a manifestation of the epithelial-to-mesenchymal transition (EMT) or the partial EMT (pEMT)5-8. However, in certain contexts this conversion has been attributed to the recently discovered unjamming transition (UJT), in which epithelial cells move collectively and cooperatively9-11. UJT and pEMT share certain aspects of collective cellular migration, but the extent to which these processes are distinct, overlapping or perhaps even identical has remained undefined. Using the confluent layer of well-differentiated primary human bronchial epithelial (HBE) cells, here we triggered UJT by exposing the sedentary layer to mechanical compression9-12. Cells thereafter migrated cooperatively, aligned into packs locally, and elongated systematically. Nevertheless, cell-cell junctions, apico-basal polarity, and barrier function remained intact in response, and mesenchymal markers remained unapparent. As such, pEMT was not evident. When we triggered pEMT and associated cellular migration by exposing the sedentary layer to TGF-{beta}1, metrics of UJT versus pEMT diverged. To account for these striking physical observations a new mathematical model attributes the effects of pEMT mainly to diminished junctional tension but attributes those of UJT mainly to augmented cellular propulsion. Together, these findings establish that UJT is sufficient to account for vigorous epithelial layer migration even in the absence of pEMT. Distinct gateways to cellular migration therefore become apparent - UJT as it might apply to migration of epithelial sheets, and EMT/pEMT as it might apply to migration of mesenchymal cells on a solitary or collective basis, activated during development, remodeling, repair or tumor invasion. Through the actions of UJT and pEMT working independently, sequentially, or interactively, living tissue is therefore seen to comprise an active engineering material whose modules for plasticity, self-repair and regeneration, are far richer than had been previously appreciated.

Engineering sophisticated behaviours in synthetic cells lacking complex biomolecular machinery remains a central challenge in synthetic biology. Here, we introduce a protein-free approach for dynamic content modulation in liposome-based synthetic cells using an internal gelation strategy. By crosslinking a polymer hydrogel within the lumen of giant vesicles and tethering it to the inner membrane leaflet, we create a composite architecture that enables controlled and reversible membrane permeabilisation via osmotic swelling and shrinking, facilitating externally gated material exchange without reconstituted protein pores or electroporation. Simultaneously, the hydrogel matrix affords control over membrane fluidity and the diffusion of cytoplasmic clients. We deploy the transport-regulation platform to construct a synthetic-cell bioreactor whereby reversible membrane permeabilisation enables content supplementation and fuels a biocatalytic reaction. The composite gel-GUV chassis provides an adaptive, robust and expandable solution for engineering increasingly modular and functional synthetic cellular systems. These findings may echo how primordial cells harnessed environmental fluctuations for content exchange through chemically distinct pathways.

Directed cell migration underlies many biological phenomena, from embryonic development to tumor metastasis and organ fibrosis. Most cells typically migrate toward stiffer regions of their extracellular matrix -a behavior known as positive durotaxis. Here we show that culture on rigid plastic reinforces this response, whereas preconditioning in soft 3D physiomimetic environments reprograms migration towards softer environments, a phenomenon known as negative durotaxis. Fetal rat lung fibroblasts preconditioned in 3D physiomimetic hydrogels exhibited negative durotaxis and accumulated near [~]5 kPa, corresponding to the physiological stiffness of the lung. In contrast, genetically identical cells maintained on conventional 2D plastic substrates migrated up stiffness gradients, toward stiffer regions. Although both populations displayed a biphasic force-stiffness relationship, they differed in force magnitude and cytoskeletal organization. Molecular-clutch modeling revealed that durotaxis reversal emerges from two distinct mechanical regimes: a mechanosensitive, high-motor-clutch state that stabilizes adhesions on stiff substrates and drives positive durotaxis, and a low-motor, weak-adhesion state in which clutch slippage on the stiff side causes negative durotaxis. Our results show that durotaxis direction is not an intrinsic cellular property. Rather, it emerges from the interplay between motor activity and adhesion dynamics and can be tuned by culture conditions.

Mechanical interactions between cells and their three-dimensional environment govern fundamental processes in tissue development and disease. Yet, how cells interrogate and respond to mechanical cues remains incompletely understood. Reproducing the mechanical properties of natural tissues in vitro is a key challenge, as these tissues exhibit complex viscoelastic behaviors and undergo continuous remodeling throughout an organisms development. Here, we leverage principles of dynamic DNA nanotechnology to introduce programmable mechanical cues into a synthetic hydrogel matrix that guides and interrogates the development of embedded cells. By systematically modulating matrix stress relaxation and stiffness, we uncover two distinct timescales of mechano-sensitive processes controlling apical-basal polarity in Madin-Darby Canine Kidney (MDCK) cells: the fast timescale (1-3 min) is linked to rapid integrin-mediated signaling, while the slower process (3-9 h) is associated with cytoskeletal reinforcements. Our analysis highlights that the commonly reported matrix "stiffness" value, often measured as the storage modulus at [~]1 Hz, has limited physiological relevance. These findings prompted us to develop a novel switchable DNA crosslinker that enables dynamic changes in viscoelasticity during ongoing cell culture. This controlled matrix reconfiguration induces reversible cell polarity inversions and guides the morphogenesis of complex multicellular structures. The material platform therefore offers unprecedented control over the mechanical microenvironment, opening new avenues for advanced applications in biophysics, tissue engineering, and disease modeling.

The ability to dynamically control organelle movement and position is essential for cellular function. Yet the underlying mechanisms driving this organization have not been fully resolved. Here, we draw from recent experimental observations and theoretical models of enzyme chemotaxis to demonstrate the chemotaxis of a bacterial organelle, the 1,2 propanediol (1,2-PD) utilization bacterial microcompartment (MCP) from Salmonella enterica. Upon encapsulating MCPs in a cell-like, biomimetic compartment, we observed the directed movement of MCPs along an external gradient of substrate. Our analysis shows that MCPs not only chemotax towards their substrate but also that enzymatic activity and substrate turnover protect them against large-scale aggregation. Our results provide a first experimental demonstration of organelle chemotaxis in a synthetic cellular system and support a recent theoretical model of chemotaxis. Together this work reveals a potentially significant driver of organelle organization while contributing to the construction of synthetic cell-like materials.

The mechanical properties of the extracellular matrix (ECM) determine cell differentiation, proliferation and migration through mechanoresponsive proteins including YAP. However, how different mechanical signals cooperate, synergize or compete to steer cell behavior remains poorly understood. Here, we have examined competition between the two major ECM mechanical cues, i.e. rigidity, which activates cell mechanosensing, and viscous energy dissipation, which reduces stiffness blunting cell mechanotransduction. To trigger competition, we have engineered protein hydrogels allowing concomitant modulation of stiffness and viscosity by mechanisms characteristic of native ECM. Culturing cells on these hydrogels, we have found that substrate energy dissipation attenuates YAP mechanosensing prevailing over stiffness cues. Hampered YAP activation on more dissipative substrates correlates with faster actin flow and smaller focal adhesions. Mechanistically, inhibition of actomyosin contractility reverses the outcome of the competition between rigidity and energy dissipation. Our results highlight the dominating contribution of substrate viscosity to the biology of the cell.

Engineered living materials (ELMs) promise genetically programmable functions by coupling biological regulation to synthetic material responses. Here, we introduce genetically encoded, reversible shape-morphing in a peptide-crosslinked polyethylene glycol (PEG) hydrogel whose network density is modulated by opposing enzymatic pairs that induce crosslinking or hydrolysis. This molecular programmability alternates the hydrogel between deswelling and swelling/disintegration and produces 2 - 5-fold changes in mechanical properties. By fabricating a bilayer hydrogel with an inert layer, these molecular modulations are translated into a reversible and directional motion with angular bending motions exceeding 80{degrees}. Further, by embedding genetically engineered bacteria or interfacing mammalian cells, producing the relevant enzymatic cues, the reversible shape-morphing of these ELMs is programmed at the genetic level. We further demonstrate genetically programmed, autonomous reversible bending in a bilayer hydrogel controlled by out-of-equilibrium counteracting biochemical reactions with dynamically changing respective reaction rates. This work establishes a concept where coordinated polymer/peptide material engineering and synthetic biology yield autonomous shape-morphing ELMs, opening avenues toward biohybrid soft robotics, adaptive microfluidic systems, and dynamic biomedical interfaces.

Stem cells exhibit prominent clusters controlling the transcription of genes into RNA. These clusters form by a phase-separation mechanism, and their size and shape are controlled via an amphiphilic effect of transcribed genes. Here, we construct amphiphile-nanomotifs purely from DNA, and achieve similar size and shape control for phase-separated droplets formed from fully synthetic, self-interacting DNA-nanomotifs. Low amphiphile concentrations induce rounding of droplets, followed by splitting and, ultimately, full dispersal at higher concentrations. Super-resolution microscopy data obtained from zebrafish embryo stem cells reveal a comparable transition for transcriptional clusters with increasing transcription levels. Brownian dynamics and lattice simulations further confirm that addition of amphiphilic particles is sufficient to explain the observed changes in shape and size. Our work reproduces key aspects of the complex organization of transcription in biological cells using relatively simple, DNA sequence-programmable nanostructures, opening novel ways to control mesoscopic organization of synthetic nanomaterials. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/525851v2_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1a8cc4borg.highwire.dtl.DTLVardef@fc6651org.highwire.dtl.DTLVardef@a17c74org.highwire.dtl.DTLVardef@1f1cf14_HPS_FORMAT_FIGEXP M_FIG C_FIG

Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands. We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.

Bone endures millions of cycles throughout its lifetime by accumulating damage at a rate slow enough to allow for cell-mediated repair, but the mechanisms that delay this fatigue failure remain poorly understood. While prior studies have focused on the fatigue response of macroscale architecture of bone, the role of its nanoscale structure in resisting fatigue has been experimentally inaccessible. Here, we combine in-situ fatigue loading with synchrotron X-ray tomography and radiography to directly observe crack propagation in human bone with [~] 21 nm spatial and 100 ms temporal resolution. We find that mineralized collagen fibrils decelerate crack growth through branching along the fibril axes, while orthogonal cracks are intermittently decelerated by nanoscale interfibrillar interfaces. These mechanisms suppress damage accumulation under physiological loads by an order of magnitude. Our findings uncover a previously unobserved toughening strategy at the nanoscale, providing insight as to how the hierarchical structure of bone bridges the timescale gap between mechanical damage and biological repair.

Tissue morphogenesis and repair, as well as organ homeostasis, require cells to constantly monitor their 3D microenvironment and adapt their behaviors in response to local biochemical and mechanical cues1-6. In vitro studies have shown that substrate stiffness and stress relaxation are important mechanical parameters in the control of cell proliferation and differentiation, stem cell maintenance, cell migration 7-11, as well as tumor progression and metastasis12,13. Yet, the mechanical parameters of the microenvironment that cells perceive in vivo, within 3D tissues, remain unknown. In complex materials with strain- and time-dependent material properties, the perceived mechanical parameters depend both on the strain and timescales at which the material is mechanically probed14. Here, we quantify in vivo and in situ the mechanics of the cellular microenvironment that cells probe during vertebrate presomitic mesoderm (PSM) specification. By analyzing the magnitude and dynamics of endogenous, cell-generated strains, we show that individual cells preferentially probe the stiffness associated with deformations of the supracellular, foam-like tissue architecture. We reveal how stress relaxation leads to a perceived microenvironment stiffness that decreases over time, with cells probing the softest regime. While stress relaxation timescales are spatially uniform in the tissue, most mechanical parameters, including those probed by cells, vary along the anteroposterior axis, as mesodermal progenitors commit to different lineages. Understanding the mechanical parameters that cells probe in their native 3D environment is important for quantitative studies of mechanosensation in vivo2-4,6,15 and can help design scaffolds for tissue engineering applications16-18.

Collective cell migration drives tissue morphogenesis, repair and remodeling, and is often accompanied by transitions from solid-like to fluid-like states. While such tissue fluidization has been linked to physical parameters such as cell density, shape and activity, how it is actively regulated by mechano-chemical interplay remains unclear. Previous research has shown that transient attenuation of actomyosin contractility induces a transition from pulsatile, spatially confined motion to coherent, persistent long-range collective flow; however, the underlying cellular and signaling mechanisms remain unclear. Here we uncover the mechanistic basis by which transient perturbation of cell contractility reprograms the migration mode of confluent epithelial cells into a leader-like, fluidizing state, by combining kinase-reporter live imaging, force measurements and mathematical modeling. This transition arises from coordinated changes in cell morphology, mechanics, and signaling, including reduced cortical tension, enhanced cell-substrate adhesion and traction forces, and increased tissue deformability. At the signaling level, this process is accompanied by a rewiring of extracellular signal-regulated kinase (ERK)-mediated mechanotransduction toward a protrusion-coupled mode that sustains migration even under fully confluent conditions. Consistently, a multicellular computational model further demonstrates that protrusion-driven migration is sufficient to promote shape-velocity alignment and drive a transition from caged to flocking-like collective states. Together, our results identify transient mechanical relaxation as a trigger for an intrinsic leader-like state that fluidizes epithelial confluent tissues through coordinated remodeling of cytoskeletal, adhesive, and signaling systems.

Biological tissues achieve proper shape and ordered structures during development through responses to internal and external signals, with mechanical cues playing a crucial role. These forces guide cellular organization, leading to complex self-organizing structures that are foundational to embryonic patterns. Emerging theories and experiments suggest that "topological morphogens" drive these processes. Despite the predominance of three-dimensional (3D) structures in biology, studying 3D tissues remains challenging due to limited model systems and the complexity of modeling. Here, we address these challenges by using self-organized cellular aggregates, specifically spindle-shaped C2C12 myoblasts, subjected to controlled mechanical stretching. Our findings reveal that these cells form a multilayered, actin-oriented tissue structure, where mechanical forces drive long-range 3D organization and muscle differentiation. Notably, tissue surface emerges as a hotspot for differentiation, correlating with directional order as shown by single molecule fluorescent in situ hybridization. Significance StatementWe explore how cells work together to form complex structures, particularly in 3D, using muscle precursors cells (C2C12 myoblasts) as a model. By applying controlled stretching forces, we found that these cells self-organize into layered tissues that guide their transformation into muscle. This research highlights the critical role of physical forces in shaping tissues, suggesting that the way cells are physically arranged and stretched in three dimensions can significantly influence their behavior and function. Our findings offer new insights into how tissues develop and could have implications for tissue engineering, where creating the right 3D environment is key to successful tissue growth and repair.

Living systems achieve robust self-assembly across length scales. Meanwhile, nanofabrication strategies such as DNA origami have enabled robust self-assembly of submicron-scale shapes.However, erroneous and missing linkages restrict the number of unique origami that can be practically combined into a single supershape. We introduce crisscross polymerization of DNA-origami slats for strictly seed-dependent growth of custom multi-micron shapes with user-defined nanoscale surface patterning. Using a library of ~2000 strands that can be combinatorially assembled to yield any of ~1e48 distinct DNA origami slats, we realize five-gigadalton structures composed of >1000 uniquely addressable slats, and periodic structures incorporating >10,000 slats. Thus crisscross growth provides a generalizable route for prototyping and scalable production of devices integrating thousands of unique components that each are sophisticated and molecularly precise. One-sentence summaryCrisscross polymerization of DNA-origami slats can yield micron-scale structures with uniquely addressable nanoscale features.