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Kidney explants are traditionally cultured at air-liquid interfaces, which disrupts 3D tissue structure and limits interpretation of developmental data. Here we develop a 3D culture technique using hydrogel embedding to capture kidney morphogenesis in real time. 3D culture better approximates in vivo-like niche spacing and tubule dynamics, as well as branching defects under control conditions and GDNF-RET signaling perturbations. To isolate the effect of material properties on explant development, we apply acrylated hyaluronic acid hydrogels that allow independent tuning of stiffness and adhesion. We find that sufficient stiffness and adhesive ligands are both required to maintain kidney shape. More adhesive hydrogels increase nephrons per ureteric bud (UB) tip while matrix stiffness has a “Goldilocks effect” centered at ~2 kPa. Our technique captures large-scale, in vivo-like tissue morphogenesis in 3D, improving insight into congenital disease phenotypes. Moreover, understanding the impact of boundary condition mechanics on kidney development benefits fundamental research and renal engineering. Huang et al. show that 3D hydrogel embedding supports more organotypic kidney development in culture. Matrix stiffness and adhesion properties were found to regulate nephron formation, highlighting the intervention potential of physical boundary conditions.

Urinary collecting tubules form during kidney embryogenesis through the branching of the ureteric bud epithelium. A travelling mesenchyme niche of nephron progenitor cells caps each branching ureteric bud tip. These ‘tip domain’ niches pack more closely over developmental time and their number relates to nephron endowment at birth. Yet, how the crowded tissue environment impacts niche number and cell decision-making remains unclear. Here, through experiments and mathematical modelling, we show that niche packing conforms to physical limitations imposed by kidney curvature. We relate packing geometries to rigidity theory to predict a stiffening transition starting at embryonic day 15 in the mouse, validated by micromechanical analysis. Using a method to estimate tip domain ‘ages’ relative to their most recent branch events, we find that new niches overcome mechanical resistance as they branch and displace neighbours. This creates rhythmic mechanical stress in the niche. These findings expand our understanding of kidney development and inform engineering strategies for synthetic regenerative tissues. Geometric packing of tubules in the developing kidney urinary collecting system leads to tissue stiffening and rhythmic mechanical stresses local to nephron-forming niches that synchronize with tubule branching.

Kidney explant cultures are traditionally carried out at air-liquid interfaces, which disrupts 3D tissue structure and limits interpretation of developmental data. To overcome this limitation, we developed a 3D culture technique using hydrogel embedding to capture morphogenesis in real time. We show that 3D culture better approximates -like niche spacing and dynamic tubule tip rearrangement, as well as -like presentation of branching defects under perturbations to glial cell-derived neurotrophic factor (GDNF)- RE arranged during T ransfection (RET) tyrosine kinase signaling. We find that the concentration of the embedding matrix influences the number of nephrons per ureteric bud (UB) tip and the spacing between tips. To isolate the effect of specific material properties on explant development, we introduce engineered acrylated hyaluronic acid hydrogels that allow independent tuning of stiffness and adhesion. We find that sufficient stiffness and adhesion are both required to maintain kidney shape. Matrix stiffness has a \"Goldilocks effect\" on the nephron per UB tip balance centered at ∼2 kPa, while higher matrix adhesion increases nephron per UB tip ratio. Our technique captures large-scale, -like tissue morphogenesis in 3D, providing a platform suited to contrasting normal and congenital disease contexts. Moreover, understanding the impact of boundary condition mechanics on kidney development benefits fundamental renal research and advances the engineering of next-generation kidney replacement tissues.

Engineering of embryonic strategies for tissue-building has extraordinary promise for regenerative medicine. This has led to a resurgence in interest in the relationship between cell biophysical properties and morphological transitions. However, mapping gene or protein expression data to cell biophysical properties to physical morphogenesis remains challenging with current techniques. Here we present MATCHY (multiplexed adhesion and traction of cells at high yield). MATCHY advances the multiplexing and throughput capabilities of existing traction force and cell-cell adhesion assays using microfabrication and an automated computation scheme with machine learning-driven cell segmentation. Both biophysical assays are coupled with serial downstream immunofluorescence to extract cell type/signaling state information. MATCHY is especially suited to complex primary tissue-, organoid-, or biopsy-derived cell mixtures since it does not rely on knowledge of cell surface markers, cell sorting, or use of lineage-specific reporter animals. We first validate MATCHY on canine kidney epithelial cells engineered for RET tyrosine kinase expression and quantify a relationship between downstream signaling and cell traction. We go on to create a biophysical atlas of primary cells dissociated from the mouse embryonic kidney and use MATCHY to identify distinct biophysical states along the nephron differentiation trajectory. Our data complement expression-level knowledge of adhesion molecule changes that accompany nephron differentiation with quantitative biophysical information. These data reveal an 'energetic ratchet' that explains spatial nephron progenitor cell condensation from the niche as they differentiate, which we validate through agent-based computational simulation. MATCHY offers automated cell biophysical characterization at >10 -cell throughput, a highly enabling advance for fundamental studies and new synthetic tissue design strategies for regenerative medicine.

Controlling the time and place of nephron formation would improve nephron density and connectivity in next-generation kidney replacement tissues. Recent developments in kidney organoid technology have paved the way to achieving self-sustaining nephrogenic niches . The physical and geometric structure of the niche are key control parameters in tissue engineering approaches. However, their relationship to nephron differentiation is unclear. Here we investigate the relationship between niche geometry, cell compartment mixing, and nephron differentiation by targeting the Rho/ROCK pathway, a master regulator of the actin cytoskeleton. We find that the ROCK inhibitor Y-27632 increases mixing between nephron progenitor and stromal compartments in native mouse embryonic kidney niches, and also increases nephrogenesis. Similar increases are also seen in reductionist mouse primary cell and human induced pluripotent stem cell (iPSC)-derived organoids perturbed by Y-27632, dependent on the presence of stromal cells. Our data indicate that niche organization is a determinant of nephron formation rate, bringing renewed focus to the spatial context of cell-cell interactions in kidney tissue engineering efforts.

The physiological functions of several organs rely on branched tubular networks, but little is known about conflicts in development between building enough tubules for adequate function and geometric constraints imposed by organ size. We show that the mouse embryonic kidney epithelium negotiates a physical packing conflict between tubule tip duplication and limited area at the organ surface. Imaging, computational, and soft material modeling of tubule 'families' identifies six geometric packing phases, including two defective ones. Experiments in kidney explants show that a retrograde tension on tubule families is necessary and sufficient for them to avoid defects by switching to a vertical orientation that increases packing density. These results reveal developmental contingencies in response to physical limitations, and create a framework for classifying kidney defects. Competing Interest Statement The authors have declared no competing interest.

Current methods for building tissues usually start with a non-biological blueprint, or rely on self-organization, which does not extend to organ-scales. This has limited the construction of large tissues that simultaneously encode fine-scale cell organization. Here we bridge scales by mimicking developmental dynamics using \"kinomorphs\", tissue scaffolds that undergo globally programmed shape and density changes to trigger local self-organization of cells in many locations at once. In this first report, we focus on mimicking the extracellular matrix (ECM) compaction and division into leaflets that occurs in kidney collecting duct development. We start by creating single-cell resolution cell patterns in ECM-mimetic hydrogels that are >10x larger than previously described, by leveraging photo-lithographic technology. These patterns are designed to mimic the branch geometry of the embryonic kidney collecting duct tree. We then predict the shape dynamics of kinomorphs driven by cell contractility-based compaction of the ECM using kinematic origami simulations. We show that these dynamics spur centimeter-scale assembly of structurally mature ~50 μm-diameter epithelial tubules that are locally self-organized, but globally programmed. Our approach prescribes tubule network geometry at ~5x smaller length-scales than currently possible using 3D printing, and at local cell densities comparable to in vivo tissues. Kinomorphs could be used to scaffold and \"plumb\" arrays of organoids in the future, by guiding the morphogenesis of epithelial networks. Such hybrid globally programmed/locally self-organized tissues address a major gap in our ability to recapitulate organ-scale tissue structure. Footnotes * https://doi.org/10.6084/m9.figshare.9751661.v1

Glioma tumor dispersion involves invading cells escaping the tumor bulk and migrating into the healthy brain parenchyma. Here, they encounter linearly aligned track-like tissue structures such as axon bundles and the perivascular space. These environments also contain micrometer-scale pores that impose mechanical confinement on invading cells. To study glioma cell migration in an in vitro system that reproduces some of these features, we used microfluidic devices with 60 μm2 cross-sectional area channels that confine cells into one-dimensional (1D) tracks. Individual cell tracking revealed strongly persistent migration at a mean rate of 8.5 ± 0.33 nm s-1. Notably, a 1D computational cell migration simulator predicts migration behaviors of glioma cells without significant adjustment of parameters estimated from previous experiments on two-dimensional (2D) substrates. Pharmacological inhibitors of integrin-mediated adhesions, myosin II activation, or drugs targeting F-actin assembly or microtubule dynamics influence migration consistent with simulations where relevant parameters are changed. These results suggest that cell parameters calibrated to a motor-clutch model on 2D substrates effectively predict 1D confined migration behaviors a priori. Our results outline a method for testing biophysical mechanisms of tumor cell migration in confined spaces and predicting the effects of anti-motility therapy.

Integrin-based adhesion complexes link the cytoskeleton to the extracellular matrix (ECM) and are central to the construction of multicellular animal tissues. How biological function emerges from the 10s-1000s of proteins present within a single adhesion complex has remained unclear. We used fluorescent molecular tension sensors to visualize force transmission by individual integrins in living cells. These measurements revealed an underlying functional modularity in which integrin class controlled adhesion size and ECM ligand specificity, while the number and type of connections between integrins and F-actin determined the force per individual integrin. In addition, we found that most integrins existed in a state of near-mechanical equilibrium, a result not predicted by existing models of cytoskeletal force transduction. A revised model that includes reversible crosslinks within the F-actin network accounts for this result, and suggests how cellular mechanical homeostasis can arise at the molecular level.

Replicating organizational principles that establish fine-scale tissue structure is critical to our capacity for building functional replacement tissues. Tissue boundaries such as epithelial-mesenchymal interfaces are engines for morphogenesis in vivo. However, despite a wealth of micropatterning approaches available to control tissue size, shape, and mechanical environment in vitro, fine-scale spatial control of cell composition within tissue constructs remains an engineering challenge. To address this, we augment DNA velcro technology for selective patterning of ssDNA-labeled cells with long-term culture on mechanically defined polyacrylamide hydrogels. We co-functionalize photoactive benzophenone-containing polyacrylamide gels (BP-PA gels) with spatially precise ssDNA features that confer temporary cell adhesion and with extracellular matrix (ECM) proteins that confer long-term adhesion. We find that co-functionalization does not compromise ssDNA patterning fidelity or cell capture, nor hydrogel mechanical properties or mechanosensitive fibroblast spreading, enabling mechanobiology studies of precise cell interfaces. We then co-pattern colonies of fibroblasts and epithelial cells to study interface formation and extracellular signal-related kinase (ERK) activity at cellular contacts. Combining DNA velcro and ECM functionalization approaches provides independent control of initial cell placement, adhesion, and mechanical environment, constituting a new tool for studying biological interfaces and for programming multicellular interactions in engineered tissues.Competing Interest StatementThe authors have declared no competing interest.