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Organoids recapitulate many aspects of the complex three-dimensional (3D) organization found within native tissues and even display tissue and organ-level functionality. Traditional approaches to organoid culture have largely employed a top-down tissue engineering strategy, whereby cells are encapsulated in a 3D matrix, such as Matrigel, alongside well-defined biochemical cues that direct morphogenesis. However, the lack of spatiotemporal control over niche properties renders cellular processes largely stochastic. Therefore, bottom-up tissue engineering approaches have evolved to address some of these limitations and focus on strategies to assemble tissue building blocks with defined multi-scale spatial organization. However, bottom-up design reduces the capacity for self-organization that underpins organoid morphogenesis. Here, we introduce an emerging framework, which we term middle-out strategies, that relies on existing design principles and combines top-down design of defined synthetic matrices that support proliferation and self-organization with bottom-up modular engineered intervention to limit the degrees of freedom in the dynamic process of organoid morphogenesis. We posit that this strategy will provide key advances to guide the growth of organoids with precise geometries, structures and function, thereby facilitating an unprecedented level of biomimicry to accelerate the utility of organoids to more translationally relevant applications. 

Spatiotemporally coordinated transformations in epithelial curvature are necessary to generate crypt-villus structures during intestinal development. However, the temporal regulation of mechanotransduction pathways that drive crypt morphogenesis remains understudied. Intestinal organoids have proven useful to study crypt morphogenesis in vitro, yet the reliance on static culture scaffolds limits the ability to assess the temporal effects of changing curvature. Here, a photoinduced hydrogel cross-link exchange reaction is used to spatiotemporally alter epithelial curvature and study how dynamic changes in curvature influence mechanotransduction pathways to instruct crypt morphogenesis. Photopatterned curvature increased membrane tension and depolarization, which was required for subsequent nuclear localization of yes-associated protein 1 (YAP) observed 24 hours following curvature change. Curvature-directed crypt morphogenesis only occurred following a delay in the induction of differentiation that coincided with the delay in spatially restricted YAP localization, indicating that dynamic changes in curvature initiate epithelial curvature–dependent mechanotransduction pathways that temporally regulate crypt morphogenesis.

 

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell–matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6–6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling. 

Stiffness and forces are two fundamental quantities essential to living cells and tissues. However, it has been a challenge to quantify both 3D traction forces and stiffness (or modulus) using the same probe in vivo. Here, we describe an approach that overcomes this challenge by creating a magnetic microrobot probe with controllable functionality. Biocompatible ferromagnetic cobalt-platinum microcrosses were fabricated, and each microcross (about 30 micrometers) was trapped inside an arginine–glycine–aspartic acid–conjugated stiff poly(ethylene glycol) (PEG) round microgel (about 50 micrometers) using a microfluidic device. The stiff magnetic microrobot was seeded inside a cell colony and acted as a stiffness probe by rigidly rotating in response to an oscillatory magnetic field. Then, brief episodes of ultraviolet light exposure were applied to dynamically photodegrade and soften the fluorescent nanoparticle–embedded PEG microgel, whose deformation and 3D traction forces were quantified. Using the microrobot probe, we show that malignant tumor–repopulating cell colonies altered their modulus but not traction forces in response to different 3D substrate elasticities. Stiffness and 3D traction forces were measured, and both normal and shear traction force oscillations were observed in zebrafish embryos from blastula to gastrula. Mouse embryos generated larger tensile and compressive traction force oscillations than shear traction force oscillations during blastocyst. The microrobot probe with controllable functionality via magnetic fields could potentially be useful for studying the mechanoregulation of cells, tissues, and embryos.