Spatial and temporal organoid control Stem cell–derived organoids form through self-organization and serve as models for organ development, function, and disease, with potential applications in drug development and personalized medicine. However, in the absence of external guidance, developmental processes are stochastic, resulting in variable end products that differ significantly from the native organ. Gjorevski et al . developed approaches for specifying the initial organoid geometry to build intestinal organoids of defined shape, size, and cell distributions, forming structures that are predictable, more similar to normal organs, and reproducible (see the Perspective by Huycke and Gartner). These methods identify symmetry-breaking mechanisms in intestinal morphogenesis and have potential for standardizing organoid-based therapies and facilitating the refinement of mechanistic studies. —BAP

Intestinal organoid protocols rely on the use of extracellular scaffolds, typically Matrigel, and upon switching from growth to differentiation promoting media, a symmetry breaking event takes place. During this stage, the first bud like structures analogous to crypts protrude from the central body and differentiation ensues. While organoids provide unparalleled architectural and functional complexity, this sophistication is also responsible for the high variability and lack of reproducibility of uniform crypt‐villus structures. If function follows form in organoids, such structural variability carries potential limitations for translational applications (e.g., drug screening). Consequently, there is interest in developing synthetic biomaterials to direct organoid growth and differentiation. It has been hypothesized that synthetic scaffold softening is necessary for crypt development, and these mechanical requirements raise the question, what compressive forces and subsequent relaxation are necessary for organoid maturation? To that end, allyl sulfide hydrogels are employed as a synthetic extracellular matrix mimic, but with photocleavable bonds that temporally regulate the material's bulk modulus. By varying the extent of matrix softening, it is demonstrated that crypt formation, size, and number per colony are functions of matrix softening. An understanding of the mechanical dependence of crypt architecture is necessary to instruct homogenous, reproducible organoids for clinical applications.

Intestinal organoids are useful in vitro models for basic and translational studies aimed at understanding and treating disease. However, their routine culture relies on animal‐derived matrices that limit translation to clinical applications. In fact, there are few fully defined, synthetic hydrogel systems that allow for the expansion of intestinal organoids. Here, an allyl sulfide photodegradable hydrogel is presented, achieving rapid degradation through radical addition‐fragmentation chain transfer (AFCT) reactions, to support routine passaging of intestinal organoids. Shear rheology to first characterize the effect of thiol and allyl sulfide crosslink structures on degradation kinetics is used. Irradiation with 365 nm light (5 mW cm−2) in the presence of a soluble thiol (glutathione at 15 × 10−3 m), and a photoinitiator (lithium phenyl‐2,4,6‐trimethylbenzoylphosphinate at 1 × 10−3 m), leads to complete hydrogel degradation in less than 15 s. Allyl sulfide hydrogels are used to support the formation of epithelial colonies from single intestinal stem cells, and rapid photodegradation is used to achieve repetitive passaging of stem cell colonies without loss in morphology or organoid formation potential. This platform could support long‐term culture of intestinal organoids, potentially replacing the need for animal‐derived matrices, while also allowing systematic variations to the hydrogel properties tailored for the organoid of interest.

Objective: Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified that 2 cytokines in M1 media attributed to the observed antifibrotic effects were TNF (tumor necrosis factor)-α) and IL (interleukin) 1β). After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. Conclusions: Proinflammatory cytokines in M1 conditioned media, notably TNF-α, IL-1β, and IL-6, inhibit the myofibroblast response in VICs and promote their osteogenic differentiation. Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.

Muscle cells sense the mechanical properties of their microenvironment, and these properties can change in response to injury or disease. Hydrogels with dynamic material properties can be used to study the effect of such varying mechanical signals. Here, we report the ability of azadibenzocyclooctyne to undergo a cytocompatible, photoinitiated crosslinking reaction. This reaction is exploited as a strategy for on-demand stiffening of three-dimensional cell scaffolds formed through an initial strain-promoted azide-alkyne cycloaddition. Myoblasts encapsulated in these networks respond to increased matrix stiffness through decreased cell spreading and nuclear localization of Yes-associated protein 1 (YAP). However, when the photocrosslinking reaction is delayed to allow cell spreading, elongated myoblasts display increased YAP nuclear localization.