Search for: All records

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. 

The ability to engineer complex multicellular systems has enormous potential to inform our understanding of biological processes and disease and alter the drug development process. Engineering living systems to emulate natural processes or to incorporate new functions relies on a detailed understanding of the biochemical, mechanical, and other cues between cells and between cells and their environment that result in the coordinated action of multicellular systems. On April 3–6, 2022, experts in the field met at the Keystone symposium “Engineering Multicellular Living Systems” to discuss recent advances in understanding how cells cooperate within a multicellular system, as well as recent efforts to engineer systems like organ-on-a-chip models, biological robots, and organoids. Given the similarities and common themes, this meeting was held in conjunction with the symposium “Organoids as Tools for Fundamental Discovery and Translation”. 

Abstract 3D organoid models have recently seen a boom in popularity, as they can better recapitulate the complexity of multicellular organs compared to other in vitro culture systems. However, organoids are difficult to image because of the limited penetration depth of high‐resolution microscopes and depth‐dependent light attenuation, which can limit the understanding of signal transduction pathways and characterization of intimate cell‐extracellular matrix (ECM) interactions. To overcome these challenges, phototransfer by allyl sulfide exchange‐expansion microscopy (PhASE‐ExM) is developed, enabling optical clearance and super‐resolution imaging of organoids and their ECM in 3D. PhASE‐ExM uses hydrogels prepared via photoinitiated polymerization, which is advantageous as it decouples monomer diffusion into thick organoid cultures from the hydrogel fabrication. Apart from compatibility with organoids cultured in Matrigel, PhASE‐ExM enables 3.25× expansion and super‐resolution imaging of organoids cultured in synthetic poly(ethylene glycol) (PEG) hydrogels crosslinked via allyl‐sulfide groups (PEG‐AlS) through simultaneous photopolymerization and radical‐mediated chain‐transfer reactions that complete in <70 s. Further, PEG‐AlS hydrogels can be in situ softened to promote organoid crypt formation, providing a super‐resolution imaging platform both for pre‐ and post‐differentiated organoids. Overall, PhASE‐ExM is a useful tool to decipher organoid behavior by enabling sub‐micrometer scale, 3D visualization of proteins and signal transduction pathways.