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Epithelial organoids are most efficiently grown from mouse-tumour-derived, reconstituted extracellular matrix hydrogels, whose poorly defined composition, batch-to-batch variability and immunogenicity limit clinical applications. Efforts to replace such ill-defined matrices for organoid culture have largely focused on non-adaptable hydrogels composed of covalently crosslinked hydrophilic macromolecules. However, the excessive forces caused by tissue expansion in such elastic gels severely restrict organoid growth and morphogenesis. Chemical or enzymatic degradation schemes can partially alleviate this problem, but due to their irreversibility, long-term applicability is limited. Here we report a family of synthetic hydrogels that promote extensive organoid morphogenesis through dynamic rearrangements mediated by reversible hydrogen bonding. These tunable matrices are stress relaxing and thus promote efficient crypt budding in intestinal stem-cell epithelia through increased symmetry breaking and Paneth cell formation dependent on yes-associated protein 1. As such, these well-defined gels provide promising versatile matrices for fostering elaborate in vitro morphogenesis. The influence of stress relaxation of the extracellular matrix on the formation of intestinal organoids was investigated. It was shown that a stress-relaxing synthetic matrix promotes crypt budding through increased symmetry breaking and niche cell formation.

Organotypic models of patient-specific tumours are revolutionizing our understanding of cancer heterogeneity and its implications for personalized medicine. These advancements are, in part, attributed to the ability of organoid models to stably preserve genetic, proteomic, morphological and pharmacotypic features of the parent tumour in vitro, while also offering unprecedented genomic and environmental manipulation. Despite recent innovations in organoid protocols, current techniques for cancer organoid culture are inherently uncontrolled and irreproducible, owing to several non-standardized facets including cancer tissue sources and subsequent processing, medium formulations, and animal-derived three-dimensional matrices. Given the potential for cancer organoids to accurately recapitulate the intra- and intertumoral biological heterogeneity associated with patient-specific cancers, eliminating the undesirable technical variability accompanying cancer organoid culture is necessary to establish reproducible platforms that accelerate translatable insights into patient care. Here we describe the current challenges and recent multidisciplinary advancements and opportunities for standardizing next-generation cancer organoid systems. This Review summarizes limitations in the current techniques used for patient-derived cancer organoid culture and highlights recent advancements and future opportunities for their standardization.

Epithelial organoids, such as those derived from stem cells of the intestine, have great potential for modelling tissue and disease biology 1 , 2 , 3 – 4 . However, the approaches that are used at present to derive these organoids in three-dimensional matrices 5 , 6 result in stochastically developing tissues with a closed, cystic architecture that restricts lifespan and size, limits experimental manipulation and prohibits homeostasis. Here, by using tissue engineering and the intrinsic self-organization properties of cells, we induce intestinal stem cells to form tube-shaped epithelia with an accessible lumen and a similar spatial arrangement of crypt- and villus-like domains to that in vivo. When connected to an external pumping system, the mini-gut tubes are perfusable; this allows the continuous removal of dead cells to prolong tissue lifespan by several weeks, and also enables the tubes to be colonized with microorganisms for modelling host–microorganism interactions. The mini-intestines include rare, specialized cell types that are seldom found in conventional organoids. They retain key physiological hallmarks of the intestine and have a notable capacity to regenerate. Our concept for extrinsically guiding the self-organization of stem cells into functional organoids-on-a-chip is broadly applicable and will enable the attainment of more physiologically relevant organoid shapes, sizes and functions. Miniature gut tubes grown in vitro from mouse intestinal stem cells are perfusable, can be colonized with microorganisms and exhibit a similar arrangement and diversity of specialized cell types to intestines in vivo.

The difficulty of studying post-implantation development in mammals has sparked a flurry of activity to develop in vitro models, termed embryoids, based on self-organizing pluripotent stem cells. Previous approaches to derive embryoids either lack the physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here, we report a bioengineering-inspired approach aimed at addressing this gap. We employ a high-throughput cell aggregation approach to simultaneously coax mouse embryonic stem cells into hundreds of uniform epiblast-like aggregates in a solid matrix-free manner. When co-cultured with mouse trophoblast stem cell aggregates, the resulting hybrid structures initiate gastrulation-like events and undergo axial morphogenesis to yield structures, termed EpiTS embryoids , with a pronounced anterior development, including brain-like regions. We identify the presence of an epithelium in EPI aggregates as the major determinant for the axial morphogenesis and anterior development seen in EpiTS embryoids . Our results demonstrate the potential of EpiTS embryoids to study peri-gastrulation development in vitro. Previous approaches to derive embryoids either lack physiological morphology and signaling interactions, or are unconducive to model post-gastrulation development. Here the authors use a high-throughput approach to induce mouse embryonic stem cells into epiblast-like aggregates, which are then co-cultured with mouse trophoblast stem cell aggregates, to yield embryoids with axial morphogenesis and anterior development.

Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures 1 , 2 – 3 . This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time 4 . However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan. As a consequence, oncogenesis and tumour formation studies are not possible in vitro and instead require the extensive use of animal models, which provide limited spatiotemporal resolution of cellular dynamics and come at a considerable cost in terms of resources and animal lives. Here we developed topobiologically complex mini-colons that are able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emergent colon tumours can be tracked in real-time at the single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intratumoural and intertumoural diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumours in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, mini-colons can be used to identify tumorigenic determinants and pharmacological opportunities. As a whole, our study paves the way for cancer initiation research outside living organisms. Topobiologically complex mini-colons—which enable the faithful in vitro recapitulation of colorectal cancer tumorigenesis and its environmental determinants—offer the possibility to reduce animal use in a wide range of experimental applications.

Despite the high prevalence of gastric diseases like gastric cancer and peptic ulcer disease attributed to Helicobacter pylori infections, there is still only a limited understanding of the underlying mechanisms. Existing in vitro models are either two-dimensional systems lacking the structural complexity of the gastric architecture, or complex three-dimensional systems that pose challenges for experimental access. In this study, we introduce a patterned homeostatic human gastric organoid-on-a-chip system with bilateral access that is capable of modeling H. pylori niche establishment and persistent colonization of the gastric epithelium. We show that in physiological apical acidic conditions, our organ-on-a-chip can generate pit cells of higher maturity in contrast to traditionally grown organoids. Upon infection with H. pylori for up to 6 days, these mature pit cells exhibit a distinctive response from other cell types, which was previously uncharacterized. Beyond its application in studying H. pylori infection, the increased structural and functional relevance of our model offers broader significance as a versatile platform for advancing our understanding of gastric epithelial cell interactions, gastric mucosal immunity, and host-pathogen interactions. Current knowledge of the mechanisms that underly Helicobacter pylori ( H. pylori ) infections remains limited. Here, the authors present an in vitro gastric model that combines structural complexity with experimental accessibility, enabling improved research into H. pylori -induced pathogenesis.

Treating cartilage injuries and degenerations represents an open surgical challenge. The recent advances in cell therapies have raised the need for a potent off-the-shelf cell source. Intra-articular injections of TGF-β transduced polydactyly chondrocytes have been proposed as a chronic osteoarthritis treatment but despite promising results, the use of gene therapy still raises safety concerns. In this study, we characterized infant, polydactyly chondrocytes during in vitro expansion and chondrogenic re-differentiation. Polydactyly chondrocytes have a steady proliferative rate and re-differentiate in 3D pellet culture after up to five passages. Additionally, we demonstrated that polydactyly chondrocytes produce cartilage-like matrix in a hyaluronan-based hydrogel, namely transglutaminase cross-linked hyaluronic acid (HA-TG). We utilized the versatility of TG cross-linking to augment the hydrogels with heparin moieties. The heparin chains allowed us to load the scaffolds with TGF-β1, which induced cartilage-like matrix deposition both in vitro and in vivo in a subcutaneous mouse model. This strategy introduces the possibility to use infant, polydactyly chondrocytes for the clinical treatment of joint diseases.

Organoid tumor models have emerged as a powerful tool in the fields of biology and medicine as such 3D structures grown from tumor cells recapitulate better tumor characteristics, making these tumoroids unique for personalized cancer research. Assessment of their functional behavior, particularly protein secretion, is of significant importance to provide comprehensive insights. Here, a label‐free spectroscopic imaging platform is presented with advanced integrated optofluidic nanoplasmonic biosensor that enables real‐time secretion analysis from single tumoroids. A novel two‐layer microwell design isolates tumoroids, preventing signal interference, and the microarray configuration allows concurrent analysis of multiple tumoroids. The dual imaging capability combining time‐lapse plasmonic spectroscopy and bright‐field microscopy facilitates simultaneous observation of secretion dynamics, motility, and morphology. The integrated biosensor is demonstrated with colorectal tumoroids derived from both cell lines and patient samples to investigate their vascular endothelial growth factor A (VEGF‐A) secretion, growth, and movement under various conditions, including normoxia, hypoxia, and drug treatment. This platform, by offering a label‐free approach with nanophotonics to monitor tumoroids, can pave the way for new applications in fundamental biological studies, drug screening, and the development of therapies. Real‐time secretion analysis from single tumoroids is enabled by a label‐free spectroscopic imaging platform with an advanced optofluidic nanoplasmonic biosensor. Demonstrated with colorectal tumoroids to investigate their vascular endothelial growth factor secretion, growth, and movement under various conditions, this platform shows its capability of monitoring secretion dynamics, motility, and morphology and promises new avenues for potential screening applications.

Immunotherapy has emerged as a new standard of care for certain cancer patients with specific cellular and molecular makeups. However, there is still an unmet need for ex vivo models able to readily assess the effectiveness of immunotherapeutic treatments in a high-throughput and patient-specific manner. To address this issue, we have developed a microarrayed system of patient-derived tumoroids with recreated immune microenvironments that are optimized for the high-content evaluation of tumor-infiltrating lymphocyte functionality. Here we show that this system offers unprecedented opportunities to evaluate tumor immunogenicity, characterize the response to immunomodulators, and explore novel approaches for personalized immuno-oncology.

Neuron development and function are exquisitely sensitive to the mechanical properties of their surroundings. Three-dimensional (3D) cultures are therefore being explored as they better mimic the features of the native extracellular matrix. Limitations of existing 3D culture models include poorly defined composition, rapid degradation, and suboptimal biocompatibility. Here we show that ionically cross-linked ultrasoft hydrogels made from unmodified alginate can potently promote neuritogenesis. Alginate hydrogels were characterized mechanically and a remarkable range of stiffness (10–4000 Pa) could be produced by varying the macromer content (0.1–0.4% w/v) and CaCl 2 concentration. Dissociated rat embryonic cortical neurons encapsulated within the softest of the hydrogels (0.1% w/v, 10 mM CaCl 2 ) showed excellent viability, extensive formation of axons and dendrites, and long-term activity as determined by calcium imaging. In conclusion, alginate is an off-the-shelf, easy to handle, and inexpensive material, which can be used to make ultrasoft hydrogels for the formation of stable and functional 3D neuronal networks. This 3D culture system could have important applications in neuropharmacology, toxicology, and regenerative medicine.