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Microbiome is an integral part of the gut and is essential for its proper function. Imbalances of the microbiota can be devastating and have been linked with several gastrointestinal conditions. Current gastrointestinal models do not fully reflect the in vivo situation. Thus, it is important to establish more advanced in vitro models to study host-microbiome/pathogen interactions. Here, we developed for the first time an apical-out human small intestinal organoid model in hypoxia, where the apical surface is directly accessible and exposed to a hypoxic environment. These organoids mimic the intestinal cell composition, structure and functions and provide easy access to the apical surface. Co-cultures with the anaerobic strains Lactobacillus casei and Bifidobacterium longum showed successful colonization and probiotic benefits on the organoids. These novel hypoxia-tolerant apical-out small intestinal organoids will pave the way for unraveling unknown mechanisms related to host-microbiome interactions and serve as a tool to develop microbiome-related probiotics and therapeutics.

Intestinal organoids recapitulate many features of the in vivo gastrointestinal tract and have revolutionized in vitro studies of intestinal function and disease. However, the restricted accessibility of the apical surface of the organoids facing the central lumen (apical-in) limits studies related to nutrient uptake and drug absorption and metabolism. Here, we demonstrate that pluripotent stem cell (PSC)-derived intestinal organoids with reversed epithelial polarity (apical-out) can successfully recapitulate tissue-specific functions. In particular, these apical-out organoids show strong epithelial barrier formation with all the major junctional complexes, nutrient transport and active lipid metabolism. Furthermore, the organoids express drug-metabolizing enzymes and relevant apical and basolateral transporters. The scalable and robust generation of functional, apical-out intestinal organoids lays the foundation for a completely new range of organoid-based high-throughput/high-content in vitro applications in the fields of nutrition, metabolism and drug discovery.

The inner surface of the intestine is a dynamic system, composed of a single layer of polarized epithelial cells. The development of intestinal organoids was a major breakthrough since they robustly recapitulate intestinal architecture, regional specification and cell composition in vitro. However, the cyst-like organization hinders direct access to the apical side of the epithelium, thus limiting their use in functional assays. For the first time, we show an intestinal organoid model from pluripotent stem cells with reversed polarity where the apical side faces the surrounding culture media and the basal side faces the lumen. These inside-out organoids preserve a distinct apico-basolateral orientation for a long period and differentiate into the major intestinal cell types. This novel model lays the foundation for developing new in vitro functional assays particularly targeting the apical surface of the epithelium and thus offers a new research tool to study nutrient/drug uptake, metabolism and host-microbiome/pathogen interactions.

Intestinal organoids recapitulate many features of the in vivo gastrointestinal tract and have revolutionized in vitro studies of intestinal function and disease. However, the restricted accessibility of the apical surface of the organoids facing the central lumen (apical-in) limits studies related to nutrient uptake and drug absorption and metabolism. Here, we demonstrate that pluripotent stem cell (PSC)-derived intestinal organoids with reversed epithelial polarity (apical-out) can successfully recapitulate tissue-specific functions. In particular, these apical-out organoids show strong epithelial barrier formation with all the major junctional complexes, nutrient transport and active lipid metabolism. Furthermore, the organoids express drug-metabolizing enzymes and relevant apical and basolateral transporters. The scalable and robust generation of functional, apical-out intestinal organoids lays the foundation for a completely new range of organoid-based high-throughput/high-content in vitro applications in the fields of nutrition, metabolism and drug discovery., Competing Interests: RT and SG are founders and shareholders of 300MICRONS GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kakni, López-Iglesias, Truckenmüller, Habibović and Giselbrecht.)

Microbiome is an integral part of the gut and is essential for its proper function. Imbalances of the microbiota can be devastating and have been linked with several gastrointestinal conditions. Current gastrointestinal models do not fully reflect the in vivo situation. Thus, it is important to establish more advanced in vitro models to study host-microbiome/pathogen interactions. Here, we developed for the first time an apical-out human small intestinal organoid model in hypoxia, where the apical surface is directly accessible and exposed to a hypoxic environment. These organoids mimic the intestinal cell composition, structure and functions and provide easy access to the apical surface. Co-cultures with the anaerobic strains Lactobacillus casei and Bifidobacterium longum showed successful colonization and probiotic benefits on the organoids. These novel hypoxia-tolerant apical-out small intestinal organoids will pave the way for unraveling unknown mechanisms related to host-microbiome interactions and serve as a tool to develop microbiome-related probiotics and therapeutics., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: R.T and S.G are founders and shareholders of 300MICRONS GmbH., (© The Author(s) 2023.)

The mammalian intestinal epithelium contains more immune cells than any other tissue, and this is largely because of its constant exposure to pathogens. Macrophages are crucial for maintaining intestinal homeostasis, but they also play a central role in chronic pathologies of the digestive system. We developed a versatile microwell-based intestinal organoid-macrophage co-culture system that enables us to recapitulate features of intestinal inflammation. This microwell-based platform facilitates the controlled positioning of cells in different configurations, continuous in situ monitoring of cell interactions, and high-throughput downstream applications. Using this novel system, we compared the inflammatory response when intestinal organoids were co-cultured with macrophages versus when intestinal organoids were treated with the pro-inflammatory cytokine TNF-α. Furthermore, we demonstrated that the tissue-specific response differs according to the physical distance between the organoids and the macrophages and that the intestinal organoids show an immunomodulatory competence. Our novel microwell-based intestinal organoid model incorporating acellular and cellular components of the immune system can pave the way to unravel unknown mechanisms related to intestinal homeostasis and disorders.

For over a decade, organoids mimicking the development, physiology, and disease of the digestive system have been a topic of broad interest and intense study. Establishing organoid models that recapitulate all distinct regions of the gastrointestinal tract (GIT) has proven challenging since each tissue surrogate requires tailor-made modifications of the original protocol to generate intestinal organoids. In this review, we discuss the challenges and current advances of the GIT organoid models. Moreover, we envision the next-generation GIT organoids as integrated organoid models, able to recapitulate structural and functional characteristics of multiple regions of the digestive tube in a single in vitro model. We discuss these new trends and provide an outlook for the future of GIT in vitro models., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The inner surface of the intestine is a dynamic system, composed of a single layer of polarized epithelial cells. The development of intestinal organoids was a major breakthrough since they robustly recapitulate intestinal architecture, regional specification and cell composition in vitro . However, the cyst-like organization hinders direct access to the apical side of the epithelium, thus limiting their use in functional assays. For the first time, we show an intestinal organoid model from pluripotent stem cells with reversed polarity where the apical side faces the surrounding culture media and the basal side faces the lumen. These inside-out organoids preserve a distinct apico-basolateral orientation for a long period and differentiate into the major intestinal cell types. This novel model lays the foundation for developing new in vitro functional assays particularly targeting the apical surface of the epithelium and thus offers a new research tool to study nutrient/drug uptake, metabolism and host-microbiome/pathogen interactions., Competing Interests: RT and SG are founders and shareholders of 300MICRONS GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Kakni, López-Iglesias, Truckenmüller, Habibović and Giselbrecht.)

As organoids offer a promising tool to study cell biology and model diseases, organoid technology has rapidly evolved over the last few years. Even though intestinal organoids are one of the most well-established organoid systems, they currently rely on the embedding into an excess amount of poorly defined, tumor-derived extracellular matrix. Here, a novel suspension method is suggested to grow mouse intestinal organoids inside thermoformed microwell arrays. This platform promotes the controlled growth of organoids under matrix-reduced conditions, with Matrigel only used as medium supplement. Hence, this system provides numerous advantages over the previously established methods. Based on the findings, viable and functional mouse intestinal organoids can be preserved for longer periods than in traditional Matrigel domes. Additionally, this microwell-based technique renders a novel organoid culture system in which the heterogeneity of the organoids is significantly reduced. The method paves the way toward more controlled organoid culture systems that can also be beneficial for further downstream applications, such as automated imaging techniques and micromanipulations, which constitute valuable tools for high-throughput applications and translational studies., (© 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)