compartment occupied by continuously dividing cells at the bottom, which after differentiation migrate upward toward the villus. In the villus, six different mature cell types are found, including secretory cells, such as goblet, Paneth, enteroendocrine, and tuft cells, and also absorptive cells, such as enterocytes and microfold cells. [1-3] The intestinal epithelium serves two main functions, nutrient uptake and protection against harmful substances and pathogens. Τhe presence of strong apical-basolateral compartmentalization is of critical importance for proper intestinal function. [3-7] Despite various advances in cell and tissue culture technologies [8-11] and organ-on-chip models [12] that have been used to study intestinal physiology over the past years, numerous morphological and functional aspects remain unknown. Miniaturized 3D, multicellular, stem cell-derived constructs that mimic in vivo tissue, called organoids, along with organ-on-chip systems represent culture systems able to recapitulate the complexity of an organ closer than any previously utilized technique. [13,14] Organoids have high self-organization capacity and hold great potential as a research tool to explore unknown aspects of organ development, tissue regeneration, disease pathology, cell biology, and as drug-screening platforms. In the past few years, numerous protocols have been described to grow organoids that resemble various organs, such as liver, brain, intestine, and lung. [15-21] Intestinal organoid culture is a relatively simple system, that typically involves the embedding of small multicellular fragments (containing LGR5 + cells) in Matrigel, which serves as an extracellular matrix mimic, supplemented with the right cocktail of growth factors. [16] This results in growing and self-sustaining intestinal organoids, which contain all the above mentioned cell types found in vivo and are organized in a crypt-villus structure that surrounds a central lumen, with strong apical-basolateral polarity. [13,22] Hence, these organoids have contributed significantly to the understanding of normal intestinal function and dysfunction in the last years. Even though organoids are a powerful tool, their use still faces numerous limitations. Bioengineering approaches hold great potential in overcoming some constraints. [23-27] More specifically, mass transport in organoids is usually restricted by their size, a limitation which researchers hope to overcome with either the use of bioreactors, microfluidic chips or integration of vascular
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.
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.
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