An object is to prepare an intestinal organoid having a characteristic close to the small intestine of a living body, from a pluripotent stem cell. An intestinal organoid is prepared from a pluripotent stem cell, by the following steps of: (1) differentiating the pluripotent stem cell into an endoderm-like cell; (2) differentiating the endoderm-like cell obtained in step (1) into an intestinal stem cell-like cell; (3) culturing the intestinal stem cell-like cell obtained in step (2) in the presence of an epidermal growth factor, a fibroblast growth factor, a TGF β receptor inhibitor, a GSK-3 β inhibitor, and a ROCK inhibitor; (4) culturing the cell obtained in step (3) to form a spheroid; and (5) differentiating the spheroid formed in step (4) to form an intestinal organoid, wherein the differentiation includes culturing in the presence of an epidermal growth factor, a BMP inhibitor, and a Wnt signal activator. Also, a plane culture system is prepared by subjecting the cells constituting the intestinal organoid formed in step (5) to plane culture in the presence of an epidermal growth factor and a TGF β receptor inhibitor. A highly functional evaluation system having the villi structure is constructed by using air-liquid interface culture in the plane culture.

Described are cell culture solutions and systems for epithelial stem cell and organoid cultures, formation of epithelial constructs and uses of the same in transplantation.

The generation of complex organ tissues from human embryonic and pluripotent stem cells (PSCs) remains a major challenge for translational studies. It is shown that PSCs can be directed to differentiate into intestinal tissue in vitro by modulating the combinatorial activities of several signaling pathways in a step-wise fashion, effectively recapitulating in vivo fetal intestinal development. The resulting intestinal “organoids” were three-dimensional structures consisting of a polarized, columnar epithelium surrounded by mesenchyme that included a smooth muscle-like layer. The epithelium was patterned into crypt-like SOX9-positive proliferative zones and villus-like structures with all of the major functional cell types of the intestine. The culture system is used to demonstrate that expression of NEUROG3, a pro-endocrine transcription factor mutated in enteric anendocrinosis is sufficient to promote differentiation towards the enteroendocrine cell lineage. In conclusion, PSC-derived human intestinal tissue should allow for unprecedented studies of human intestinal development, homeostasis and disease.

The method for producing a cell construct including small intestinal epithelial cells includes: seeding stem cells onto cell culture substrate, the cell culture substrate having surface including cell culture part, wherein the cell culture part includes non-cell-adhesive part, and cell-adhesive part extending continuously or intermittently along periphery of the non-cell-adhesive part and surrounding the non-cell-adhesive part; and culturing the stem cells seeded to differentiate a part of the stem cells into small intestinal epithelial cells.

In some aspects, disclosed herein are methods and compositions for generating enterochromaffin cells. In some aspects, the methods and compositions disclosed herein relate to use of an inhibitor of IL-4/JAK3 signaling pathway. In other aspects, disclosed herein are cell compositions, pharmaceutical compositions, and medical devices that relate to pancreatic cells that are generated according to the methods disclosed herein.

Embodiments of the disclosure concern systems, methods, and/or compositions for cultivation of mammalian viruses, including at least human noroviruses and sapoviruses within the Caliciviridae family of viruses. The ex vivo culture systems include intestinal enteroids in combination with bile or a functionally active fraction or component thereof. In specific embodiments, the culture system is utilized to test inactivation compounds for therapeutic or environmental efficacy and to test contaminated comestibles and/or environmental entities for determination of the presence of infectious virus. Furthermore, antiviral compositions may be tested using systems of the disclosure, including drugs, small molecule inhibitors, and biologics such as neutralizing monoclonal antibodies.

A method of making a live cell construct is carried out by: (a) providing a non-cellular support having a top surface and a bottom surface, (b) contacting live undifferentiated cells to the non-cellular support, and then (c) propagating a gastrointestinal epithelial cell monolayer on said top surface. In some embodiments, the live cells in the monolayer include: (i) undifferentiated cells (e.g., stem or progenitor cells); and (ii) optionally, but in some embodiments preferably, differentiated cells (e.g., enterocytes, Paneth cells, enteroendocrine cells, tuft cells, microcells, intra-epithelial lymphocytes, and/or goblet cells). Constructs formed by such methods and methods of using the same (e.g., in high through-put screening) are also described.

Disclosed are methods of assessing the ability of a candidate therapeutic agent to reverse, reduce or prevent intestinal injury by a potential toxic agent using a three-dimensional, engineered, bioprinted, biological intestinal tissue model. Also disclosed are methods of assessing the effect of an agent on intestinal function, the method comprising contacting the agent with a three-dimensional, engineered, bioprinted, biological intestinal tissue model.

The invention disclosed herein generally relates to methods and systems for improving physiological growth of cultured tissues. In particular, the invention disclosed herein relates to methods and systems for promoting maintenance of cultured intestinal organoids (e.g., derived from pluripotent stem cells or from primary sources such as biopsy tissue).

A method for culturing primary cells of gastric cancer and gallbladder cancer and cholangiocarcinoma and auxiliary reagents. A method for culturing primary cells of gastric cancer and gallbladder cancer and cholangiocarcinoma and auxiliary reagents. The core of the technology is that: (1) the solid tumor tissues of gastric cancer and gallbladder cancer and cholangiocarcinoma are treated with a mild cell dissociation reagent, and the primary tumor cells of gallbladder cancer and cholangiocarcinoma in a bile sample are isolated by a mild method to ensure the vitality of cancer cells to the greatest extent; (2) a special serum-free medium is prepared, and tumor cells of gastric cancer and gallbladder cancer and cholangiocarcinoma are cultured in vitro by a suspension culture system to eliminate the interference of normal cells to the greatest extent while ensuring normal amplification of cancer cells.