Intestinal Organoid Production via Staged Differentiation
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Solution Overview
Problem
Current methods for preparing intestinal organoids from pluripotent stem cells fail to replicate the complete structure of the intestinal tract, particularly the crypt-villus structures, and are inefficient for large-scale production, limiting their application in high-throughput assays and drug screening.
Innovation Solution
A method involving specific culture conditions, including the use of low-molecular compounds and passage culture, is developed to differentiate pluripotent stem cells into intestinal organoids with characteristics closer to those of the small intestine, involving steps such as differentiating into endoderm-like and intestinal stem cells, culturing with growth factors, and forming spheroids, which enhances production efficiency and uniformity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If intestinal organoid is prepared by differentiation induction using Matrigel embedding, then the organoid structure is formed, but the crypt-villus structures of the intestinal tract are not reproduced and the culture volume is limited
Solution Approach 1:
The invention segments the differentiation process into distinct stages: first forming intestinal stem cell-like cells from endoderm-like cells, then forming spheroids, and finally differentiating into intestinal organoids with crypt-villus structures. This staged approach allows each stage to be optimized independently, achieving both structural completeness and scalability.
Solution Approach 2:
The invention transitions from traditional 2D culture to 3D spheroid formation and then to complex 3D organoid structures with crypt-villus architecture. This dimensional progression enables more realistic intestinal tract structure reproduction while maintaining culture expandability through spheroid intermediate formation.
2Reliability
If traditional differentiation induction method is used, then intestinal organoid is prepared, but the production efficiency and uniformity are insufficient for large-scale application
Solution Approach 1:
The invention performs preliminary actions by first differentiating pluripotent stem cells into endoderm-like cells, then into intestinal stem cell-like cells, and forming spheroids before final organoid differentiation. This preliminary staging ensures uniform cell populations enter the final differentiation stage, improving both uniformity and efficiency of large-scale production.
Solution Approach 2:
The invention employs specific parameter changes including adding low-molecular compounds at defined stages, controlling culture conditions (temperature, pH, oxygen concentration), and adjusting growth factor concentrations to optimize each differentiation stage. These parameter optimizations enhance both organoid uniformity and production efficiency.
3Reliability
If primary small-intestinal epithelial cells are used for pharmacokinetics evaluation, then comprehensive evaluation is achieved, but it is difficult to obtain the cells
Solution Approach 1:
The invention creates a copy of primary small-intestinal epithelial cells by differentiating pluripotent stem cells into intestinal organoids that replicate the structural and functional characteristics of native intestinal epithelium, including crypt-villus structures. This cell-free copy system maintains evaluation accuracy while eliminating the difficulty of obtaining primary cells.
Solution Approach 2:
The invention uses parameter changes in culture conditions and differentiation protocols to generate intestinal organoids with physiological relevance comparable to primary cells, achieving reliable pharmacokinetics evaluation without requiring invasive cell collection from donors.
Data Source
AI summary
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.