Intestinal Organoid Preparation via Sequential Differentiation
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Solution Overview
Problem
Current methods for preparing intestinal organoids from pluripotent stem cells face challenges in achieving functional maturity and efficiency, with existing 2D culture systems failing to sustain cells long-term and 3D cultures lacking verified pharmacokinetic functionality.
Innovation Solution
A method involving differentiation steps using MEK1/2, DNA methylation, TGF-β receptor, and γ-secretase inhibitors, along with epidermal growth factor and Wnt signal activators, to form intestinal organoids in a 3D culture with low molecular weight compounds, enhancing efficiency and functionality.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If intestinal organoids are prepared using conventional 3D culture methods with Matrigel, then three-dimensional tissue structures can be formed, but the pharmacokinetic functions remain immature and insufficiently verified
Solution Approach 1:
The patent implements preliminary action by first establishing a stable 3D organoid structure using Matrigel embedding before applying sequential differentiation protocols. The culture process begins with establishing intestinal stem cell colonies, then progressively introduces differentiation-inducing factors in a predetermined sequence to ensure proper tissue maturation before functional verification
2Quantity of substance
If intestinal epithelial cells are obtained through 2D culture, then cells can be obtained for evaluation, but they cannot be cultured long-term and do not form functional intestinal organoids
Solution Approach 1:
The patent transitions from 2D monolayer culture to 3D organoid culture by embedding intestinal stem cells in Matrigel droplets. This dimensional change enables long-term culture sustainability while maintaining cell proliferation and differentiation capabilities, producing functional three-dimensional tissue structures that mimic in vivo intestinal architecture
Solution Approach 2:
The patent uses Matrigel as an intermediary extracellular matrix that provides structural support and biochemical signals necessary for long-term organoid maintenance. The Matrigel embedding serves as a mediator between the cultured cells and the culture environment, enabling sustained growth and functional maturation that cannot be achieved in 2D culture systems
Data Source
AI summary
An object of the present invention is to prepare a functional intestinal organoid from pluripotent stem cells. An intestinal organoid is prepared from pluripotent stem cells, by the following steps (1) to (4): (1) differentiating pluripotent stem cells into endoderm-like cells; (2) differentiating the endoderm-like cells obtained in step (1) into intestinal stem cell-like cells; (3) culturing the intestinal stem cell-like cells obtained in step (2) to form spheroids; and (4) differentiating the spheroids formed in step (3) to form an intestinal organoid, the step including culture in the presence of a MEK1/2 inhibitor, a DNA methylation inhibitor, a TGF-β receptor inhibitor, and a γ-secretase inhibitor, in addition to an epidermal growth factor, a BMP inhibitor, and a Wnt signal activator.