Ciliary Marginal Zone-Like Structure Production via Wnt and FGF Modulation
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Solution Overview
Problem
There is a need for an efficient method to produce a ciliary marginal zone-like structure for retinal tissue formation and maintenance, as existing methods are inadequate in achieving high efficiency and functional relevance.
Innovation Solution
A method involving the culture of cell aggregates with Chx10 positive cells in serum-free or serum-containing media, modulating the Wnt and FGF signaling pathways to create a ciliary marginal zone-like structure, which can be used for evaluating toxicity, drug efficacy, or as a biological material for transplantation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional culture methods are used to produce ciliary marginal zone-like structure, then the production process is simple, but the production efficiency is low
Solution Approach 1:
The patent applies preliminary action by pre-determining the culture conditions and signaling pathway modulations before the actual production process. The method establishes specific culture protocols with predetermined concentrations of signaling molecules and defined culture durations, allowing the ciliary marginal zone-like structure to form efficiently without requiring complex real-time adjustments during cultivation.
Solution Approach 2:
The patent utilizes parameter changes by systematically varying culture conditions including signaling pathway activations, medium compositions, and culture durations. By optimizing parameters such as Wnt and FGF signaling levels, the method achieves high production efficiency of ciliary marginal zone-like structure while maintaining controllable process complexity.
2Reliability
If the culture period is extended to allow RPE65 gene-expressing cells to appear, then more complete retinal tissue differentiation occurs, but the production time increases
Solution Approach 1:
The patent applies partial action by culturing the cell aggregate for a specific duration that is sufficient to achieve the desired Chx10 positive cell proportion (20-100%) but stops before RPE65 gene-expressing cells appear. This partial culture approach produces a cell aggregate with neural retinal progenitor characteristics without completing full retinal differentiation, thereby reducing culture time while maintaining reliability for the intended application.
3Productivity
If substances acting on Wnt signal pathway are continuously present in culture medium, then neural retinal progenitor cell formation is enhanced, but RPE65 gene-expressing cells may appear prematurely
Solution Approach 1:
The patent applies periodic action by using time-dependent culture protocols where substances acting on Wnt signaling pathway are present only during specific culture periods. The method involves adding these substances at defined time points and removing them at predetermined stages, creating a periodic signaling environment that enhances neural retinal progenitor cell formation while preventing premature appearance of RPE65 gene-expressing cells through controlled signal timing.
Data Source
AI summary
The present invention provides a method for producing a cell aggregate containing a ciliary marginal zone-like structure, including a step of culturing a cell aggregate containing a retinal tissue in which Chx10 positive cells are present in a proportion of 20% or more and 100% or less of the tissue in a serum-free medium or serum-containing medium each containing a substance acting on the Wnt signal pathway and a substance inhibiting the FGF signal pathway for only a period before the appearance of a RPE65 gene expressing cell, followed by culturing the “cell aggregate in which a RPE65 gene-expressing cell does not appear” thus obtained in a serum-free medium or serum-containing medium each free of a substance acting on the Wnt signal pathway and so on. According to the production method of the present invention, a ciliary marginal zone-like structure can be produced with high efficiency.