Organoid Culture Medium Without EGF for Long-Term EGFR Maintenance
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing cell culture media for intestinal epithelial cells face challenges in maintaining long-term culturing due to the decrease in expression of epidermal growth factor receptor (EGFR) caused by epidermal growth factor (EGF) stimulation, leading to inhibition of differentiation or cell death, especially in human and tumor tissues.
Innovation Solution
A cell culture medium comprising insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2), and epiregulin (EREG) under hypoxic conditions, with optional components like Wnt agonists, BMP inhibitors, and TGF-β inhibitors, is used to culture epithelial stem cells and tumor cells without EGF and p38 inhibitor, forming organoids efficiently.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If epidermal growth factor (EGF) is added to the cell culture medium to promote cell proliferation, then cell growth is enhanced, but expression of epidermal growth factor receptor (EGFR) decreases leading to inhibition of differentiation or cell death
Solution Approach 1:
The patent removes EGF from the cell culture medium to prevent the harmful feedback effect where EGF stimulation causes EGFR expression decrease. By extracting this specific component, the medium maintains EGFR expression levels while avoiding the associated harmful effects on cell differentiation and survival
Solution Approach 2:
The patent changes the compositional parameters of the cell culture medium by substituting EGF with alternative growth factors (insulin-like growth factor 1, fibroblast growth factor 2, and epiregulin) at specific concentrations. This parameter change maintains cell proliferation capability while preventing EGFR downregulation
2Reliability
If p38 inhibitor is added to the cell culture medium to maintain EGFR expression, then EGFR expression is maintained, but differentiation is inhibited or cell death occurs preventing culturing of some human tissue and tumor tissue
Solution Approach 1:
The patent removes p38 inhibitor from the cell culture medium to eliminate its harmful side effects. By extracting this component, the medium avoids inhibition of differentiation and cell death that occur with p38 inhibitor treatment, thereby expanding the range of culturable tissues
Solution Approach 2:
The patent changes the medium composition by excluding p38 inhibitor and instead using a combination of growth factors (IGF1, FGF2, EREG) at optimized concentrations to maintain EGFR expression through alternative mechanisms that do not interfere with cell differentiation
3Duration of action of moving object
If conventional cell culture media are used for long-term culturing of intestinal epithelial cells, then initial cell growth is achieved, but long-term maintenance is impossible due to unknown growth factors and EGFR downregulation
Solution Approach 1:
The patent performs preliminary optimization of the cell culture medium composition before long-term culturing begins. By pre-determining the optimal combination of growth factors (IGF1, FGF2, EREG) and their concentrations, the medium is prepared in advance to support sustained EGFR expression and intestinal epithelial stem cell maintenance over extended periods
Solution Approach 2:
The patent changes multiple parameters of the cell culture medium simultaneously, including the types and concentrations of growth factors, to create an optimized formulation that enables long-term culturing. This comprehensive parameter optimization addresses the previously unknown growth factor requirements for intestinal epithelial stem cells
Data Source
Figure 1
Figure 2
Figure 3
AI summary
A cell culture medium for culturing organoid containing at least two types of components selected from the group consisting of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and epiregulin (EREG), and at least one type of component among the following components i) to iii): i) Wnt agonist, ii) bone morphogenetic protein (BMP) inhibitor, and iii) transforming growth factor-β (TGF-β) inhibitor.