Mesenchymal-like Stem Cell Isolation via 3D Culture and Magnetic Beads
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Solution Overview
Problem
Current methods for isolating and culturing mesenchymal stem cells from human pluripotent stem cells face challenges such as low cell viability, increased culture periods, high costs, and genetic instability, particularly due to the use of flow cytometers and cytokine applications, which also struggle to effectively control pluripotency and maintain genetic stability.
Innovation Solution
A method involving the differentiation of human pluripotent stem cells cultured to passage 70 or lower to produce embryoid bodies, selecting cystic embryoid bodies, and loading them onto a cell-permeable 3D culture unit to isolate mesenchymal-like stem cells, followed by uniformization of monolayer-shaped cell clusters, which express CD90 and SOX2 at 95% or greater, enhancing anti-inflammatory efficacy and immunosuppression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If flow cytometer is used to separate cells representing desired markers, then cell separation is achieved, but cell viability decreases and culture period increases
Solution Approach 1:
The patent extracts and removes the harmful laser irradiation step from the cell separation process. Instead of using flow cytometer with laser that damages cells, the invention uses antibody-conjugated magnetic beads for magnetic separation, which gently isolates target cells without exposing them to damaging electromagnetic radiation, thereby maintaining cell viability while achieving precise separation.
Solution Approach 2:
The patent introduces magnetic beads as an intermediary substance between the antibody and the cell separation process. The magnetic beads serve as a mediator that carries the antibody to bind with target cells, enabling separation through magnetic field application rather than direct laser irradiation, thus protecting cell viability during the separation process.
2Adaptability or versatility
If large amounts and large types of cytokines and chemicals are applied to induce differentiation, then differentiation is achieved, but cost increases
Solution Approach 1:
The patent extracts and removes the expensive cytokine and chemical treatment steps from the differentiation process. Instead of using large amounts of growth factors and chemicals, the invention relies on the natural differentiation capacity of pluripotent stem cells when cultured in a 3D embryoid body system, significantly reducing material costs while maintaining differentiation capability.
Solution Approach 2:
The patent enables the pluripotent stem cells to perform self-differentiation without external cytokine or chemical induction. By providing appropriate 3D culture conditions and allowing embryoid body formation, the cells naturally differentiate into various lineages including mesenchymal stem cells, eliminating the need for expensive external differentiation factors.
3Adaptability or versatility
If adult mesenchymal stem cells are used for cell therapy, then various therapeutic applications are achieved, but proliferation ability is limited to 40 passages
Solution Approach 1:
The patent performs preliminary action by deriving mesenchymal stem cells from pluripotent stem cells before they are needed for therapy. By establishing an immortal pluripotent cell line that can be continuously passaged and then differentiating it into mesenchymal stem cells when needed, the system overcomes the limited passage number of adult mesenchymal stem cells and provides an unlimited source of therapeutic cells.
Solution Approach 2:
The patent segments the cell therapy process into two distinct stages: (1) maintenance of pluripotent stem cells with unlimited proliferation capacity, and (2) differentiation into mesenchymal stem cells for therapeutic application. This segmentation allows the system to benefit from both the unlimited passage capability of pluripotent cells and the therapeutic versatility of mesenchymal stem cells.
Data Source
AI summary
Disclosed is a method for effectively preparing mesenchymal-like stem cells, the method including: preparing human pluripotent stem cells cultured to passage 70 or lower after establishment of cell lines; inducing differentiation of the human pluripotent stem cells to produce embryoid bodies and selecting cystic embryoid bodies therefrom; loading the cystic embryoid bodies on a cell-permeable three-dimensional (3D) culture unit to isolate mesenchymal-like stem cells therefrom; isolating only monolayer-shaped cell clusters from the mesenchymal-like stem cells passing through the cell-permeable 3D culture unit; and uniformizing sizes of the monolayer-shaped cell clusters, in which the mesenchymal-like stem cells have anti-inflammatory efficacy and immunosuppression. Further, disclosed are mesenchymal-like stem cells prepared by the preparation method, a transporter or a therapeutic composition including the mesenchymal-like stem cells, and a composition for prevention or treatment of diseases that includes an active ingredient or exosomes secreted from the mesenchymal-like stem cells.