Muscle-Derived Stem Cell Isolation via Micro-Biopsy
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Solution Overview
Problem
Current methods for obtaining mesenchymal stem cells (MSCs) in veterinary medicine, particularly for equine use, are invasive, inefficient, and lack standardization, with limited availability of specific antibodies and invasive sampling methods that are not suitable for living horses.
Innovation Solution
A minimally invasive process involving a small muscular biopsy (10-20 mg) from living animals, followed by centrifugation on a density gradient to enrich pluripotent MSCs, which are then cultured and characterized by specific markers (CD44, CD90, CD105, and miR expression) for differentiation into adipocytes, osteocytes, and chondrocytes without the need for enzyme digestion or external growth factors.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If bone marrow aspirate is harvested from the sternum or ilium, then mesenchymal stem cells can be obtained, but the procedure is invasive and can provoke infections
Solution Approach 1:
The invention changes the source location parameter from deep bone marrow (sternum/ilium) to superficial adipose tissue (tail head region), and changes the harvesting method parameter from aspirate to liposuction, thereby reducing invasiveness while maintaining stem cell yield
Solution Approach 2:
The invention uses adipose tissue as an intermediary source that is more accessible and less invasive than bone marrow, while still providing the required mesenchymal stem cells for treatment
2Quantity of substance
If large muscular biopsies (6g weight) are collected from horse cadavers, then muscle-derived mesenchymal stem cells can be obtained, but the method is highly invasive and not useful for living horses
Solution Approach 1:
The invention changes the biopsy size parameter from large (6g) to small (10-20 mg), and changes the source from cadaver to living animal, making the procedure minimally invasive and suitable for clinical application
Solution Approach 2:
The invention uses a small portion of muscle tissue (micro-biopsy) that is sufficient for stem cell isolation without causing significant harm to the animal, applying the principle of using minimal necessary tissue
3Quantity of substance
If enzyme digestion techniques are used to isolate stem cells from biopsies, then cells can be obtained, but the possibilities are limited and the process is complex
Solution Approach 1:
The invention extracts stem cells directly from the micro-biopsy through mechanical dissociation and density gradient centrifugation, eliminating the need for enzyme digestion and simplifying the isolation process
Solution Approach 2:
The invention replaces chemical/enzymatic digestion with mechanical methods (mincing and density gradient centrifugation) for cell isolation, reducing process complexity and avoiding enzymatic limitations
4Measurement precision
If human antibodies are used for immunophenotyping, then stem cell characterization can be performed, but cross-reactivity in animals must be tested and standard specifications are lacking
Solution Approach 1:
The invention uses equine-specific antibodies that are species-matched copies designed for equine proteins, eliminating cross-reactivity issues and simplifying immunophenotyping by providing standard specifications for equine stem cells
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method provides a high yield of MSCs that are pluripotent, minimally invasive, and suitable for autologous or xenogenic transplantation, effectively promoting tissue regeneration in equine injuries such as tendinitis and osteoarthritis, with stability through multiple freeze-thaw cycles.
Implementation Method 1
Cells that came out of the explant were centrifuged on a (discontinuous) density gradient to select the part of the cultured cell population with the greatest percentages of pluripotent cells
Data Source
Figure 1A~1B
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AI summary
The present invention provides a new method of obtaining muscle-derived mesenchymal stem cells from microbiopsies of mammalian origin. The invention provides for a minimally invasive methodology yielding high amounts of MSCs that can differentiate into different cell lineages.