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In-vitro efficient culture method for muscle stem cells

A culture method and stem cell technology, applied in the culture process, tissue culture, animal cells, etc., can solve the problems of unable to maintain the regeneration ability of skeletal muscle stem cells and unable to maintain the expression of Pax7 in cells

Pending Publication Date: 2017-06-16
卫露生物医学科技(徐州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the existing clinical trials did not realize a problem: the cell culture technology used could not maintain the regenerative ability of skeletal muscle stem cells
We found that the above methods are questionable for the maintenance of cell stemness. For example, these methods cannot maintain the expression of Pax7, which is essential for maintaining the regenerative function of muscle cells in vitro and in vivo

Method used

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  • In-vitro efficient culture method for muscle stem cells
  • In-vitro efficient culture method for muscle stem cells
  • In-vitro efficient culture method for muscle stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1. Reagent formula and preparation method:

[0022] The composition of culture medium tissue digestion solution is 0.2% type I collagenase, the balance is serum-free DMEM medium, pH=7.5-7.8; muscle stem cell medium consists of DMEM, 20% FBS, 1% penicillin and streptomycin, 1% chicken Embryo extract composition. The collagen coating contains 5 mg / ml type I rat tail collagen. DMEM is a commercial medium.

[0023] The preparation method of tissue digestion solution is: weigh 0.1 gram type I collagenase, fully dissolve in 50 milliliters of serum-free DMEM medium, use sterile filter membrane to filter, and filtrate is kept at 4 ℃.

[0024] The preparation method of primary muscle stem cell medium is: add 100 ml FBS, 5 ml penicillin and streptomycin, and 10 ml chicken embryo extract to 385 ml DMEM. Sterile membrane filtration, and the filtrate was stored in 4 embryos.

Embodiment 2

[0025] Example 2. Isolation and cultivation of mouse muscle stem cells:

[0026]Freshly obtained 8-12-week-old adult mouse limb muscles were shredded in a sterile ultra-clean bench. Then add 8ml of tissue digestion solution for each gram of tissue, incubate at 37°C for 2 hours, centrifuge at 1000rpm for 5 minutes, and remove the supernatant to obtain digested and dissociated tissues and cells; the volume ratio of digested tissues is 1:10 Add serum to terminate enzyme digestion; centrifuge at 1000rpm for 5min, and remove the supernatant. The digested tissue was placed in a collagen-coated petri dish, added muscle stem cell culture medium, and cultured in an incubator at 37°C with 5% CO2. After 3-5 days of culture, when the muscle stem cells grow to 70-80% abundance, the cells are harvested for cell passage or downstream applications, such as in vitro myotube formation or cell therapy.

[0027] The purer mouse muscle stem cells can be obtained through the technique of the inve...

Embodiment 3

[0028] Example 3. Maintenance and preservation of mouse muscle stem cells:

[0029] The mouse muscle stem cells cultured in vitro can be directly added to the cell freezing solution composed of 90% fetal bovine serum and 10% dimethyl sulfoxide DMSO to freeze and store the cells in liquid nitrogen.

[0030] In cell culture, when the degree of cell aggregation exceeds 80%, the cells will spontaneously differentiate and form myotubes. Therefore, when expanding cells, it is necessary to passage in time when the degree of cell aggregation is 60-70%, so as to avoid excessive differentiation of cells.

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Abstract

The invention discloses an in-vitro efficient culture method for muscle stem cells, and a universal muscle stem cell in-vitro culture kit comprising a muscular tissue digestive enzyme, a muscle cell culture medium and a cell culture plate coating solution. The in-vitro culture method is characterized by being capable of rapidly acquiring and amplifying muscle stem cells, maintaining regeneration activity of the muscle stem cells and culturing stem cells which are stable in cell morphology, capable of stably expressing a muscle stem cell marker Pax7 and high in proliferation activity. By adopting the technique disclosed by the invention, relatively pure mouse muscle stem cells can be obtained, and the muscle stem cell marker Pax7 can be expressed. In addition, the cells also express muscle system transcription factors Myf-5 and MyoD; after the cells are differentiated into myotubes, a distinctive muscle structure protein Desmin can be stably expressed.

Description

technical field [0001] The invention relates to a high-efficiency culture method for muscle stem cells in vitro, which relates to a general-purpose muscle stem cell in vitro culture kit including muscle tissue digestive enzymes, muscle cell culture medium and cell culture plate coating solution, belongs to the field of biotechnology, and particularly relates to a A method that can quickly obtain and expand muscle stem cells, and maintain the regenerative activity of muscle stem cells. Background technique [0002] Muscle stem cells, namely satellite cells, are small spindle-shaped flat mononuclear cells that exist between the sarcolemma and basement membrane of muscle fibers in muscle tissue. Muscle stem cells are activated when muscle tissue is damaged and muscle fibers are broken, and then muscle stem cells will multiply in value and fuse with each other to form regenerative muscle fibers, thereby supplementing and replacing damaged muscle fibers. These functions of muscl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0659C12N2500/80
Inventor 沈露露徐杰
Owner 卫露生物医学科技(徐州)有限公司
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