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Method for promoting proliferation of muscle stem cells

A stem cell and muscle technology, applied in the field of animal cell culture and biology, can solve the problems of unfavorable cell culture meat, decreased ability to maintain the stemness of muscle stem cells, etc., and achieve the effect of promoting effective proliferation

Pending Publication Date: 2021-04-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although conventional culture methods can expand muscle stem cells in vitro, the stemness maintenance ability of muscle stem cells will be greatly reduced during the expansion process. The specific performance is that the expression ratio of Pax7 protein and the efficiency of differentiation into myotubes all increase from day 0 to More than 90%, after 5 to 7 days of culture, it will drop to less than 30%, which is the so-called invalid expansion of muscle stem cells, which is very unfavorable for clinical transplantation therapy and the manufacture of cell culture meat

Method used

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  • Method for promoting proliferation of muscle stem cells
  • Method for promoting proliferation of muscle stem cells
  • Method for promoting proliferation of muscle stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The design of the culture medium of embodiment 1 different formulations

[0042] Formula A medium preparation: add 10% (v / v) FBS, 1% (v / v) penicillin and streptomycin and the following components according to the final concentration in DMEM (Gibco): L-ascorbic acid 100 μmol / L, IGF -1 100ng / mL.

[0043] Formula B medium preparation: add 10% (v / v) FBS, 1% (v / v) penicillin and streptomycin to DMEM (Gibco).

[0044]Formula C medium preparation: add 10% (v / v) FBS, 1% (v / v) penicillin and streptomycin and the following components according to the final concentration in DMEM (Gibco): L-ascorbic acid 200 μmol / L, D - Ascorbic acid 50 μmol / L, IGF-1 50 ng / mL.

Embodiment 2

[0045] Example 2 Application of media with different formulations to expand porcine muscle stem cells

[0046] The culture medium formula designed in Example 1 is used to amplify porcine muscle stem cells, and the specific steps are as follows:

[0047] Day 0: Isolate porcine muscle stem cells, digest muscle tissue with 1mg / mL collagenase XI, obtain single cells, and use flow sorting system to separate CD31-CD45-CD56+CD29+ cell populations in 4×10 4 / mL density was inoculated in cell culture plates, the experimental group was added with formula A medium, and the control group was added with formula B medium, placed at 37 ° C, 5% CO 2 cultured in an incubator.

[0048] On the third day, the cells were taken out from the incubator, the culture supernatant was sucked, the experimental group was added with fresh formula A culture medium, and the control group was added with fresh formula B culture medium, and placed in the incubator again for static culture.

[0049] Day 6: Use ...

Embodiment 3E

[0056] Embodiment 3EdU experiment detects the proliferative ability of muscle stem cells

[0057] EdU is a thymine nucleoside analogue, and its attached alkyne group is rare in natural compounds, which can replace thymine (T) and infiltrate into the DNA molecule being synthesized during the DNA replication period. Click chemistry-CuAAC (copper-catalyzed azide-alkyne cycloaddition reaction) was employed in the reaction, which allows direct measurement of DNA synthesis in the S phase of the cell cycle. The EdU Flow Cytometry Assay Kits (Cy5) kit from APE BIO Company was used for detection. Cy5 azide was connected with EdU to fluorescently label the DNA of proliferating cells, which could be detected by flow cytometry (the maximum excitation wavelength of Cy5 azide is 646nm, the maximum The emission wavelength is 662nm).

[0058] Take 1~3×10 5 Muscle stem cells were inoculated in a 6-well plate, the experimental group was added the formula A medium designed in Example 1, and th...

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Abstract

The invention discloses a method for promoting proliferation of muscle stem cells, and belongs to the technical field of cell culture and biology. The invention designs a culture medium for effectively amplifying muscle stem cells in vitro. The culture medium comprises L-ascorbic acid, D-ascorbic acid and / or insulin-like growth factor-I, After the muscle stem cells cultured by the optimized culture medium are cultured for 21 days, the total cell number is increased by more than 10<6> times, and the proportion of Pax7 <+> cells still exceeds 60%; the amplified muscle stem cells can form a largenumber of typical muscle tubes through induced differentiation, and express muscle fiber specific proteins MyHC and Actin. and effective amplification of the muscle stem cells is achieved, and the method has important significance in large-scale culture of the muscle stem cells for preparing stem cell grafts and producing cell culture meat.

Description

technical field [0001] The invention relates to a method for promoting the proliferation of muscle stem cells, which belongs to the field of animal cell culture and biotechnology. Background technique [0002] Muscle stem cells, also known as muscle satellite cells, are a type of mononuclear stem cells located between the basement membrane of muscle tissue and the muscle cell membrane. It is the most critical progenitor cell in the process of muscle tissue growth and injury repair. When the muscle is damaged, the satellite cells in the resting state will be activated, begin to proliferate rapidly, and can start the differentiation program, and finally differentiate and fuse to form New myotube cells allow for rapid repair and regeneration of skeletal muscle. In addition, satellite cells can also differentiate into osteoblasts and adipocytes, etc., which can provide nutrition and stable support for new muscle, increase the fat content of new muscle, and improve the repairing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A23L13/00A23L13/50
CPCC12N5/0659A23L13/00A23L13/50C12N2500/38C12N2501/105C12N2511/00
Inventor 周景文关欣方佳华雷庆子陈坚堵国成余世琴
Owner JIANGNAN UNIV
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