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Skeletal muscle stem cell serum-free medium and its preparation method and application

A serum-free medium and stem cell technology, applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problems of difficult purification and low expansion efficiency of skeletal muscle stem cells, and improve the expansion efficiency and purity. , the effect of high amplification efficiency and avoiding the risk of disease

Active Publication Date: 2019-10-11
北京欣博睿丰医疗科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The technical problem to be solved by the present invention is to provide an optimized serum-free medium for skeletal muscle stem cells, aiming at the problems of adding fetal bovine serum to the skeletal stem cell medium, which contains animal-derived components, resulting in difficult purification of skeletal muscle stem cells and low amplification efficiency. , used for primary culture and large-scale expansion of human skeletal muscle stem cells, providing high-quality skeletal muscle stem cells for clinical research and application

Method used

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  • Skeletal muscle stem cell serum-free medium and its preparation method and application
  • Skeletal muscle stem cell serum-free medium and its preparation method and application
  • Skeletal muscle stem cell serum-free medium and its preparation method and application

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Embodiment 1

[0037] The preparation of embodiment 1 human skeletal muscle stem cell serum-free medium

[0038] Cell basal medium (DMEM: F12 (1:1) dry powder, product number: SH30004, purchased from Thermo Company), cytokines (purchased from PEPROTECH Company), and booster (purchased from Sigma Company, with a purity of more than 98%), Human recombinant insulin (purchased from Sigma, with a purity of more than 98%), chondroitin 6-sulfate (purchased from Hainan Huluwa Pharmaceutical Co., Ltd.), vitamin C (purchased from Sigma, with a purity of 98%), serum replacement (trade name "Ultroser G" under the brand name Lonza; also available from Crescent Chemical Co., Inc.), NaHCO 3 (purchased from Sigma Company, the purity is more than 98%) and amino acid (purchased from Sigma Company, the purity is more than 98%), transferrin (purchased from Sigma Company, the purity is more than 98%), L-glutamine (purchased from Sigma Company, the purity is more than 98%), In Sigma Company, purity is more than ...

experiment example 1

[0047] Experimental example 1 Comparison of growth curves of skeletal muscle stem cells in three media

[0048] The three kinds of medium used in this experiment are the serum-free medium (hereinafter referred to as self-made medium) prepared in Example 1 of the present invention respectively, the DMEM of 20% fetal bovine serum: F12 1: 1 medium (hereinafter referred to as medium A) , SKGM-2, a special low-serum medium for skeletal muscle stem cells (hereinafter referred to as medium B), purchased from Lonza Company.

[0049] 1. Isolation and purification of skeletal muscle stem cells

[0050] (1) Surgical biopsy Aseptically take 2-5g of rectus femoris or deltoid muscle voluntarily donated by a young healthy person, transfer to a 50ml centrifuge tube containing 20ml of PBS containing 1% penicillin and streptomycin, and transport to the laboratory at 4°C.

[0051] (2) Take out the muscle tissue from the 50ml centrifuge tube, put it into a sterile plate, remove fat and connectiv...

experiment example 2

[0067] Experimental example 2 Purity identification and differentiation ability evaluation of skeletal muscle stem cells

[0068] Desmin antibody, immunohistochemical kits and auxiliary reagents were purchased from Wuhan Boster Company; PBS was phosphate buffered saline with a pH of 7.2-7.4; SABC reagent, DAB chromogenic reagent, DAB chromogenic reagent (purchased from Wuhan Boster Company ).

[0069] 1. Digest the P20 skeletal muscle stem cells, collect and use self-made medium to press 2×10 5 The density of cells / well was added to 1:10 diluted poly-lysine-coated 6-well plate for culture, and when the cells covered more than 90% of the 6-well plate, it was used for immunohistochemical experiments.

[0070] 2. Wash the cells with PBS solution 3 times, 2min each time.

[0071] 3. Add 1ml / well of 4% paraformaldehyde, fix at room temperature for 30min.

[0072] 4. Wash the cells with PBS solution 3 times, 2min each time.

[0073] 5. Add 0.5% triton x-100 1ml / well for 10min to...

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Abstract

The invention discloses a skeletal muscle stem cell serum-free medium and a preparation method and application thereof. The skeletal muscle stem cell serum-free medium, with the pH value ranging from 7.4 to 7.4, comprises DMEM(dulbecco's modified eagle medium) / F12, serum replacement, cytokines, vitamins, amino acid, recombinant human insulin, mepiquat chloride and 6-chondroitin sulfate. The invention further discloses the preparation method of the skeletal muscle stem cell serum-free medium. Specifically, the serum replacement refers to animal-free component which is defined, thus, serum caused pathogenic risk and instability of different batches and the like are avoided. The skeletal muscle stem cell serum-free medium is applicable to human skeletal muscle stem cell primary culture and scaled amplification culture and has the advantages of high purity, quick cell proliferation, high expansion efficiency, capability of realizing steady passage of at least 20 generations and the like. In addition, the skeletal muscle stem cell serum-free medium effectively improves skeletal muscle stem cell expansion efficiency and purity and can serve as an in-scale high-quality skeletal muscle stem cell culture system to meet requirements for clinical research and application.

Description

technical field [0001] The present invention relates to a cell culture medium, in particular to a serum-free medium for human skeletal muscle stem cells. The present invention further relates to a preparation method of a serum-free medium for human skeletal muscle stem cells and its application in culturing human skeletal muscle stem cells. The field of culture medium for skeletal muscle stem cells. Background technique [0002] Skeletal muscle stem cells (also known as myoblasts, myoblasts, English name: myoblast) can only be directed to differentiate into muscle tissue, and exist in skeletal muscle tissue in the form of skeletal muscle satellite cells, with natural cell fusion. Afterwards, skeletal muscle stem cells are activated, proliferate in large numbers and produce contractile proteins to repair and maintain skeletal muscle tissue. Skeletal muscle stem cell transplantation technology has been continuously reported at home and abroad in recent years in the treatment ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077
Inventor 刘谋元刘麟
Owner 北京欣博睿丰医疗科技发展有限公司
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