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

A serum-free medium, stem cell technology, applied in cell culture medium, cell culture active agent, bone/connective tissue cells, etc., can solve the problems of difficulty in purifying skeletal muscle stem cells and low expansion efficiency, and improve the expansion efficiency. and the effect of high purity, high amplification efficiency and high purity

Active Publication Date: 2017-03-15
北京欣博睿丰医疗科技发展有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Preparation of serum-free medium for human skeletal muscle stem cells

[0038] Cell basal medium (DMEM: F12 (1:1) dry powder, product number: SH30004, purchased from Thermo Company), cytokines (purchased from PEPROTECH Company), metrophin (purchased from Sigma Company, the purity is more than 98%), Human Recombinant Insulin (purchased from Sigma, above 98% purity), Chondroitin 6-sulfate (purchased from Hainan Culuwa Pharmaceutical Co., Ltd.), Vitamin C (purchased from Sigma, above 98% purity), serum replacement (trade name "Ultroser G", brand Lonza; also available from Crescent Chemical Co., Inc.), NaHCO 3 (purity of 98% or more purchased from Sigma) and amino acids (purity of 98% or more purchased from Sigma), transferrin (purity of 98% or more purchased from Sigma), L-glutamine (purity of 98% or more purchased from Sigma) from Sigma company, the purity is more than 98%), vitamin B6 (purchased from Sigma company, the purity is more than 98%), thiamine hydroc...

experiment example 1

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

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

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

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

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

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 pH 7.2-7.4; SABC reagent, DAB chromogenic reagent, DAB chromogenic reagent (purchased from Wuhan Boster Company) ).

[0069] 1. Digest P20 skeletal muscle stem cells and collect them with homemade medium at 2×10 5 The density of cells / well was added to a 1:10 dilution of polylysine-coated 6-well plate for culture. When the cells were more than 90% of the 6-well plate, it was used for immunohistochemical experiments.

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

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

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

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

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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 invention relates to a cell culture medium, in particular to a serum-free culture medium for human skeletal muscle stem cells. The invention further relates to a preparation method of a serum-free culture medium for human skeletal muscle stem cells and its application in culturing human skeletal muscle stem cells. 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. After that, skeletal muscle stem cells are activated, proliferate 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 of muscle atrophy, heart disease, diabetes, tum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0037C12N5/0658C12N2500/24C12N2500/25C12N2500/32C12N2500/35C12N2500/36C12N2500/38C12N2500/46C12N2500/92C12N2501/105C12N2501/11C12N2501/115C12N2501/90C12N2501/999
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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