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Method for efficiently separating and purifying porcine skeletal muscle satellite cells

A satellite cell and skeletal muscle technology, which is applied in the field of high-efficiency separation and purification of porcine skeletal muscle satellite cells, can solve the problems of low cell purity, small number of cells, and low purity of satellite cells, achieving high cell purity, easy operation, The effect of a large number of cells

Pending Publication Date: 2020-12-01
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] According to the separation and culture method reported, such as the pig skeletal muscle single muscle fiber transplantation method "porcine skeletal muscle satellite cell in vitro culture method", the purity of satellite cells obtained is low; according to the publicly reported skeletal muscle satellite cell separation method "an animal muscle satellite cell Cell Separation and Culture Technology "Separation method of skeletal muscle cells

Method used

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  • Method for efficiently separating and purifying porcine skeletal muscle satellite cells
  • Method for efficiently separating and purifying porcine skeletal muscle satellite cells
  • Method for efficiently separating and purifying porcine skeletal muscle satellite cells

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Embodiment 1

[0027] A method for culturing pig skeletal muscle satellite cells in vitro, comprising the following steps:

[0028] (1) Preparation of experimental reagents and instruments

[0029] Now prepare 0.1% type II collagenase solution: dissolve 20mg collagenase in 20ml PBS without calcium and magnesium ions, use after filtering with a 0.22um filter, store at -20°C, dissolve at 37°C;

[0030] Complete culture medium: DMEM high sugar + 20% FBS; 2 tubes of 50ml centrifuge tubes containing 2% double-antibody PBS; appropriate amount of 0.25% trypsin; 100-mesh 300-mesh cell screen, ophthalmic scissors, tweezers autoclaved; petri dish.

[0031] (2) Collect samples

[0032] 1-day-old healthy large white pigs were bled to death; after the epidermis was sterilized, the longissimus dorsi muscle tissue between the third and sixth ribs of the pig was taken out with sterile surgical instruments, and the time was controlled within 5 minutes. The removed muscle tissue was wiped with an alcohol co...

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Abstract

The invention discloses a porcine skeletal muscle satellite cell in-vitro culture method which comprises the following steps: digesting porcine skeletal muscle tissue blocks by using a type II collagenase solution and trypsin in sequence, performing sieving, and carrying out solid-liquid separation to obtain a precipitate; and preparing a cell suspension from the precipitate, carrying out differential adherent purification culture until the cell density reaches 70% or above, performing digesting with pancreatin, terminating digestion with a complete culture medium, performing centrifugation, discarding a supernatant, adding a culture medium, performing re-suspending, and carrying out subculture. For identifying the skeletal muscle satellite cells, PAX7 and MyoD gene immunofluorescence staining identification is performed on the skeletal muscle satellite cells subjected to subculture, positive expression is shown, and it is indicated that the separated cells are the skeletal muscle satellite cells if the cells are completely overlapped with cell nucleuses subjected to DAPI staining. The pig skeletal muscle satellite cell in-vitro culture method is easy and convenient to operate, thenumber of obtained cells is large, the cell purity is high, and a material is provided for growth and development research of pig skeletal muscles.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to a method for efficiently separating, purifying and culturing pig skeletal muscle satellite cells. Background technique [0002] Pork is the main meat consumed in daily life. The meat production rate of pigs is closely related to the growth and development of skeletal muscle. The research on the myogenic differentiation of skeletal muscle satellite cells requires high-purity skeletal muscle satellite cell lines. [0003] According to the separation and culture method reported, such as the pig skeletal muscle single muscle fiber transplantation method "porcine skeletal muscle satellite cell in vitro culture method", the purity of satellite cells obtained is low; according to the publicly reported skeletal muscle satellite cell separation method "an animal muscle satellite cell Cell Separation and Culture Technology "Separation method of skeletal muscle cells, the number of skeletal muscl...

Claims

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Application Information

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IPC IPC(8): C12N5/077
CPCC12N5/0659C12N2509/00
Inventor 胡悦旻鞠辉明
Owner YANGZHOU UNIV
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