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Isolated culture method of muscle satellite cells

A satellite cell, separation and culture technology, applied in the field of muscle satellite cell separation and culture, can solve the problems of inconsistent understanding of muscle stem cells, immature muscle satellite cell separation and culture, etc. wall effect

Inactive Publication Date: 2021-04-06
BEIJING YULONG SHENGSHI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far people's understanding of muscle stem cells is still not unified
[0005] However, the isolation and culture of muscle satellite cells is still immature

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] In order to achieve the above object, the technical scheme adopted in the present invention is:

[0064] A method for isolating and culturing muscle satellite cells in vitro, using a muscle tissue digestion method for isolating and culturing, specifically comprising the following steps:

[0065] (1) Cut off the skin of the legs under aseptic conditions (mammals are used as the test object, such as dogs), and take about 1 cm of the leg muscles 3 size, immersed in 20ml PBS buffer.

[0066] (2) Remove fat, bones and tendons, and wash the muscle tissue repeatedly 8 to 10 times with PBS buffer.

[0067] (3) Cut the muscle tissue into 1mm with sterile surgical scissors 3 Small piece, awaiting further processing.

[0068] (4) Digest the shredded muscle tissue with 1% collagenase containing 2.5mmol / L calcium chloride, at 37°C for 1 hour, and blow once every 15 minutes to separate the cells.

[0069] (5) Then add an equal volume of DMEM / F12 medium containing 10% FBS. After ...

Embodiment 2

[0074] A kind of muscle satellite cell isolated and cultured in vitro, similar to Example 1, the difference is that in step (4), the shredded muscle tissue is digested with 0.8% collagenase containing 2.0mmol / L calcium chloride, at 35°C, 90min, pipetting once every 15min to separate the cells.

Embodiment 3

[0076] A kind of muscle satellite cell isolated and cultured in vitro, similar to Example 1, the difference is that in step (4), the shredded muscle tissue is digested with 1.2% collagenase containing 3.0mmol / L calcium chloride, at 38°C, 45min, pipetting once every 15min to separate the cells.

[0077] Cell Viability Detection

[0078] The cell viability was determined by the traditional trypan blue method in the laboratory. Use the addition of complete medium in step (8) to terminate the digested cell solution, centrifuge and resuspend, draw a small amount of cell suspension and trypan blue to mix at 9:1, and use a cell counting plate to calculate the staining and concentration within 3 minutes. Number of unstained cells.

[0079] Cell viability = number of unstained cells / total number of cells × 100%

[0080] The experimental results were averaged four times successively. According to the formula, the average values ​​of Examples 1-3 were 93.08%, 88.17%, and 89.25%, and t...

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Abstract

The invention discloses an isolated culture method of muscle satellite cells, which comprises the following steps of cutting off leg skin under aseptic conditions, taking leg muscles, and immersing in a buffer solution, removing fat, bones and tendons, and repeatedly cleaning muscle tissues with a buffer solution, shearing muscle tissues into small blocks under a sterile condition, and digesting with collagenase to separate cells, adding an isopyknic culture medium, blowing, beating, filtering, collecting filtrate, centrifuging, discarding supernate, and resuspending cells by using the culture medium, adding the cell suspension into a culture dish, and culturing in a cell incubator, carrying out cell purification culture by a differential attachment method, and when the cell confluence degree reaches 80%, using trypsin for digestion subculture, and when the cell confluence degree reaches 90% or above, carrying out cell cryopreservation. The chicken muscle satellite cells cultured by the isolated culture method disclosed by the invention are higher in activity, more in quantity, easier to adhere to the wall, stronger in passage and higher in differentiation rate, and the culture method is simple, convenient to operate, time-saving and labor-saving.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating and culturing muscle satellite cells. Background technique [0002] Muscle stem cells are present in embryos and adults, and muscle satellite cells are abundant in muscle. With the development of tissue engineering technology, the use of muscle tissue engineering technology to repair dynamic paralysis by surgical methods has created a new research prospect with its unique advantages. In particular, muscle stem cells have attracted worldwide attention because they are directly involved in the differentiation of skeletal muscle. [0003] Although the existence of muscle satellite cells has been discovered for more than 50 years and some attempts have been made in vitro, the methods for the isolation and identification of muscle satellite cells are still uncertain. [0004] Muscle satellite cells are small mononuclear spindle-shaped cells that are stem cells de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0659C12N2509/00
Inventor 张晓南吴芳春谷涌泉侍晓云张斌
Owner BEIJING YULONG SHENGSHI BIOTECH CO LTD
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