Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells

A muscle satellite cell and differentiation-inducing technology, which is applied in the field of in vitro isolation and differentiation of cells, can solve the problems of no contractile function, poor passage stability, and small number of myotubes, so as to save culture costs and improve differentiation For the effect of strong myotube ability and proliferative activity

Inactive Publication Date: 2010-11-17
NORTHEAST AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0005] The purpose of the present invention is to solve the problems of high cost of separation and culture of existing bovine muscle satellite cells, poor passage stability, and the existence of a sm...

Method used

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  • Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells
  • Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells
  • Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells

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specific Embodiment approach 1

[0012] Specific embodiment one: the in vitro separation and culture method of bovine muscle satellite cells of the present embodiment is realized by the following steps: one, clean bovine muscle tissue with D-Hanks liquid and cut into 1mm 3 The small pieces were placed in D-Hanks solution containing 0.25g trypsin, digested at 37°C for 30min, then centrifuged to discard the supernatant, then added collagenase solution, digested at 37°C for 2-3h to obtain cells Suspension; 2. Use a 400-mesh sieve to filter the cell suspension, then centrifuge, and resuspend the resulting cell pellet with D-Hanks solution and centrifuge, then resuspend the cells with cell growth medium, and then inoculate in Polymeric Lyme In the cell culture flask coated with amino acid, the cells ppl to pp6 were cultivated by differential attachment method; 3. The cells in pp6 were cultivated to a confluence rate of 95%, and then passaged with trypsin with a mass concentration of 0.25%. Promptly complete the in...

specific Embodiment approach 2

[0026] Embodiment 2: This embodiment differs from Embodiment 1 in that the centrifugation in steps 1 and 2 is performed at a speed of 1000 r / min for 5 minutes. Other steps and parameters are the same as those in Embodiment 1.

specific Embodiment approach 3

[0027] Embodiment 3: The difference between this embodiment and Embodiment 1 is that in step 1, collagenase solution is added and digested at 37° C. for 2 hours to obtain a cell suspension. Other steps and parameters are the same as those in Embodiment 1.

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Abstract

Methods for in vitro isolation and culture and induced differentiation of bovine muscle satellite cells relate to the methods for in vitro isolation and culture and induced differentiation of cells. The invention solves the following problems: the cost is high and the heredity stability is not good in the existing method for isolation and culture of bovine muscle satellite cells, and the obtained myotubes are fewer and do not have contracting function in the existing method for induced differentiation of bovine muscle satellite cells. The method for in vitro isolation and culture of bovine muscle satellite cells is characterized by cleaning the tissues and digesting the tissues with trypsin and collagenase solution; carrying out centrifuging after filtering, re-suspending the cells and culturing the cells by a differential velocity adherent method after inoculation; and culturing the cells until the merging rate is 95% and carrying out heredity with pancreatin. The method for induced differentiation of bovine muscle satellite cells is characterized by obtaining PEI-plasmid complexes; culturing the bovine muscle satellite cells until the merging rate is 75%, adding high-glucose DMEM culture solution after cleaning, adding the PEI-plasmid complexes to the surfaces of the cells, changing culture solution A for culturing and then changing culture solution B for induced differentiation. In the method, the cost for isolation and culture of the bovine muscle satellite cells is low, the heredity stability is good and the myotubes obtained through induced differentiation are large in quantity and have contracting function.

Description

technical field [0001] The invention relates to a method for separating and culturing cells in vitro and inducing differentiation. Background technique [0002] Myoblasts in skeletal muscle exist in the form of muscle satellite cells (satellite cells, SC). It is called "muscle stem cell", which is a kind of precursor cell directed to the myoblast line; the muscle regeneration process is mainly completed by muscle satellite cells; muscle satellite cells are located between the sarcolemma and basement membrane of muscle fibers , generally small spindle-shaped flat mononuclear cells; when muscle tissue is stimulated, such as when skeletal muscle is damaged, satellite cells will be activated, a large number of divisions, proliferation and mutual fusion to form regenerative muscle fibers; activated satellite cells and damaged Cells fuse to repair damaged cells, or several myoblasts fuse to form myotubes, which in turn generate new muscle cells. Activation, proliferation, and di...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/10
Inventor 严云勤佟慧丽李树峰兴孝友高学军陆黎敏李光鹏金慧然
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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