Separation and purification method of high-purity porcine muscle stem cells

A technology for separation and purification of stem cells, applied in the field of separation and purification of high-purity porcine muscle stem cells, can solve the problems of poor experiment repeatability and difficulty in ensuring the purity of muscle stem cells, and achieve the effect of less pollution, reduced proportion and reduced use.

Inactive Publication Date: 2019-12-31
NANJING AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods are not based on the molecular characteristics of porcine muscle stem cells, it is difficult to guarantee the purity of muscle stem cells, and the experiment repeatability is poor

Method used

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  • Separation and purification method of high-purity porcine muscle stem cells
  • Separation and purification method of high-purity porcine muscle stem cells
  • Separation and purification method of high-purity porcine muscle stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] The isolation method of mononuclear cell in the pig muscle of embodiment 1:

[0047] Get the muscles such as pig biceps femoris under aseptic conditions, place 1-2 minute in 70% (volume percentage) ethanol solution; In the anti-culture solution; proceed to the next step within 24 hours.

[0048] Cut into 0.5-1.5mm with scissors under sterile conditions 3 Fragments are placed in DMEM culture fluid containing penicillin-streptomycin double antibody, the mass fraction of penicillin-streptomycin is 3% (volume percentage), wherein, in the penicillin-streptomycin double antibody solution, the content of penicillin is 10000U / ml, the content of streptomycin is 10mg / ml. Minced muscle is added collagenase and neutral protease solution, and the ratio of described collagenase and neutral protease is 1:1-2:1 (mass ratio), and minced muscle and mixed enzyme liquid volume ratio are 0.05-0.5% (mass ratio). volume ratio), digested at 37°C for 30-90min. During the digestion process, ...

Embodiment 2

[0053] Example 2 Porcine muscle mononuclear cell flow cytometry sorting method and purity identification

[0054] The mononuclear cells of the resuscitated pig were cultured for 2-4 days in a collagen pretreated cell culture dish, and the medium was basal medium and supplements, and the basal medium was composed of 79% F-10, 20% fetal bovine serum, 1 % penicillin-streptomycin double antibody (both volume percent), the supplement includes fibroblast growth factor 2, and the concentration of the fibroblast growth factor 2 is 1-10 ng / ml. After culturing, a large number of cells adhered to the wall. There is also a large amount of suspended cells and tissue debris. Washing 1-2 times with phosphate buffer can remove a large number of suspended cells and tissue debris and achieve the purpose of preliminary purification. Afterwards, trypsin was used for digestion, and the cell pellet was collected by centrifugation. The cells were resuspended in PBS containing 1.5% (mass volume pe...

Embodiment 3

[0058] Example 3 Induced Differentiation of Porcine Muscle Stem Cells

[0059] The muscle stem cells are cultured on matrigel-pretreated cell culture dishes for 3-5 days, the culture medium is basal medium and supplements, and the basal medium is 79% F-10, 20% fetal bovine serum, 1% penicillin - the culture medium of streptomycin double antibody (both volume percent), the supplement includes fibroblast growth factor 2, and the concentration of the fibroblast growth factor 2 is 1-10 ng / ml. When the cells reached 95% density, the differentiation experiment was carried out. It was washed once with PBS preheated at 37° C., and a differentiation medium was added, which included 97% DMEM medium, 2% horse serum, and 1% penicillin-streptomycin double antibody (both volume percentages). After culturing at 37°C for 3-4 days, observed under a microscope, myotubes with a large number of cell fusions were completely differentiated.

[0060] The obtained differentiated cells were washed 1...

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Abstract

The invention relates to a separation and purification method of high-purity porcine muscle stem cells, for research and production of cultured meat. The separation and purification method disclosed by the invention comprises the steps of obtaining sterile muscular tissue, efficiently digesting the muscular tissue to obtain a large quantity of muscle mononuclear cells, and finally performing steaming cell sorting to obtain the high-purity porcine muscle stem cells. The invention further discloses a method for efficiently digesting the porcine muscular tissue. By the method, the digestion speedand the digestion intensity can be increased, and a large quantity of muscle mononuclear cells can be obtained. When the purification method disclosed by the invention is applied, the porcine musclestem cells of which the purity exceeds 92% (by PAX7) can be stably obtained. The porcine muscle stem cells obtained through separation have efficient disintegration efficiency, and MYOSIN expression quantity can reach 85%-90%. When the method disclosed by the invention is applied, a large quantity of seed cell sources can be provided for research and actual production of the cultured meat.

Description

technical field [0001] The invention belongs to the technical field of stem cells and animal cell cultured meat, and in particular relates to a method for separating and purifying high-purity pig muscle stem cells for the research and production of cultured meat. Background technique [0002] Meat is one of the most important food components in human evolution. With the improvement of people's living standards, my country's meat consumption demand is increasing year by year. However, traditional animal husbandry consumes a lot of natural resources and easily causes environmental pollution. In recent years, problems such as bird flu and African swine fever have also sounded the alarm for the current animal husbandry industry. Cultured meat is a kind of meat production method in which different kinds of cells such as animal muscle stem cells and fat stem cells are cultivated in vitro, and differentiated and reorganized in vitro into animal muscle, fat and other tissues that ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/077
CPCC12N5/0659C12N5/0658C12N2509/00C12N2509/10
Inventor 周光宏丁世杰朱浩哲李春保徐幸莲
Owner NANJING AGRICULTURAL UNIVERSITY
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