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Efficient and economical porcine muscle stem cell separation method and application thereof

A stem cell and muscle technology, applied in the biological field, can solve the problems of high cost of flow sorting instruments and follow-up maintenance costs, difficulty in popularization, and difficulty in industrialization, etc., achieves efficient and economical separation and extraction methods, less chance of contamination, and improves cell survival rate effect

Active Publication Date: 2022-07-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the cost of flow sorting instruments and follow-up maintenance is expensive, not all laboratories can be equipped
Muscle stem cell isolation methods that rely on flow cytometry often fall into a situation where promotion is difficult and industrialization is difficult

Method used

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  • Efficient and economical porcine muscle stem cell separation method and application thereof
  • Efficient and economical porcine muscle stem cell separation method and application thereof
  • Efficient and economical porcine muscle stem cell separation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1: Determination of pronase digestibility

[0043] All experiments were performed in a sterile environment (biosafety cabinet or ultra-clean bench), and the surgical instruments were all autoclaved.

[0044] (1) Obtain porcine muscle tissue

[0045] 3-day-old young pigs were selected, and CO was used 2 After being euthanized, the pigs were washed with sterile water and then soaked in 75% ethanol for 3 min. This was followed by two washes with sterile PBS buffer. Then, the pigs were aseptically dissected using autoclaved surgical instruments in a biological safety cabinet to remove the internal organs; then the back and leg muscles of the pigs were taken out with a scalpel and surgical scissors. The removed muscle tissue was washed twice with PBS buffer containing 1% penicillin-streptomycin-amphotericin B tertiary antibody (Antibiotic-Antimycotic, AA) and 1% gentamicin (GEN), and then stored in PBS containing 1%. %AA and 1% GEN in PBS buffer.

[0046](2) Cut...

Embodiment 2

[0064] Example 2 Determination of Final Enzyme Combination Digestion Mode

[0065] After the pronase was determined, the pronase was combined with trypsin, collagenase, and dispase into G4, G5, G6, and G7, respectively (see Table 1). Except for the G4 group, which required trypsin secondary digestion, the other three groups did not require secondary digestion. The remaining steps are the same as G1-G4. Specific steps are as follows:

[0066] (2) Cut muscle tissue

[0067] Put the preserved muscle tissue into a petri dish in a biological safety cabinet / clean bench, chop the muscle tissue with a scalpel, and remove fat, connective and other tissues during the process, and cut the muscle tissue in a short time as much as possible. Shred to 1mm 3 .

[0068] (3) Digestion to obtain a single cell population of muscle stem cells

[0069] First, 5 g of crushed muscle tissue was placed in 4 groups of 50 mL centrifuge tubes containing DMEM medium containing different digestive enz...

Embodiment 3

[0082] Example 3: Expression identification of Pax7 and MyoD in muscle stem cells of each group

[0083] For the cell suspension (G4-G7) obtained after differential purification in step (7) in Example 2, 5000 cells were taken and seeded in a 96-well plate, and Pax7 and MyoD were detected after the cells adhered for 48 hours. Another 10,000 cells were seeded in a 96-well plate, and the medium was complete medium, and the formula was as follows: DMEM medium containing 15% FBS, 10 ng / mL bFGF, 1% AA and 1% GEN.

[0084] 1) Fix with 50 μL of 4% paraformaldehyde (pre-cooling at 4°C), remove the paraformaldehyde after permeabilization at room temperature for 15 min, and carefully wash with PBS buffer 3 times;

[0085] 2) After adding 50 μL of 0.5% TritonX-100 for 15 min, remove the solution and carefully wash it three times with PBS buffer;

[0086] 3) Add blocking solution and incubate at room temperature for 30 min; remove the solution and carefully wash 3 times with PBS buffer; ...

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Abstract

The invention discloses an efficient and economical pig muscle stem cell separation method and application thereof, and belongs to the technical field of biology. According to the method, different enzymes for digesting muscle tissues are combined, the single cell releasing capacity of different combined enzymes is determined, the most efficient and economical enzyme combined digestion mode (the combination of streptavidin and dispersozyme) is selected, and finally a large number of muscle stem cells are obtained through a differential purification method. The invention further discloses a method for efficiently digesting the pig muscle tissue, and the muscle stem cells can be maximally added while the cost is reduced. By applying the enzyme combined digestion and separation method, the total single cell number of 4.6-6 * 10 < 6 > cells / g and the pig muscle stem cell number of 8-15 * 10 < 5 > cells / g (in a double positive proportion of CD29 and CD56) can be stably obtained. The muscle stem cells obtained through separation have high differentiation capacity, and an efficient and economical separation method is provided for research and actual production of cell culture meat seed cells.

Description

technical field [0001] The invention relates to an efficient and economical method for separating porcine muscle stem cells and application thereof, and belongs to the field of biotechnology. Background technique [0002] In recent years, with the rapid development of my country's social economy, there has been a serious imbalance between the supply and demand of meat and agricultural products. The traditional animal husbandry consumes a lot of resources, causes serious environmental pollution, and also has a series of problems such as animal welfare and animal diseases. Cell cultured meat is a subversive meat production technology that has emerged in recent years. It is a technology that uses in vitro culture to obtain meat based on the mechanism of animal muscle growth and repair. The production of cultured meat first requires obtaining seed cells such as muscle stem cells, embryonic stem cells, and mesenchymal stem cells. Among the most promising are muscle stem cells, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/94C12N9/52C12N9/50C12N5/0775
CPCC12N9/94C12N9/52C12N9/50C12N5/0662C12N2509/00C12N2509/10
Inventor 关欣堵国成李妹陈坚周景文
Owner JIANGNAN UNIV
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