Purification method of livestock and poultry animal muscle stem cells

A purification method and stem cell technology, applied in animal cells, vertebrate cells, non-embryonic pluripotent stem cells, etc., can solve the problems of difficulty in guaranteeing the purity of muscle stem cells, high flow sorting requirements, and high sorting costs, and achieve maintenance. Cell differentiation potential, excellent purification effect, and the effect of ensuring purification effect

Pending Publication Date: 2022-07-22
NANJING JOES FUTURE FOOD TECH CO LTD
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  • Summary
  • 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
However, although the positive rate of PAX7 in the flow sorting method can reach 92%, the flow sorting has high requirements on experimental equipment, experimental technology and other links, and the cost of each sorting is relatively expensive. Therefore, the development and use of low-cost, convenient and efficient Purification method of porcine muscle stem cells is very important

Method used

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  • Purification method of livestock and poultry animal muscle stem cells
  • Purification method of livestock and poultry animal muscle stem cells
  • Purification method of livestock and poultry animal muscle stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Isolation of pig cell samples to be purified:

[0043] Under sterile conditions, muscles such as biceps femoris and rectus dorsi of young pigs within one week of birth were removed and placed in 70% (volume percent) ethanol solution for 1-2 minutes; the obtained porcine muscle tissue was stored in a 3% (volume percent) in the penicillin-streptomycin double-antibody culture medium; proceed to the next step within 24h.

[0044] Stir the pieces of meat to 0.5-1.5mm with a meat grinder under aseptic conditions 3 Fragments, placed in DMEM / F12 medium containing penicillin-streptomycin double antibody, the mass fraction of penicillin-streptomycin is 3% (volume percentage), wherein, in the penicillin-streptomycin double antibody solution, the penicillin The content is 10000U / ml, and the content of streptomycin is 10mg / ml. Add the crushed muscle to the collagenase and neutral protease solution, the ratio of the collagenase and neutral protease is 1:1-2:1 (mass ratio)...

Embodiment 2

[0047] Example 2 Separation of chicken cell samples to be purified:

[0048] Take 18-20d fertilized eggs under sterile conditions, break the tip of the egg, cut the egg membrane to take out the chicken embryo, cut the chicken breast, leg muscles, etc., and place it in 70% (volume percent) ethanol solution for 1-2min ; Preserve the obtained chicken muscle tissue in a penicillin-streptomycin double-antibody culture solution containing 3% (volume percentage); carry out the next step (within 24h). figure 1 A).

[0049] Cut the meat pieces to 0.5-1.5mm with scissors under sterile conditions 3 Fragments, placed in DMEM / F12 medium containing penicillin-streptomycin double antibody, the mass fraction of penicillin-streptomycin is 3% (volume percentage), wherein, in the penicillin-streptomycin double antibody solution, the penicillin The content is 10000U / ml, and the content of streptomycin is 10mg / ml. Add the crushed muscle to the collagenase and neutral protease solution, the rati...

Embodiment 3

[0052] Example 3 Muscle tissue primary cell purification

[0053] S1: Inoculate the pig cell samples to be purified and the chicken cell samples to be purified obtained from Example 1 and Example 2 into a 10 cm rat tail collagen culture dish, add 8 mL of growth medium, and adhere overnight for 20 hours;

[0054] S2: Add 8mL PBS to wash the adherent cells in the petri dish, wash twice, remove the PBS in the petri dish, add 4mL 4 ℃ refrigerator pre-cooled basal medium DMEM / F12 medium to the petri dish and place it at 0 ℃ In the middle ice bath, the ice bath time of the chicken cell sample to be purified is 20 min, and the exfoliated cells are collected at the 20 min of the ice bath; the ice bath time of the pig cell sample to be purified is 45 min, and the exfoliated cells are collected every 15 min during the ice bath. The collected two groups of exfoliated cell systems were centrifuged at 330g for 5 min, respectively, and the exfoliated cells were separated to obtain purified ...

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Abstract

The invention provides a livestock and poultry animal muscle stem cell purification method which comprises the following steps: S1, inoculating a to-be-purified cell sample into a culture dish, adding a growth culture medium, and culturing until the cell sample adheres to the wall; the cell sample to be purified comprises muscle stem cells; s2, cleaning the adherent cells obtained in the step S1 with PBS, removing the PBS, adding a basic culture medium, carrying out ice bath, and collecting the fallen adherent cells in batches, namely purified muscle stem cells; the purification method provided by the invention is rapid, simple and convenient, greatly simplifies operation steps, effectively reduces the proportion of non-muscle stem cells, improves the purification efficiency, obtains an excellent purification effect, and provides an effective means for industrial expanded production of muscle stem cell related products.

Description

technical field [0001] The invention belongs to the technical field of stem cells and animal cell cultured meat, in particular to a method for purifying muscle stem cells of livestock and poultry. Background technique [0002] Meat food is the main source of human protein intake, and animal husbandry is the main way of meat production, but animal husbandry production will cause serious environmental problems. Meat consumption is projected to grow by more than 50% in 2050 as the global population grows and meat consumption per capita increases. The search for new greener and more efficient ways of producing meat has become a common global goal. Cultured meat technology is a very potential meat production technology. Studies have shown that this technology can reduce energy consumption by 7-45%, reduce greenhouse gas emissions by 78-96%, reduce land use by 99%, and reduce water consumption by 82-96% compared to traditional breeding. At present, cultured meat has ushered in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077C12N5/074
CPCC12N5/0658C12N5/0662C12N2509/00C12N2509/10C12N2501/115
Inventor 周光宏蒋翊宸丁世杰朱浩哲孙志远吴中元唐长波
Owner NANJING JOES FUTURE FOOD TECH CO LTD
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