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A high-efficiency method for culturing primary cells of sheep embryonic skeletal muscle

A primary cell and culture method technology, applied in the field of high-efficiency sheep embryo skeletal muscle primary cell culture, can solve the problems of slow culture process, slow tissue loss, susceptibility to fungi, etc., to speed up the cell culture process and improve cell The effect of purity

Active Publication Date: 2021-10-29
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The two methods have their own advantages and disadvantages: the tissue block method is easy to operate in the early stage, and the loss of tissue volume is the least during the experimental culture process, but the culture process is relatively slow and it is very susceptible to fungal infection
The single-cell suspension can maximize the contact area between the tissue and the culture dish, and increase the attachment efficiency, but the whole process is still relatively slow compared with the tissue particle method, and the loss of the tissue is large, which is not conducive to the full use of the sample

Method used

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  • A high-efficiency method for culturing primary cells of sheep embryonic skeletal muscle
  • A high-efficiency method for culturing primary cells of sheep embryonic skeletal muscle
  • A high-efficiency method for culturing primary cells of sheep embryonic skeletal muscle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] (1) Select the lake sheep palace and intrauterine 72 germical embryos, and 75% of the collected sheep are repeatedly sprayed, cut the uterus and remove the embryo to place the embryo, cut down the limbs of the fetus and soak 75 % Alcohol is about 10 minutes, cutting muscle tissue to 1 mm 3The irregular small blocks are separated from scratches, and immersed in PBS buffers containing dual-resistant PBS buffer, remove muscle membranes, fibers, etc., and then cut muscle tissue and rinse 4 to 5 times with ophthalmic shear.

[0038] (2) The collagenase powder is diluted with PBS buffer, adding a concentration of a type I collaborase, and the liquid surface is not tissue, placed in a cellular incubator for 1 h, and set the incubator condition to temperature 37 ° C, Volume fraction is 5% CO 2 And saturated humidity, rocking every 15 min once. After the digestion is completed, the collagenase mixture containing muscle tissue, 800 rpm is centrifuged for 10min, and the supernatant is...

Embodiment 2

[0047] (1) Select the lake's sheep palace and intrauterine 39 gestational age embryos, 75% of the collected sheep, 75% alcohol repeatedly spray, cut the uterus to remove the embryo in the ultra-clean station, cut the limbs of the fetus, and tissue muscle tissue Cut by about 1 mm 3 Irregular small pieces and soaked in 75% alcohol about 10 min, separated by forceps with tweezers, immersed in dual-resistant PBS buffer, remove muscle membrane, fiber, etc., then cut muscle tissue with eye shelter Rinse 4 ~ 5 times.

[0048] (2) The collagenase powder is diluted with PBS buffer, adding a concentration of a type I collaborase, and the liquid surface is not tissue, placed in a cellular incubator for 1 h, and set the incubator condition to temperature 37 ° C, Volume fraction is 5% CO 2 And saturated humidity, rocking every 15 min once. After the digestion is completed, the collagenase mixture containing muscle tissue, 800 rpm is centrifuged for 10min, and the supernatant is abandoned. Afte...

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Abstract

The invention belongs to the technical field of animal cell culture, in particular to a method for primary culture of sheep embryonic skeletal muscle cells. The method of the present invention comprises four steps of taking materials, digesting, filtering and cultivating. A 40-mesh cell sieve is used to filter and grind and then settle down. The lower tissue particles are collected and placed in a culture dish for cultivation. Between block and single cell suspension, it not only reduces the probability of fungal and bacterial contamination, but also greatly improves the purity and quantity of muscle cells. This method combines the advantages of tissue block method and single cell suspension, and can obtain skeletal muscle cells with high purity, high viability, high activity and extremely low risk of contamination.

Description

Technical field [0001] The present invention belongs to the field of animal cell culture, and more particularly to a highly efficient sheep embryonic skeleton myogenesis method. Background technique [0002] China is the production and consumption of meat sheep. In recent years, with the improvement of the living standards of the residents, the demand for lamb markets is strong. China's meat sheep has a low production efficiency, and the production capacity is extremely mismatched. Studying sheep skeletal muscle growth and development mechanisms can increase the efficiency of meat and sheep production, and can cultivate and build in vitro growth models of sheep skeletal muscle tradition cells, laying the foundation for sheep production. [0003] Senior cell culture refers to the first cultivation of in vitro after obtaining the tissue from the animal body. Senior cells still retain the biological and genetic properties of the original tissue, and also close to and reflect the gro...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/077C12N5/073
CPCC12N5/0603C12N5/0658C12N2509/00C12N2509/10
Inventor 张莉胡文萍刘璐璐尚明玉王欣悦冯勉熊金珂
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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