A kind of culture method of mammalian embryonic cell

A technology for mammals and culture methods, applied to animal cells, embryonic cells, vertebrate cells, etc., can solve the problems of uterine fetal fluid interference, differences in culture medium components, unfavorable cost control, etc., and achieve high cleavage rate and high birth rate Effect

Active Publication Date: 2021-03-30
SICHUAN AGRI UNIV
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Problems solved by technology

However, this method relies on the extraction of uterine fetal fluid during pregnancy, which is unfavorable in terms of cost control, and the uterine fetal fluid is interfered by various factors during the extraction process, so it is difficult to ensure the stability of the obtained uterine fetal fluid quality
[0006] However, from the current research, the scope of application of the existing embryo culture medium and its culture method is extremely limited.
By studying the culture method in CN 103333855 A, its effect on the culture of embryonic cells of other mammals (such as dogs and cattle) is relatively poor
The possible reason is that although the addition of uterine fetal fluid during pregnancy improves the culture effect to a certain extent, other substances in the culture medium are the key to the final culture effect, and for different embryonic cells, the required Differences in culture medium composition can be substantial

Method used

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  • A kind of culture method of mammalian embryonic cell
  • A kind of culture method of mammalian embryonic cell

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] For culture of mammalian embryonic cells:

[0027] When culturing, the fetal fluid of the pregnant uterus of the mammal itself is not added, and the culture is divided into two stages, the first stage is:

[0028] When the method is cultivated, the fetal fluid of the pregnant uterus of the mammal itself is not added, and the cultivation is divided into two stages, the first stage is:

[0029] Mammalian embryonic cells are placed in the first culture medium and cultivated for 6 hours; the formula of the first culture medium, in parts by weight, is:

[0030] 0.4 parts of glucose, 3.3 parts of sodium lactate, 0.10 parts of potassium dihydrogen phosphate, 2.2 parts of sodium bicarbonate, 3.3 parts of hyaluronic acid glycosaminoglycan, 0.8 parts of calcium citrate, 0.1 parts of IGFⅡ, 0.9 parts of taurine, chlorine dihydrate Calcium 0.3 parts;

[0031] When preparing the first culture medium, in addition to the above-mentioned components, water is added to make up to 100 pa...

Embodiment 2

[0038] For culture of mammalian embryonic cells:

[0039] When culturing, the fetal fluid of the pregnant uterus of the mammal itself is not added, and the culture is divided into two stages, the first stage is:

[0040] Mammalian embryonic cells are placed in the first culture medium and cultivated for 5 hours; the formula of the first culture medium, in parts by weight, is:

[0041] 0.6 parts of glucose, 3.0 parts of sodium lactate, 0.12 parts of potassium dihydrogen phosphate, 1.9 parts of sodium bicarbonate, 3.2 parts of hyaluronic acid glycosaminoglycan, 0.9 parts of calcium citrate, 0.2 parts of IGFⅡ, 0.3 parts of taurine, chlorine dihydrate Calcium 0.2 parts;

[0042] When preparing the first culture medium, in addition to the above-mentioned components, water is added to make up to 100 parts by weight;

[0043] The second stage is:

[0044] After the end of the first stage, remove the first culture medium, change to the second culture medium and cultivate for 3.0 ho...

Embodiment 3

[0049] For culture of mammalian embryonic cells:

[0050] When culturing, the fetal fluid of the pregnant uterus of the mammal itself is not added, and the culture is divided into two stages, the first stage is:

[0051] Mammalian embryonic cells are placed in the first culture medium and cultivated for 5.5 hours; the formula of the first culture medium, in parts by weight, is:

[0052] 0.5 parts of glucose, 3.2 parts of sodium lactate, 0.11 parts of potassium dihydrogen phosphate, 2.0 parts of sodium bicarbonate, 3.3 parts of hyaluronic acid glycosaminoglycan, 0.8 parts of calcium citrate, 0.2 parts of IGFⅡ, 0.6 parts of taurine, chlorine dihydrate Calcium 0.3 parts;

[0053] When preparing the first culture medium, in addition to the above-mentioned components, water is added to make up to 100 parts by weight;

[0054] The second stage is:

[0055]After the end of the first stage, remove the first culture medium and transfer to the second culture medium to cultivate for 3...

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Abstract

The invention provides a culture method of mammal embryonic cells. During culture, uterine fetal fluid of mammals during pregnancy is not added; the culture is divided into two stages, and during the two stages, culture times and medium formulas are different. The culture method provided by the invention is suitable for culture of the embryonic cells of the multiple mammals and has higher cleavage rate, fusion rate and blastocyst rate. After an embryo obtained by the culture method provided by the invention is directly transplanted, the birth rate is high.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for culturing mammalian embryonic cells. Background technique [0002] With the development of embryo engineering technology, the study of embryo culture medium has become increasingly important. At present, in the culture of animal embryos, the existing synthetic culture medium (such as TCM199 or mTCM199) contains a high concentration of glucose, which has certain toxicity to early embryos. Therefore, many researchers improved the culture medium by adding serum, amino acids, and growth factors. [0003] In terms of mouse embryo culture, KSOM culture medium is more mature, which has obtained relatively high blastocyst formation rate and blastomere number. [0004] However, it has been found that a single culture solution cannot achieve very satisfactory results. Therefore, it has become a research hotspot to cultivate in vitro cultured embryos accordi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/073
CPCC12N5/0603C12N2500/12C12N2500/14C12N2500/30C12N2500/32C12N2500/34C12N2501/11C12N2501/33
Inventor 罗擎英刘蜀坤黎杉珊刘耀文张志清申光辉吴贺君陈安均刘兴艳李美良苏赵
Owner SICHUAN AGRI UNIV
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