CN114350597A - Culture medium for mouse embryo body epitaxy - Google Patents
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- CN114350597A CN114350597A CN202111664644.7A CN202111664644A CN114350597A CN 114350597 A CN114350597 A CN 114350597A CN 202111664644 A CN202111664644 A CN 202111664644A CN 114350597 A CN114350597 A CN 114350597A
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Abstract
The invention discloses a culture medium for mouse embryo body epitaxy, which contains a basic culture medium, fetal calf serum, human umbilical serum, sodium pyruvate, glutamine, penicillin-streptomycin, N2 additive and B27 additive, and can promote the in vitro mouse embryo continuous culture stage to E8.0 on the basis of improving the culture success rate. The method provides a basis for the development research of embryo before implantation and embryo in implantation period, the improvement of the culture success rate and the extension of the in vitro culture time can expand the selection range of relevant experimental contents and experimental period of mouse embryo, and also can provide a basis for the in vitro culture of other mammal embryos.
Description
Technical Field
The invention relates to the field of in-vitro culture of mammalian embryos, in particular to a culture medium for mouse embryo body epitaxy.
Background
Mouse embryos are one of the most commonly used mammalian embryo models, and become good embryo research models due to the advantages of mammalian embryo characteristics, short development time and the like.
The establishment of an embryo in-vitro continuous culture system is an important platform for clarifying the development mechanism of mammalian embryos, and the embryo development before implantation of a mouse has certain research results at present, but the research on the implantation period and the post implantation period is very limited. The main reasons are that the survival rate of the embryo in vitro is not high, the promotion of various factors in vivo is not realized, the culture time of the embryo in vitro is short, and the method for continuously culturing the mouse embryo body outside before implantation is not mature, and is continuously supplemented and perfected, so that the development of related mechanism research experiments is slow or impossible.
The factors influencing the success rate of in vitro culture of mouse embryo bodies are many, and the embryo sources and self conditions, and the components and the proportion of culture solutions such as growth factors, energy substances, serum and the like have great influence on the success rate. In order to research the mechanism related to the development of mouse embryo and provide reference for the in vitro culture of mammalian embryo, researchers have been exploring methods for prolonging the in vitro culture time of embryo.
The existing in vitro embryo culture technology can only culture the embryo of a mouse to reach E6.5, cannot meet the requirements of in vitro embryo experiments, has low success rate of only 36.7 percent, and possibly has influence on subsequent researches in actual experiments. Therefore, prolonging the survival time and survival rate of in vitro embryos is the most basic requirement for establishing animal embryo models.
Disclosure of Invention
The invention aims to provide a culture medium additive for mouse embryo body epitaxy, which can improve the survival rate of mouse embryos and prolong the in vitro culture time.
Another object of the present invention is to provide a culture medium for mouse embryo body epitaxy, which can promote the continuous culture stage of in vitro mouse embryo to E8.0 on the basis of improving the culture success rate.
The invention further aims to provide the application of the culture medium in the embryo related experiment during the mouse embryo body epitaxy.
The invention discloses a culture medium additive for culturing mouse embryo body in an epitaxial manner, which comprises fetal calf serum, human umbilical serum, sodium pyruvate, glutamine and penicillin-streptomycin.
Furthermore, the culture medium additive also comprises an N2 additive and a B27 additive.
The invention also discloses a culture medium for mouse embryo body epitaxy, which consists of a basic culture medium and the culture medium additive, and contains 10-20% of fetal calf serum, 10% of human umbilical serum, 1mM of sodium pyruvate, 2mM of glutamine and 100 units/mL of penicillin-streptomycin.
Further, it also contains 100 XN 2 additive agent with total volume of 1% and 50 XB 27 additive agent with total volume of 2%.
The basic culture medium is one or more of CMRL1066 culture medium, DMEM culture medium, 1640 culture medium, MEM culture medium, F10 culture medium, M199 culture medium and F12 culture medium.
The invention also discloses application of the culture medium as a culture medium for mouse embryo body epitaxy.
The application is to add the culture medium of claim 3 or 4 into a matrigel culture environment inoculated with mouse embryos, wherein the matrigel culture environment inoculated with the mouse embryos is inoculated in the matrigel-paved culture environment by a product of mixing the mouse blastocysts digested with zona pellucida and matrigel.
In order to protect the embryo and establish a 3D bionic culture system, the volume concentration of the matrix colloid is 30 percent.
The invention also discloses application of the culture medium as a culture medium in an embryo culture experiment.
When mouse embryos are cultured by using the culture medium, the culture medium used firstly contains 10 percent of fetal calf serum, 10 percent of human umbilical serum, 1 percent of 100 XN 2 additive, 2 percent of 50 XB 27 additive, 1mM sodium pyruvate, 2mM glutamine and 100 unit/mL penicillin-streptomycin. The same composition and ratio were used during the medium change.
After 48 hours of culture, the medium was changed to a medium containing 20% by volume fetal bovine serum, 10% by volume human umbilical serum, 1% by volume 100 XN 2 additive, 2% by volume 50 XB 27 additive, 1mM sodium pyruvate, 2mM glutamine, 100 units/mL penicillin-streptomycin.
The culture method of the invention is based on matrigel, the components and the proportion of the culture medium are adjusted, the blastocyst before implantation can be continuously cultured to E6.5 in vitro, the formation rate of the egg column reaches 81.8 percent and is far higher than the prior culture level; the embryo can be continuously cultured to the seventh day in an in vitro environment by the method, and the embryo can be developed to the stage of E8.0, so that the existing culture stage is broken through, and a foundation can be provided for the development research of the embryo before implantation and the embryo in the implantation period; the improvement of the culture success rate and the extension of the in vitro culture time can expand the selection range of relevant experimental contents and experimental periods of mouse embryos and also provide a basis for in vitro culture of other mammalian embryos.
Drawings
FIG. 1 is a morphological diagram of an embryo cultured to an egg column (E6.5).
FIG. 2 is a morphological diagram of in vitro continuous culture of pre-implantation blastocysts to E8.0, where A is the hatched blastocyst just beginning to be cultured, B is the expanded blastocyst 2 days after in vitro culture, C is the pre-columella embryo 3 days after in vitro culture, D is the columella-like embryo 4 days after in vitro culture, E is the E7.5-like pro-intestinal embryo 5 days after in vitro culture, and F is the E8.0-like neuro-embryo 7 days after in vitro culture.
Detailed Description
The following describes in detail a specific embodiment of the present invention with reference to the drawings, examples and comparative examples. The following examples and comparative examples are only for more clearly illustrating the technical aspects of the present invention so that those skilled in the art can well understand and utilize the present invention, and do not limit the scope of the present invention.
The names and abbreviations of the experimental methods, production processes, instruments and equipment related to the examples and comparative examples of the present invention are all conventional names in the art, and are clearly and clearly understood in the related fields of use, and those skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the names, and implement the process according to the conventional conditions or the conditions suggested by the manufacturers.
The raw materials and reagents used in the examples of the present invention are not particularly limited in terms of their sources, and are all conventional products commercially available. In the examples described below, matrigel was used from BD Biocoat, the media used were CMRL medium, fetal bovine serum, penicillin-streptomycin from Gibico, acidic Taiwan fluid from Sigma, glutamine from Gibico, sodium pyruvate from Merck, N2 additive (17502001) and B27 (17504044) additive from Gibco.
In addition, the human umbilical cord serum used in the following examples is prepared by the laboratory, 10 umbilical cord blood of volunteer lying-in women are taken after informed consent and ethical examination, the human umbilical cord serum is separated and obtained according to the serum separation procedure of conventional blood, and independent experiments are respectively carried out, so that the umbilical cord serum from different human sources has no significant influence on the culture result, the culture result has no significant difference and is stable and reliable, and the explanation shows that the umbilical cord serum of healthy people can be separated according to the basic process and meets the culture requirement.
Example 1 preparation of culture Medium.
The first medium was prepared with the following ingredients:
to 7.4mL of CMRL1066 medium were added 1mL of fetal bovine serum, 1mL of human umbilical serum, 100. mu.L of sodium pyruvate at a concentration of 100mM, 100. mu.L of glutamine at a concentration of 200mM, 100. mu.L of penicillin-streptomycin at a concentration of 10000 units, 100. mu.L of N2 additive at a concentration of 100X and 200. mu.L of B27 additive at a concentration of 50X, and the mixture was uniformly mixed and stored at 4 ℃ for further use.
The second culture medium after 48h of culture was prepared with the following components:
2mL of fetal bovine serum, 1mL of human umbilical serum, 100. mu.L of sodium pyruvate at a concentration of 100mM, 100. mu.L of glutamine at a concentration of 200mM, 100. mu.L of penicillin-streptomycin at a concentration of 10000 units, 100. mu.L of N2 additive at a concentration of 100 Xand 200. mu.L of B27 additive at a concentration of 50 Xare added to 6.4 mL of CMRL1066 medium, and the mixture is uniformly mixed and stored at 4 ℃ for later use.
Example 2 in vitro culture of mouse embryos.
Mouse embryos were cultured as follows:
1) extracting E3.5 mouse blastocysts from the uterus of a pregnant female ICR mouse with the age of 6-7 weeks, taking 25 of the blastocysts, washing the blastocysts in fresh and preheated M2 culture medium for 3-5 times, transferring the blastocysts into 30 mu L of acid typhoid liquid microdroplets by using a glass tube, immediately transferring the blastocysts into 30 mu L of fresh M2 culture medium microdroplets after a zona pellucida is digested, mixing the blastocysts after the zona pellucida is digested with 100 mu L of matrix glue with the volume concentration of 30 percent, inoculating the mixture onto a 24-hole culture plate which is paved with extracellular matrix glue (100 mu L/hole) in advance, enabling the glue to wrap the embryos, inoculating about 10 embryos into each hole, and establishing a 3D in-vitro culture platform;
2) adding 500 μ L of the first medium prepared in example 1 to each well of the culture plate, and after culturing for 48 hours, replacing the second medium prepared in example 1 to continue culturing;
3) place the plates in an ambient of 5% CO2The culture box is used for culturing at 37 ℃, the culture medium is changed every day, the embryo state is continuously observed, as shown in figure 1, 22 embryos reach the E6.5 egg column stage at the 4 th day, the culture success rate reaches 87.6%, and the repeatability is realized; after the end of day 7 culture, the embryos reached the E8.0 neural plate stage as shown in FIG. 2.
Claims (9)
1. A culture medium additive for mouse embryo body epitaxy contains fetal calf serum, human umbilical serum, sodium pyruvate, glutamine and penicillin-streptomycin.
2. The media supplement of claim 1, further comprising N2 supplement and B27 supplement.
3. A culture medium for mouse embryo body epitaxy, which comprises a basal medium and the medium additive of claim 1 or 2, and comprises 10-20% of fetal bovine serum, 10% of human umbilical serum, 1mM of sodium pyruvate, 2mM of glutamine, and 100 units/mL of penicillin-streptomycin.
4. The culture medium of claim 3, further comprising 100 XN 2 additives in a total volume of 1% and 50 XB 27 additives in a total volume of 2%.
5. The medium according to claim 3 or 4, wherein the basal medium is one or more of CMRL1066 medium, DMEM medium, 1640 medium, MEM medium, F10 medium, M199 medium, F12 medium.
6. Use of the medium according to claim 3 or 4 as a medium for culturing mouse embryo body in epitaxy.
7. The use according to claim 6, wherein the culture medium according to claim 3 or 4 is added to a matrigel culture environment inoculated with mouse embryos, wherein the matrigel culture environment inoculated with mouse embryos is inoculated with a matrigel-plated culture environment from a product of mixing of mouse blastocysts digested with zona pellucida and matrigel.
8. The use according to claim 7, wherein the matrix colloid has a volume concentration of 30%.
9. Use of the culture medium according to claim 3 or 4 as a culture medium in embryo culture experiments.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107361020A (en) * | 2017-08-28 | 2017-11-21 | 山西医科大学 | NTDs mouse embryo animal models and its construction method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107361020A (en) * | 2017-08-28 | 2017-11-21 | 山西医科大学 | NTDs mouse embryo animal models and its construction method |
Non-Patent Citations (2)
| Title |
|---|
| 邵红莲: "小鼠植入后胚胎体外延时培养体系的建立以及应用", 中国优秀硕士学位论文全文数据库（电子期刊）, no. 05, pages 656 - 657 * |
| 邵红莲等: "小鼠植入后胚胎体外延时培养体系的应用进展", 中国比较医学杂志, vol. 30, no. 8, pages 2 - 3 * |
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