CN105238741A - Complete medium for culturing ruminal epithelium cells of dairy cows and application thereof - Google Patents
Complete medium for culturing ruminal epithelium cells of dairy cows and application thereof Download PDFInfo
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- CN105238741A CN105238741A CN201510774237.XA CN201510774237A CN105238741A CN 105238741 A CN105238741 A CN 105238741A CN 201510774237 A CN201510774237 A CN 201510774237A CN 105238741 A CN105238741 A CN 105238741A
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Abstract
The invention discloses a complete medium for culturing ruminal epithelium cells of dairy cows. Every 1000 mL of the complete media are prepared from EGF with the concentration of 1-5 ng/mL, IGF-1 with the concentration of 0.5-1.5 ug/mL, hydrocortisone with the concentration of 0.5-1.5 ug/mL, mycillin with the concentration of 100-300 IU/mL, 100 mL of fetal calf serum and the balance DMEM/F12 powder. The complete medium for culturing the ruminal epithelium cells of the dairy cows has the advantages that a commercial DMEM/F12 formula is improved, and therefore the purposes that the ruminal epithelium cells of the dairy cows are rapid in wall adhering and growing; cells cultured by a common DMEM/F12 medium start to be adhered to the wall and grow cells after being inoculated for 5-6 days, but the cells cultured by the complete medium start to be adhered to the wall and grow cells after being inoculated for 3-4 days, and therefore 2-3 days are shortened.
Description
Technical field
The present invention relates to a kind of cultivation epithelial perfect medium of cow rumen and application thereof.
Background technology
Along with the development of biotechnology, the cow rumen epithelial cell of vitro culture can be used as the important means of cud nutrition regulation, but adherent and growth encounters problem during cow rumen epithelial cell vitro culture, current investigator mostly uses DMEM/F12 substratum when cultivating cow rumen epithelial cell, under use DMEM/F12 culture medium condition, the adherent required time length of cow rumen epithelial cell, poor growth, illustrate that this kind of substratum is bad for the effect of cow rumen epithelial cell vitro culture.
Summary of the invention
The object of the invention is to set up the perfect medium cultivated for cow rumen epithelial cell.
Technical scheme of the present invention is: the epithelial perfect medium of a kind of cultivation cow rumen, 1000mL perfect medium is made up of following raw material: concentration is the EGF of 1 ~ 5ng/mL, concentration is the IGF-1 of 0.5 ~ 1.5ug/mL, concentration is the hydrocortisone of 0.5 ~ 1.5ug/mL, concentration is the mycillin of 100 ~ 300IU/mL, 100mL foetal calf serum, surplus is DMEM/F12 pulvis.
The preparation method of the described epithelial perfect medium of cultivation cow rumen, its step is as follows:
(1) DMEM/F12 pulvis is made DMEM/F12 basic culture solution;
(2) in 500 ~ 800mLDMEM/F12 basic culture solution, add 100mL foetal calf serum, make DMEM/F12 nutrient solution;
(3) add EGF, IGF-1, mycillin, then add DMEM/F12 basic culture solution and be settled to 1000mL, make and cultivate the epithelial perfect medium of cow rumen.
The pH of described substratum is 7.0 ~ 7.4.
The invention has the beneficial effects as follows: the present invention improves business DMEM/F12 formula, so reach cow rumen epithelial cell adherent fast, grow fast object.The cell of common DMEM/F12 culture medium culturing after inoculation 5 ~ 6d starts adherently to grow cell, and described perfect medium cultured cells after inoculation 3 ~ 4d start the adherent cell that grows, shorten 2 ~ 3d.Adherent rear common DMEM/F12 culture medium culturing cell 4 ~ 5d still poor growth, can not confluent culture bottle, and described perfect medium culturing cell only needs 3 ~ 4d can confluent culture bottle.
Accompanying drawing explanation
Fig. 1 is that common DMEM/F12 cultivates cud epithelial cell figure;
Fig. 2 is that perfect medium cultivates cud epithelial cell figure.
Embodiment
The epithelial perfect medium of a kind of cultivation cow rumen, 1000mL perfect medium is made up of following raw material: concentration is the EGF of 1 ~ 5ng/mL, concentration is the IGF-1 of 0.5 ~ 1.5ug/mL, concentration is the hydrocortisone of 0.5 ~ 1.5ug/mL, concentration is the mycillin of 100 ~ 300IU/mL, 100mL foetal calf serum, surplus is DMEM/F12 pulvis.
Cultivate the preparation method of the epithelial perfect medium of cow rumen, its step is as follows:
(1) DMEM/F12 pulvis is made 1000mLDMEM/F12 basic culture solution;
(2) in 500 ~ 800mLDMEM/F12 basic culture solution, add 100mL foetal calf serum, make DMEM/F12 nutrient solution;
(3) add EGF, IGF-1, mycillin, then add DMEM/F12 basic culture solution and be settled to 1000mL, make and cultivate the epithelial perfect medium of cow rumen.
The pH of described substratum is 7.0 ~ 7.4.
A kind of application of cultivating the epithelial perfect medium of cow rumen, cow rumen epithelium is shredded rear enzyme-addedly to digest, the cultivation of cud epithelial cell proliferation is carried out after the sieved filter of cell, last collecting cell, described cultivation is glue primordial covering adherent culture, and described glue primordial covering adherent culture is I type Collagen type-I bag quilt.
Primary cud epithelial cell culturing step is as follows:
(1) sample: under aseptic condition, take out newborn holstein cow cud tissue, cut off cud and by external for its stomach internal layer, then organize 10min with 75% alcohol immersion with scissors, finally use contains dual anti-HBSS cleansing tissue 5 times gently.Repeat 75% alcohol immersion and HBSS cleaning step three times;
(2) digest: cut stomach epithelium gently with scissors, be then cut into 1mm with scissors
3size, contains the digestion of 0.5%PronaseE+0.5%CollagenaseII enzyme liquid with 50mL centrifuge tube and shreds and organize about 1.5h, shake centrifuge tube for several times, fully digest to be used in tissue between the period of digestion by 37 DEG C, in 5%CO2 incubator;
(3) filter: after seeing cell detachment under the microscope, with the sieved filter Digestive system of 100 order cell, for several times, the centrifugal 10mim of 1500rpm obtains cell precipitation afterwards Digestive system after filtration to be positioned over piping and druming in centrifuge tube;
(4) bag quilt: by culturing bottle I type Collagen type-I bag quilt used.
(5) cultivate: by the common DMEM/F12 substratum of cell precipitation that obtains or perfect medium resuspended, and be placed on 37 DEG C, 5%CO
2cultivate in cell culture incubator;
(6) liquid is changed: use fresh complete medium after 24h instead and continue to cultivate.
Use inverted microscope to observe the foster form of cud epithelial cells of primary culture, common DMEM/F12 substratum and described culture medium culturing cell results are shown in Fig. 1 and Fig. 2, and what wherein grow in paving stone sample is cud epithelial cell.
The cell of common DMEM/F12 culture medium culturing after inoculation 5 ~ 6d starts adherently to grow cell, and described perfect medium cultured cells after inoculation 3 ~ 4d start the adherent cell that grows, shorten 2 ~ 3d.Adherent rear common DMEM/F12 culture medium culturing cell 4 ~ 5d still poor growth, can not confluent culture bottle, and described perfect medium culturing cell only needs 3 ~ 4d can confluent culture bottle.
Claims (4)
1. cultivate the epithelial perfect medium of cow rumen for one kind, it is characterized in that: 1000mL perfect medium is made up of following raw material: concentration is the EGF of 1 ~ 5ng/mL, concentration is the IGF-1 of 0.5 ~ 1.5ug/mL, concentration is the hydrocortisone of 0.5 ~ 1.5ug/mL, concentration is the mycillin of 100 ~ 300IU/mL, 100mL foetal calf serum, surplus is DMEM/F12 pulvis.
2. the preparation method of the epithelial perfect medium of cultivation cow rumen according to claim 1, it is characterized in that, its step is as follows:
(1) DMEM/F12 pulvis is made 1000mLDMEM/F12 basic culture solution;
(2) in 500 ~ 800mLDMEM/F12 basic culture solution, add 100mL foetal calf serum, make DMEM/F12 nutrient solution;
(3) add EGF, IGF-1, mycillin, then add DMEM/F12 basic culture solution and be settled to 1000mL, make and cultivate the epithelial perfect medium of cow rumen.
3. the preparation method of the epithelial perfect medium of cultivation cow rumen according to claim 1, is characterized in that: the pH of described substratum is 7.0 ~ 7.4.
4. cultivate the application of the epithelial perfect medium of cow rumen for one kind, it is characterized in that: cow rumen epithelium is shredded rear enzyme-addedly to digest, the cultivation of cud epithelial cell proliferation is carried out after the sieved filter of cell, last collecting cell, described cultivation is glue primordial covering adherent culture, and described glue primordial covering adherent culture is I type Collagen type-I bag quilt.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967674A (en) * | 2017-05-22 | 2017-07-21 | 江西农业大学 | A kind of isolated culture method of sheep rumen epithelial cell |
| CN108103004A (en) * | 2017-12-20 | 2018-06-01 | 中国农业科学院特产研究所 | A kind of method of sika deer cud epithelial cell culture |
| CN109097320A (en) * | 2018-07-23 | 2018-12-28 | 南京农业大学 | A kind of sheep lamb cud epithelial cell cultural method |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN113088482A (en) * | 2021-04-16 | 2021-07-09 | 四川农业大学 | Method for separating and culturing rumen epithelial cells of calves |
| CN113388570A (en) * | 2021-06-16 | 2021-09-14 | 浙江大学 | Bovine rumen epithelial tissue dissociation method for single cell sequencing |
| CN114058592A (en) * | 2021-11-16 | 2022-02-18 | 四川农业大学 | Immortalized yak rumen epithelial cell line and construction method thereof |
| CN117503800A (en) * | 2024-01-04 | 2024-02-06 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
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Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106967674A (en) * | 2017-05-22 | 2017-07-21 | 江西农业大学 | A kind of isolated culture method of sheep rumen epithelial cell |
| CN106967674B (en) * | 2017-05-22 | 2020-07-10 | 江西农业大学 | Separation culture method of sheep rumen epithelial cells |
| CN108103004A (en) * | 2017-12-20 | 2018-06-01 | 中国农业科学院特产研究所 | A kind of method of sika deer cud epithelial cell culture |
| CN109097320A (en) * | 2018-07-23 | 2018-12-28 | 南京农业大学 | A kind of sheep lamb cud epithelial cell cultural method |
| CN112458040A (en) * | 2020-12-23 | 2021-03-09 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN112458040B (en) * | 2020-12-23 | 2022-07-05 | 四川农业大学 | Isolation culture method of rumen epithelial cells of adult yaks |
| CN113088482A (en) * | 2021-04-16 | 2021-07-09 | 四川农业大学 | Method for separating and culturing rumen epithelial cells of calves |
| CN113388570A (en) * | 2021-06-16 | 2021-09-14 | 浙江大学 | Bovine rumen epithelial tissue dissociation method for single cell sequencing |
| CN113388570B (en) * | 2021-06-16 | 2023-02-14 | 浙江大学 | Bovine rumen epithelial tissue dissociation method for single cell sequencing |
| CN114058592A (en) * | 2021-11-16 | 2022-02-18 | 四川农业大学 | Immortalized yak rumen epithelial cell line and construction method thereof |
| CN117503800A (en) * | 2024-01-04 | 2024-02-06 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
| CN117503800B (en) * | 2024-01-04 | 2024-04-05 | 北京益华生物科技有限公司 | Gastric mucosa epithelial cell extract and preparation method and application thereof |
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Application publication date: 20160113 |