CN107236699A - A kind of culture medium and its application for adhere-wall culture cell - Google Patents
A kind of culture medium and its application for adhere-wall culture cell Download PDFInfo
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- CN107236699A CN107236699A CN201710352458.7A CN201710352458A CN107236699A CN 107236699 A CN107236699 A CN 107236699A CN 201710352458 A CN201710352458 A CN 201710352458A CN 107236699 A CN107236699 A CN 107236699A
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- culture
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- culture medium
- wall
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
Abstract
The present invention relates to a kind of culture medium for adhere-wall culture cell and its application, the culture medium contains adherent inducing component on the basis of basal medium, and the adherent inducing component is by following material composition：Hydrocortisone, insulin, prostaglandin E1, transferrins, trilute.Further relate to a kind of method of adhere-wall culture mdck cell and thus obtain mdck cell culture.Serum is free of in the culture medium of the present invention, but still there is the adherent function of inducing cell, and it is especially suitable for adhere-wall culture mdck cell, cultivate obtained attached cell form complete, obvious epithelial cell sample is presented, cell refractivity is good, and inside can play bigger effect without obvious particle sediment in vaccine research.
Description
Technical field
The present invention relates to field of cell culture, more specifically it relates to a kind of culture medium for adhere-wall culture cell and its
Using further relating to a kind of method of adhere-wall culture mdck cell and thus obtain mdck cell culture.
Background technology
Mdck cell system (MDCK Cell Lines) is by Madin and Darby in 1958 from U.S. Cocker Spaniel
The renal tissue separation of female song frame dog, which is cultivated, sets up, and is typically the epithelioid cell grown with adherent manner.Mdck cell system is wide
It is general to be used for a variety of viral amplifications and purifying, such as：Reovirus, adenovirus, canine parvovirus, agranulocytosis virus of cats
And avian influenza virus etc..Because its viral efficiency of infection is high, propagation is fast, and variation is difficult, mdck cell system is acknowledged as most suitable
One of 3 kinds of cell lines produced in first, influenza B virus.
Traditional mdck cell culture is used mostly serum adhere-wall culture mode.The application of serum is asked there is also many
Topic：The pollution of susceptible viral, mycoplasma or other pathogens；Differences between batches cause the quality between product batches to be difficult to strict control
System；The presence of a large amount of haemocyanins adds the difficulty of downstream separation purifying, and Partial Protein is difficult to thorough by isolating and purifying means
Bottom is removed, and have impact on the final mass of product；In addition, serum origin is difficult, expensive, extensive animal cell culture process
Middle use serum will greatly increase production cost.If however, not increase serum in culture medium, can cause cell without the choice specimen of calligraphy
Wall.Even if in addition, the mdck cell of existing medium culture is also that form differs in same batch, sprawl not exclusively, and
And cell interior refractivity is poor, precipitation particle is more.
Accordingly, it would be desirable to which a kind of new culture medium is used to cultivate mdck cell.
The content of the invention
To solve problem above, the invention provides a kind of culture medium for adhere-wall culture cell, it is cultivated on basis
Adherent inducing component is contained on the basis of base, the adherent inducing component is by following material composition：Hydrocortisone, pancreas islet
Element, prostaglandin E1, transferrins, trilute.
Further, the adherent inducing component is by following material composition：1-10×10-8MM hydrocortisones, 1-10 μ g/
ML insulin, 1-10 × 10-8MM prostaglandin E1s, 1-10 μ g/mL transferrins, 1-10 × 10-12MM triiodos thyroid gland original ammonia
Acid.
In a preferred embodiment, the basal medium is DMEM/F12 culture mediums.
In a preferred embodiment, the cell is mdck cell.
Present invention also offers application of the above-mentioned culture medium in adhere-wall culture mdck cell.
Present invention also offers a kind of method of adhere-wall culture mdck cell, it is including the use of above-mentioned medium culture MDCK
The step of cell.
Present invention also offers a kind of mdck cell culture, it is obtained by the above method.
Serum is free of in the culture medium of the present invention, but still there is the adherent function of inducing cell, and is especially suitable for
For adhere-wall culture mdck cell, cultivate obtained attached cell form completely, obvious epithelial cell sample, cell refractive power is presented
Property it is good, inside without obvious particle sediment, bigger effect can be played in vaccine research.
Brief description of the drawings
Fig. 1 is the microphoto after using embodiment 1-4 and the mdck cell of conventional medium culture adherent respectively.
Embodiment
The principle and feature of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the present invention.
1. the preparation of serum free medium
The culture medium of the present invention is obtained by the way that the composition shown in table 1 to be added to prepare in DMEM/F12 culture mediums.
The composition that table 1 is added into DMEM/F12 culture mediums
| Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | |
| Hydrocortisone (mM) | 1×10-8 | 4×10-8 | 8×10-8 | 10×10-8 |
| Insulin (μ g/mL) | 1 | 4 | 8 | 10 |
| MM prostaglandin E1s (mM) | 1×10-8 | 4×10-8 | 8×10-8 | 10×10-8 |
| Transferrins (μ g/mL) | 1 | 4 | 8 | 10 |
| Trilute (mM) | 1×10-8 | 4×10-8 | 8×10-8 | 10×10-8 |
The culture of 2.MDCK cells
The medium culture mdck cell obtained respectively with being prepared in above-described embodiment, with traditional DMEM+10% serum
To compare, cultural method is as follows：Supernatant is abandoned in recovery cell, centrifugation, adds K-1 culture mediums, cultivates cell, treats that cell density is given birth to
Length is passed on to 80%.K-1 culture mediums are serum free medium, it is necessary to centrifuge removal tryptose after Trypsin Induced
Enzyme, because K-1 does not have serum, it is impossible to suppress the effect of trypsase, therefore need centrifugation.
3. microscope inspection observation checking after cell attachment
The attached cell that above-mentioned culture is obtained is put to be observed under the microscope, as a result as shown in figure 1, using conventional medium
The mdck cell form heterogeneity of culture, is sprawled not exclusively, cell interior refractivity is poor, and precipitation particle is more, and is used above-mentioned
The mdck cell form that embodiment culture is obtained is homogeneous, and adherent rear form is complete, and obvious epithelial cell sample, cell refractive power is presented
Property it is good, inside without obvious particle sediment.
The foregoing is only presently preferred embodiments of the present invention, be not intended to limit the invention, it is all the present invention spirit and
Within principle, any modification, equivalent substitution and improvements made etc. should be included in the scope of the protection.
Claims (7)
1. a kind of culture medium for adhere-wall culture cell, it is characterised in that contained on the basis of basal medium adherent
Inducing component, the adherent inducing component is by following material composition：Hydrocortisone, insulin, prostaglandin E1, turn iron egg
In vain, trilute.
2. culture medium according to claim 1, it is characterised in that the adherent inducing component is by following material composition：1-
10×10-8MM hydrocortisones, 1-10 μ g/mL insulin, 1-10 × 10-8MM prostaglandin E1s, 1-10 μ g/mL transferrins,
1-10×10-12MM trilutes.
3. culture medium according to claim 1, it is characterised in that the basal medium is DMEM/F12 culture mediums.
4. the culture medium according to any one of claim 1-3, it is characterised in that the cell is mdck cell.
5. application of the culture medium in adhere-wall culture mdck cell any one of claim 1-4.
6. a kind of method of adhere-wall culture mdck cell, it is characterised in that including the use of any one of claim 1-4
The step of medium culture mdck cell.
7. a kind of mdck cell culture, it is characterised in that obtained by the method described in claim 6.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710352458.7A CN107236699A (en) | 2017-05-18 | 2017-05-18 | A kind of culture medium and its application for adhere-wall culture cell |
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| CN201710352458.7A CN107236699A (en) | 2017-05-18 | 2017-05-18 | A kind of culture medium and its application for adhere-wall culture cell |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101189326B (en) * | 2004-12-23 | 2013-06-12 | 米迪缪尼有限公司 | Non-tumorigenic MDCK cell line for propagating viruses |
| CN105543163A (en) * | 2016-01-30 | 2016-05-04 | 马忠仁 | Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells |
-
2017
- 2017-05-18 CN CN201710352458.7A patent/CN107236699A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101189326B (en) * | 2004-12-23 | 2013-06-12 | 米迪缪尼有限公司 | Non-tumorigenic MDCK cell line for propagating viruses |
| CN105543163A (en) * | 2016-01-30 | 2016-05-04 | 马忠仁 | Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells |
Non-Patent Citations (1)
| Title |
|---|
| 黄锭等: "犬肾细胞 MDCK 无血清贴壁及单细胞悬浮培养", 《生物工程学报》 * |
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