CN106047788A - Culture medium for high-density cell culture system - Google Patents
Culture medium for high-density cell culture system Download PDFInfo
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- CN106047788A CN106047788A CN201610702787.5A CN201610702787A CN106047788A CN 106047788 A CN106047788 A CN 106047788A CN 201610702787 A CN201610702787 A CN 201610702787A CN 106047788 A CN106047788 A CN 106047788A
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- culture medium
- concentration
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- cultivation system
- concentration cultivation
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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
- C12N5/0018—Culture media for cell or tissue culture
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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
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- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
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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
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
Abstract
The present invention relates to a culture medium for a high-density cell culture system. The culture medium comprises a base culture medium and added components, wherein the base culture medium is a DMEM culture medium, and the added components comprise D-glucose with a concentration of 5-10 g/L, L-glutamine with a concentration of 5-7 g/L, HEPES with a concentration of 5-7 g/L, and sodium pyruvate with a concentration of 6-10 g/L. According to the present invention, the combination of the DMEM adopted as the base culture medium and the specific added components is used, and particularly the D-glucose, the L-glutamine and the sodium pyruvate simultaneously meet the specific concentration ranges, such that the nutrition requirements of the rapid and large-scale cell proliferation are met, and the culture medium is particularly suitable for the high-density cell culture system.
Description
Technical field
The present invention relates to a kind of culture medium being applicable to concentration cultivation system, belong to technical field of cell culture.
Background technology
Cell is cultivated and has been widely used in the every field such as biology, medical science, new drug development, becomes most important basis
One of science.
About Cell culture invitro system, except two dimensions such as conventional culture plate, culture dish, culture bottles in prior art
Cell culture system, has been developed that dimensional culture system, the most presently commercially available concentration cultivation bucket now, be one both
Go for attached cell, go for again the dimensional culture system of suspension cell, a large amount of cell can be cultivated rapidly,
And the long term cell culture of half a year by a definite date more than can be carried out, it is not necessary to often change consumptive material, with traditional culture plate, culture dish
Compare with culture bottle, more convenient, cost is lower, therefore suffers from the extensive concern of this area research staff.
But, traditional culture medium of the prior art is applicable to medium scale cell culture system mostly, typically comes
Say that cannot be applicable to cell large-scale, highdensity cultivates；On the other hand, existing culture medium is only applicable to culture plate, training
Support the cell culture system of the two dimension such as ware, culture bottle, it is impossible to be applicable to concentration cultivation bucket such dimensional culture system；
When traditional culture medium is applied to the cultivation of concentration cultivation bucket, conventional medium nutrition can not meet the quick increasing of cell
Growing, cell poor growth, culture effect is the best.
The most urgently exploitation is exclusively used in the culture medium of concentration cultivation system.
Summary of the invention
In view of the problems referred to above and/or the other problems of correlation technique, one aspect of the present invention provides one and is applicable to highly dense
Spending the culture medium of cell culture system, it includes basal medium and addO-on therapy, and wherein, described basal medium is DMEM training
Support base；Described addO-on therapy and concentration thereof are as follows: D-Glucose, 5-10g/L；L-glutaminate, 5-7g/L；HEPES, 5-7g/
L；Sodium Pyruvate, 6-10g/L；The concentration of each addO-on therapy is total with the described culture medium being applicable to concentration cultivation system
On the basis of volume.
Preferably, on the basis of the cumulative volume of the described culture medium being applicable to concentration cultivation system, described DMEM
The dry powder amount of culture medium is 17～22g/L.
Preferably, on the basis of the cumulative volume of the described culture medium being applicable to concentration cultivation system, described DMEM
The dry powder amount of culture medium is 19.5g/L.
Preferably, the concentration of described D-Glucose is 7.5g/L, and the concentration of described L-glutaminate is 6g/L, described acetone
The concentration of acid sodium is 8g/L.
Preferably, the pH value of the culture medium being applicable to concentration cultivation system described in is 7.2～7.6；Described interpolation
Component also includes that phenol red sodium, the concentration of described phenol red sodium are 15-20mg/L.
Preferably, the osmotic pressure of the culture medium being applicable to concentration cultivation system described in is 320-360mOsm/L.
Another aspect of the present invention provides the above-mentioned culture medium of concentration cultivation system that is applicable at high-density cells
Application in cultivating system.
Preferably, described concentration cultivation system is concentration cultivation bucket.
The culture medium being applicable to concentration cultivation system of the present invention, uses culture medium and spy based on DMEM
The combination of fixed addO-on therapy, especially D-Glucose, L-glutaminate and Sodium Pyruvate three meet specific concentration simultaneously
During scope, it is possible to meet the nutritional need required for cell propagation quick, substantial amounts of, be particularly suitable for concentration cultivation body
System.
Detailed description of the invention
The present invention is further illustrated by the following examples, but the present invention is not limited to these specific embodiment parties
Formula.
Embodiment 1
The process for preparation of the culture medium being applicable to concentration cultivation system of embodiment 1 is as follows:
Step 1): aseptically the DMEM dry powder of synthesis is weighed 195g on electronic balance and be placed on disinfecting container
In, stir after adding the dilution of 9L redistilled water acquisition basal medium；
Step 2): by addO-on therapy: 75g D-Glucose, 60g L-glutaminate, 64g HEPES, 8g Sodium Pyruvate,
The phenol red sodium of 170mg is sequentially added into step 1) in the basal medium that obtains, fully mix；
Step 3): filtration sterilization, then regulate pH value to 7.4 ± 0.2, it is eventually adding redistilled water by volume regulation to 10L；
Step 4): after culture medium has been prepared, filtration sterilization, subpackage, sealing, last cold preservation (+4 DEG C of environment) is standby.
Embodiment 2
The process for preparation of the peripheral blood cells culture medium being applicable to concentration cultivation system of embodiment 2 is as follows:
Step 1): aseptically the DMEM dry powder of synthesis is weighed 195g on electronic balance and be placed on disinfecting container
In, stir after adding the dilution of 9L redistilled water acquisition basal medium；
Step 2): by addO-on therapy: 70g D-Glucose, 62g L-glutaminate, 66g HEPES, 7.8g Sodium Pyruvate,
The phenol red sodium of 180mg is sequentially added into step 1) in the basal medium that obtains, fully mix；
Step 3): filtration sterilization, then regulate pH value to 7.4 ± 0.2, it is eventually adding redistilled water by volume regulation to 10L；
Step 4): after culture medium has been prepared, filtration sterilization, subpackage, sealing, last cold preservation (+4 DEG C of environment) is standby.
Application examples 1
The concentration cultivation bucket using commercially available FiberCell brand C2025 model (sees FiberCell cell
Culture systems production marketing webpage), and the serum-free medium that the embodiment of the present invention 1 obtains carries out the cultivation of 293T cell.
Being transferred in concentration cultivation bucket by 293T cell, cell-seeding-density is 2.5 × 106Individual/ml, liquid volume added
About 50ml；Specifically, the culture medium in liquid containing bottle is cultivated bucket by silica gel tube with cell and is connected, under the effect of peristaltic pump,
Persistent loop flows, and the doughnut that culture medium cultivates bucket by cell swaps, and the cell cultivated in bucket to cell is continuous
Offer nutrient substance；Whole concentration cultivation bucket is put into 5%CO2, the incubator of 37 DEG C is cultivated.Cultivate 1 day, 2
My god, 3 days, 4 days, after 5 days, record cell quantity.
Comparative example 1: use the concentration cultivation bucket of commercially available FiberCell brand C2025 model (to see
FiberCell cell culture system production marketing webpage), and the commercially available conventional DMEM culture medium of BI brand (01-055-1A),
Incubation is identical with application examples 1, specifically repeats no more.
Detection cell proliferation rate
Application examples 1 and comparative example 1, all after cultivating 1,2,3,4 and 5 days, separately sampled culture fluid, carry out cell concentration
Detection, testing result sees table 1 below.)
Table 1
| Embodiment 1 (cell/ml) | Comparative example 1 (cell/ml) | |
| 1st day | 5.00E+04 | 5.00E+04 |
| 2nd day | 1.12E+05 | 8.17E+04 |
| 3rd day | 1.93E+05 | 1.30E+05 |
| 4th day | 3.40E+05 | 1.47E+05 |
| 5th day | 3.83E+05 | 3.00E+05 |
Carry out contrasting by above-mentioned result it can be seen that the cell proliferation rate of application examples of the present invention 1 is far above comparative example 1
, this explanation: the cell culture medium of the embodiment of the present invention is compared the DMEM culture medium of routine and is more suitable for concentration cultivation
System.
It is to be understood that, although this specification is been described by according to embodiment, but the most each embodiment only comprises one
Individual independent technical scheme, this narrating mode of description is only that for clarity sake those skilled in the art should will say
Bright book is as an entirety, and the technical scheme in each embodiment can also be through appropriately combined, and forming those skilled in the art can
With other embodiments understood.
The a series of detailed description of those listed above is only for the feasibility embodiment of the present invention specifically
Bright, they also are not used to limit the scope of the invention, all equivalent implementations made without departing from skill of the present invention spirit
Or change should be included within the scope of the present invention.
Claims (8)
1. being applicable to a culture medium for concentration cultivation system, it includes basal medium and addO-on therapy, wherein,
Described basal medium is DMEM culture medium；
Described addO-on therapy and concentration thereof are as follows:
D-Glucose, 5-10g/L；
L-glutaminate, 5-7g/L；
HEPES, 5-7g/L；
Sodium Pyruvate, 6-10g/L；
The concentration of each addO-on therapy is on the basis of the cumulative volume of the described culture medium being applicable to concentration cultivation system.
It is applicable to the culture medium of concentration cultivation system the most as claimed in claim 1, it is characterised in that:
On the basis of the cumulative volume of the described culture medium being applicable to concentration cultivation system, the dry powder of described DMEM culture medium
Amount is 17～22g/L.
It is applicable to the culture medium of concentration cultivation system the most as claimed in claim 2, it is characterised in that:
On the basis of the cumulative volume of the described culture medium being applicable to concentration cultivation system, the dry powder of described DMEM culture medium
Amount is 19.5g/L.
It is applicable to the culture medium of concentration cultivation system the most as claimed in claim 1, it is characterised in that:
The concentration of described D-Glucose is 7.5g/L, and the concentration of described L-glutaminate is 6g/L, the concentration of described Sodium Pyruvate
For 8g/L.
5. the culture medium being applicable to concentration cultivation system as described in any one in Claims 1-4, its feature exists
In:
The pH value of the described culture medium being applicable to concentration cultivation system is 7.2～7.6；
Described addO-on therapy also includes that phenol red sodium, the concentration of described phenol red sodium are 15-20mg/L.
6. the culture medium being applicable to concentration cultivation system as described in any one in Claims 1-4, its feature exists
In:
The osmotic pressure of the described culture medium being applicable to concentration cultivation system is 320-360mOsm/L.
7. the culture medium being applicable to concentration cultivation system as described in any one in claim 1 to 6 is in high density
Application in cell culture system.
Apply the most as claimed in claim 7, it is characterised in that: described concentration cultivation system is concentration cultivation
Bucket.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610702787.5A CN106047788A (en) | 2016-08-22 | 2016-08-22 | Culture medium for high-density cell culture system |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999049014A1 (en) * | 1998-03-25 | 1999-09-30 | Cornell Research Foundation, Inc. | Localization and propagation of neural and neuronal progenitor cells |
| CN101984051A (en) * | 2010-11-19 | 2011-03-09 | 中山大学 | Serum-free cell culture fluid suitable for enriching and culturing tumour stem cells |
| WO2013050962A1 (en) * | 2011-10-04 | 2013-04-11 | Mitra Biotech Private Limited | Ecm composition, tumor microenvironment platform and methods thereof |
-
2016
- 2016-08-22 CN CN201610702787.5A patent/CN106047788A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999049014A1 (en) * | 1998-03-25 | 1999-09-30 | Cornell Research Foundation, Inc. | Localization and propagation of neural and neuronal progenitor cells |
| CN101984051A (en) * | 2010-11-19 | 2011-03-09 | 中山大学 | Serum-free cell culture fluid suitable for enriching and culturing tumour stem cells |
| WO2013050962A1 (en) * | 2011-10-04 | 2013-04-11 | Mitra Biotech Private Limited | Ecm composition, tumor microenvironment platform and methods thereof |
Non-Patent Citations (1)
| Title |
|---|
| 汪多仁: "《绿色医药化学品》", 31 July 2008, 科技文献出版社 * |
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