CN106011063A - DC cell culture medium and preparing method thereof - Google Patents
DC cell culture medium and preparing method thereof Download PDFInfo
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- CN106011063A CN106011063A CN201610483002.XA CN201610483002A CN106011063A CN 106011063 A CN106011063 A CN 106011063A CN 201610483002 A CN201610483002 A CN 201610483002A CN 106011063 A CN106011063 A CN 106011063A
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- 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
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- C12N2500/00—Specific components of cell culture medium
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Abstract
The invention discloses a DC cell culture medium. The DC cell culture medium is composed of RPMI-1640 culture medium, interleukin-4, endothelial cell growth factor, transferrin, granulocyte-macrophage colony stimulating factor and tumor necrosis factor alpha, wherein the final concentration of interleukin-4 is 5-20 microgrammes/liter, the final concentration of endothelial cell growth factor is 0.1-0.5 microgramme/liter, the final concentration of transferrin is 30-100 microgrammes/liter, the final concentration of granulocyte-macrophage colony stimulating factor is 20-100 microgrammes/liter, and the final concentration of tumor necrosis factor alpha is 10-50 microgrammes/liter. The invention further discloses a method for preparing the DC cell culture medium. The method comprises the steps of adding interleukin-4, endothelial cell growth factor, transferrin, granulocyte-macrophage colony stimulating factor and tumor necrosis factor alpha to the RPMI-1640 culture medium in sequence, wherein one component needs to be added after the last component is completely dissolved and standing is conducted; then regulating the pH value; and finally conducting membrane filtration to obtain the DC cell culture medium.
Description
Technical field
The present invention relates to culture medium technical field, particularly relate to a kind of DC cell culture medium and preparation method thereof.
Background technology
DC cell full name is dendritic cell, be professional antigen the most of concern be delivery cell,
Can picked-up, processing and antigen-presenting, the immunoreation of startup T cell mediation.By American scholar Steinman
Found in mouse lymph nodes first in 1973.
Multiplex RPMI-1640 or the DMEM culture medium of DC cell cultivation at present, but due to DC cell certainly
Body multiplication capacity is not strong, and simple RPMI-1640 or DMEM culture medium cannot improve the growth of DC cell
Necessary nutritional labeling, cultivates DC cell multiplex containing 10～20wt% hyclones the most at present
RPMI-1640 or DMEM culture medium.But use hyclone to cultivate DC cell and there is various problems,
Not only increase the cost of culture medium, and differ greatly between the hyclone of different brands, additionally, due to
Complicated component in hyclone, these become branch to have a strong impact on accuracy and the reliability of subsequent experimental, cultivate
Base repeatability is poor, and culture efficiency is only 15～20%.And owing to the clinical cell product used is forbidden
Residual animal serum albumen, and conventional medium exists hyclone, technique needs increase cell and make
Bovine serum albumin residue detection in product, adds production difficulty and cost, promotes people to proceed by serum-free
The exploitation of culture medium.
Therefore at present DC cell culture medium have that culture efficiency is the highest, cytoactive low, subsequent experimental interference,
The bigger problems such as animal serum protein residue.
Summary of the invention
The technical problem existed based on background technology, the present invention proposes a kind of DC cell culture medium and preparation thereof
Method, improves DC cell culture efficiency, increases cytoactive, it is ensured that the repeatability of subsequent experimental, fall
The low use cost of DC cell culture medium, and avoid the albuminised incorporation of animal blood, save DC thin
The arrangement step of born of the same parents' culture medium and cost.
A kind of DC cell culture medium that the present invention proposes, its component includes: RPMI-1640 culture medium, Bai Jie
Element-4, endothelial cell growth factor (ECGF), transferrins, granulocyte-macrophage colony stimutaing factor and tumor are bad
Necrosis factor α.
Preferably, final concentration of 5～20 μ g/L of interleukin-4, endothelial cell growth factor (ECGF) final concentration of
0.1～0.5 μ g/L, final concentration of the 30 of transferrins～100mg/L, granulocyte-macrophage colony stimulate because of
The final concentration of 20～100 μ g/L, final concentration of 10～50 μ g/L of tumor necrosis factor α of son.
Preferably, the pH value of described DC cell culture medium is 7.35～7.40.
Preferably, the final concentration of 10 μ g/L of interleukin-4, endothelial cell growth factor (ECGF) final concentration of
0.2 μ g/L, the final concentration of 50mg/L of transferrins, the end of granulocyte-macrophage colony stimutaing factor is dense
Degree is 50 μ g/L, the final concentration of 20 μ g/L of tumor necrosis factor α.
The preparation method of the above-mentioned DC cell culture medium that the present invention also proposes, in RPMI-1640 culture medium
Add after interleukin-4 is completely dissolved, stand, add after endothelial cell growth factor (ECGF) is completely dissolved, stand,
It is subsequently added into after transferrins is completely dissolved, stands, continuously add granulocyte-macrophage colony stimutaing factor
After being completely dissolved, stand, be subsequently adding after tumor necrosis factor α is completely dissolved, stand, then regulate pH
Value, then membrane filtration obtains DC cell culture medium.
Preferably, time of repose is 4～6min.
Preferably, Sodium Pyruvate regulation pH value is used.
Preferably, the aperture of filter membrane is 0.1 μm, for removing the impurity in culture medium and the micro-life that may contain
Thing.
The present invention is directed to the feature that the nutrition of RPMI-1640 culture medium own is poor, in RPMI-1640 culture medium
Basis on add multiple nutrients material innovatively, including interleukin-4, endothelial cell growth factor (ECGF), turn
Ferritin, granulocyte-macrophage colony stimutaing factor and tumor necrosis factor α, be added without tire Sanguis Bovis seu Bubali completely
Clearly, make the various constituent concentrations in gained culture medium fix, improve DC cell culture efficiency, increase cell
Activity, it is ensured that the repeatability of subsequent experimental, reduces the use cost of DC cell culture medium, and avoids
The albuminised incorporation of animal blood, saves arrangement step and the cost of DC cell culture medium.
Detailed description of the invention
Below, by specific embodiment, technical scheme is described in detail.
Embodiment 1
A kind of DC cell culture medium that the present invention proposes, its component includes: RPMI-1640 culture medium, Bai Jie
Element-4, endothelial cell growth factor (ECGF), transferrins, granulocyte-macrophage colony stimutaing factor and tumor are bad
Necrosis factor α；The wherein final concentration of 5 μ g/L of interleukin-4, endothelial cell growth factor (ECGF) final concentration of
0.5 μ g/L, the final concentration of 30mg/L of transferrins, the end of granulocyte-macrophage colony stimutaing factor is dense
Degree is 100 μ g/L, the final concentration of 10 μ g/L of tumor necrosis factor α.
The preparation method of the above-mentioned DC cell culture medium that the present invention also proposes, in RPMI-1640 culture medium
Add after interleukin-4 is completely dissolved, stand 4min, add after endothelial cell growth factor (ECGF) is completely dissolved,
Stand 6min, be subsequently added into after transferrins is completely dissolved, stand 4min, continuously add granulocyte-huge and bite
After colony-stimulating factor is completely dissolved, stands 6min, be subsequently adding tumor necrosis factor α and be completely dissolved
After, stand 4min, then use Sodium Pyruvate regulation pH value to 7.38, then using aperture is 0.1 μm
Membrane filtration obtain DC cell culture medium.
Embodiment 2
A kind of DC cell culture medium that the present invention proposes, its component includes: RPMI-1640 culture medium, Bai Jie
Element-4, endothelial cell growth factor (ECGF), transferrins, granulocyte-macrophage colony stimutaing factor and tumor are bad
Necrosis factor α；The wherein final concentration of 20 μ g/L of interleukin-4, endothelial cell growth factor (ECGF) final concentration of
0.1 μ g/L, the final concentration of 100mg/L of transferrins, the end of granulocyte-macrophage colony stimutaing factor is dense
Degree is 20 μ g/L, the final concentration of 50 μ g/L of tumor necrosis factor α.
The preparation method of the above-mentioned DC cell culture medium that the present invention also proposes, in RPMI-1640 culture medium
Add after interleukin-4 is completely dissolved, stand 6min, add after endothelial cell growth factor (ECGF) is completely dissolved,
Stand 4min, be subsequently added into after transferrins is completely dissolved, stand 6min, continuously add granulocyte-huge and bite
After colony-stimulating factor is completely dissolved, stands 4min, be subsequently adding tumor necrosis factor α and be completely dissolved
After, stand 6min, then employing Sodium Pyruvate and 4-hydroxyethyl piperazine ethanesulfonic acid regulation pH value are to 7.35,
Then membrane filtration obtains DC cell culture medium.
Embodiment 3
A kind of DC cell culture medium that the present invention proposes, its component includes: RPMI-1640 culture medium, Bai Jie
Element-4, endothelial cell growth factor (ECGF), transferrins, granulocyte-macrophage colony stimutaing factor and tumor are bad
Necrosis factor α；The wherein final concentration of 10 μ g/L of interleukin-4, endothelial cell growth factor (ECGF) final concentration of
0.2 μ g/L, the final concentration of 50mg/L of transferrins, the end of granulocyte-macrophage colony stimutaing factor is dense
Degree is 50 μ g/L, the final concentration of 20 μ g/L of tumor necrosis factor α.
The preparation method of the above-mentioned DC cell culture medium that the present invention also proposes, room temperature be 20～25 DEG C, wet
Degree be 40～60%, be in the two stage biological safety cabinet of atmospheric pressure state, cultivate to 500mLRPMI-1640
Base adds after 5 μ g interleukin-4s are completely dissolved, stands 5min, add 0.1 μ g endothelial cell growth because of
After son is completely dissolved, stand 5min, be subsequently added into after 25mg transferrins is completely dissolved, stand 5min,
Continuously add after 25 μ g granulocyte-macrophage colony stimutaing factors are completely dissolved, stand 5min, then add
Enter after 10 μ g tumor necrosis factor αs are completely dissolved, stand 5min, then use Sodium Pyruvate regulation pH value
To 7.40, after the membrane filtration then using aperture to be 0.1 μm, obtain DC cell culture medium.
Embodiment 3 gained DC cell culture medium is phenol red liquid, and clarification is without muddy.
Example 3 gained DC cell culture medium is at 37 DEG C, 5%CO2Under the conditions of empty training 96h, outward appearance without
Significant change, has no muddy thing under microscope.
Example 3 gained DC cell culture medium is at 37 DEG C, 5%CO2Under the conditions of cultivate DC cell, 48h
Using cell counting and MTS method detection cytoactive, cell culture efficiency reaches more than 50%, has propagation and lives
The DC cell of property > 90%.
The above, the only present invention preferably detailed description of the invention, but protection scope of the present invention not office
Being limited to this, any those familiar with the art is in the technical scope that the invention discloses, according to this
The technical scheme of invention and inventive concept thereof in addition equivalent or change, all should contain the protection in the present invention
Within the scope of.
Claims (8)
1. a DC cell culture medium, it is characterised in that its component includes: RPMI-1640 culture medium,
Interleukin-4, endothelial cell growth factor (ECGF), transferrins, granulocyte-macrophage colony stimutaing factor and swollen
Tumor necrosis factor α.
DC cell culture medium the most according to claim 1, it is characterised in that the final concentration of interleukin-4
It is 5～20 μ g/L, final concentration of 0.1～0.5 μ g/L of endothelial cell growth factor (ECGF), the final concentration of transferrins
It is 30～100mg/L, final concentration of 20～100 μ g/L of granulocyte-macrophage colony stimutaing factor, swollen
The final concentration of 10～50 μ g/L of tumor necrosis factor α.
DC cell culture medium the most according to claim 1 or claim 2, it is characterised in that described DC cell
The pH value of culture medium is 7.35～7.40.
4. according to DC cell culture medium described in any one of claim 1-3, it is characterised in that interleukin-4
Final concentration of 10 μ g/L, the final concentration of 0.2 μ g/L of endothelial cell growth factor (ECGF), the final concentration of transferrins
For 50mg/L, the final concentration of 50 μ g/L of granulocyte-macrophage colony stimutaing factor, tumor necrosis factor α
Final concentration of 20 μ g/L.
5. a preparation method for DC cell culture medium as described in any one of claim 1-4, its feature exists
In, after addition interleukin-4 is completely dissolved in RPMI-1640 culture medium, stands, add endotheliocyte
After somatomedin is completely dissolved, stand, be subsequently added into after transferrins is completely dissolved, stand, continuously add
After granulocyte-macrophage colony stimutaing factor is completely dissolved, stands, be subsequently adding tumor necrosis factor α complete
After CL, standing, then regulate pH value, then membrane filtration obtains DC cell culture medium.
The preparation method of DC cell culture medium the most according to claim 5, it is characterised in that during standing
Between be 4～6min.
7. according to the preparation method of DC cell culture medium described in claim 5 or 6, it is characterised in that adopt
PH value is regulated with Sodium Pyruvate.
8. according to the preparation method of DC cell culture medium described in any one of claim 5-7, it is characterised in that
The aperture of filter membrane is 0.1 μm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109983116A (en) * | 2016-11-25 | 2019-07-05 | 株式会社日立制作所 | Cell culture culture medium and the cell culture apparatus and cell culture processes for using it |
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| CN105483085A (en) * | 2016-02-16 | 2016-04-13 | 广州赛莱拉干细胞科技股份有限公司 | DC cell culture method and culture medium |
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2016
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Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103173411A (en) * | 2013-04-08 | 2013-06-26 | 宁波高新区世纪开元生物技术有限公司 | Method and kit for preparing dendritic cells |
| CN104450615A (en) * | 2013-09-25 | 2015-03-25 | 深圳市北科生物科技有限公司 | Cell CTL (BiAT) with bispecific antibody as well as preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109983116A (en) * | 2016-11-25 | 2019-07-05 | 株式会社日立制作所 | Cell culture culture medium and the cell culture apparatus and cell culture processes for using it |
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