CN105969729A - T cell culture medium and preparation method thereof - Google Patents

T cell culture medium and preparation method thereof Download PDF

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Publication number
CN105969729A
CN105969729A CN201610482947.XA CN201610482947A CN105969729A CN 105969729 A CN105969729 A CN 105969729A CN 201610482947 A CN201610482947 A CN 201610482947A CN 105969729 A CN105969729 A CN 105969729A
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China
Prior art keywords
culture medium
final concentration
cell culture
cholesterol
completely dissolved
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Pending
Application number
CN201610482947.XA
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Chinese (zh)
Inventor
丁贵鹏
冯振卿
许国贞
刘振云
唐奇
朱进
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Sinobioway Cell Therapy Co Ltd
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Sinobioway Cell Therapy Co Ltd
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Priority to CN201610482947.XA priority Critical patent/CN105969729A/en
Publication of CN105969729A publication Critical patent/CN105969729A/en
Pending legal-status Critical Current

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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
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • 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
    • 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
    • C12N2500/36—Lipids
    • 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
    • C12N2501/10—Growth factors
    • C12N2501/165—Vascular endothelial growth factor [VEGF]
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)

Abstract

The invention discloses a T cell culture medium. The T cell culture medium comprises an RPMI-1640 culture medium, interleukin-2, an endothelial cell growth factor, cholesterol, triglyceride and transferrin, wherein the final concentration of the interleukin-2 is 2-10 microgram/L, the final concentration of the endothelial cell growth factor is 0.1-0.5 microgram/L, the final concentration of the cholesterol is 30-100mg/L, the final concentration of the triglyceride is 80-200mg/L and the final concentration of the transferrin is 30-100mg/L. The invention further discloses a preparation method of the T cell culture medium. The preparation method includes: sequentially adding the interleukin-2, the endothelial cell growth factor, the cholesterol, the triglyceride and the transferrin into the RPMI-1640 culture medium, wherein the next component is added after the previous component is completely dissolved and standing is performed; regulating pH, and filtering through a filter membrane to obtain the T cell culture medium.

Description

A kind of T cell culture medium and preparation method thereof
Technical field
The present invention relates to culture medium technical field, particularly relate to a kind of T cell culture medium and preparation method thereof.
Background technology
T cell cultivates multiplex RPMI-1640 or DMEM culture medium at present, but due to T cell self Multiplication capacity is not strong, and simple RPMI-1640 or DMEM culture medium cannot improve T cell growth must The nutritional labeling of palpus, cultivates the multiplex RPMI-1640 containing 10～20wt% hyclones of T cell the most at present Or DMEM culture medium.But use hyclone to cultivate T 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 in hyclone Complicated component, these become branch to have a strong impact on accuracy and the reliability of subsequent experimental, culture medium repeatability Difference, culture efficiency is only 15～20%.And owing to the clinical cell product used is forbidden to remain animal blood Albumin, and conventional medium exists hyclone, technique needs increase Ox blood serum in cell manufactured goods Vestiges of protein detects, and adds production difficulty and cost, promotes people to proceed by opening of serum-free medium Send out.
Therefore at present T 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 T cell culture medium and preparation thereof Method, improves T cell culture efficiency, increases cytoactive, it is ensured that the repeatability of subsequent experimental, fall The low use cost of T cell culture medium, and avoid the albuminised incorporation of animal blood, save T cell The arrangement step of culture medium and cost.
A kind of T cell culture medium that the present invention proposes, its component includes: RPMI-1640 culture medium, Bai Jie Element-2, endothelial cell growth factor (ECGF), cholesterol, triglyceride and transferrins.
Preferably, final concentration of 2～10 μ g/L of interleukin-2, endothelial cell growth factor (ECGF) final concentration of 0.1～0.5 μ g/L, final concentration of the 30 of cholesterol～100mg/L, triglyceride final concentration of 80～ 200mg/L, final concentration of the 30 of transferrins～100mg/L.
Preferably, the pH value of described T cell culture medium is 7.35～7.40.
Preferably, the final concentration of 5 μ g/L of interleukin-2, the final concentration of 0.2 μ g/L of endothelial cell growth factor (ECGF), The final concentration of 50mg/L of cholesterol, the final concentration of 100mg/L of triglyceride, the final concentration of transferrins For 50mg/L.
The preparation method of the above-mentioned T cell culture medium that the present invention also proposes, adds in RPMI-1640 culture medium Enter after interleukin-2 is completely dissolved, stand, add after endothelial cell growth factor (ECGF) is completely dissolved, stand, It is subsequently added into after cholesterol is completely dissolved, stands, continuously add after triglyceride is completely dissolved, stand, so After rear addition transferrins is completely dissolved, standing, then regulate pH value, then membrane filtration obtains T cell Culture medium.
Preferably, time of repose is 4～6min.
Preferably, Sodium Pyruvate regulation pH 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 adds multiple nutrients material on the basis of RPMI-1640 culture medium innovatively, including Interleukin-2, endothelial cell growth factor (ECGF), cholesterol, triglyceride and transferrins, make in gained culture medium Various constituent concentrations fix, improve T cell culture efficiency, increase cytoactive, it is ensured that subsequent experimental Repeatability, reduce the use cost of T cell culture medium, and avoid the albuminised incorporation of animal blood, Save arrangement step and the cost of T cell culture medium.
Detailed description of the invention
Below, by specific embodiment, technical scheme is described in detail.
Embodiment 1
A kind of T cell culture medium that the present invention proposes, its component includes: RPMI-1640 culture medium, Bai Jie Element-2, endothelial cell growth factor (ECGF), cholesterol, triglyceride and transferrins；Wherein the end of interleukin-2 is dense Degree is 2 μ g/L, final concentration of 0.5 μ g/L, the final concentration of 30mg/L of cholesterol of endothelial cell growth factor (ECGF), The final concentration of 200mg/L of triglyceride, the final concentration of 30mg/L of transferrins.
The preparation method of the above-mentioned T cell culture medium that the present invention also proposes, adds in RPMI-1640 culture medium Enter after interleukin-2 is completely dissolved, stand 4min, add after endothelial cell growth factor (ECGF) is completely dissolved, quiet Put 6min, be subsequently added into after cholesterol is completely dissolved, stand 4min, continuously add triglyceride and be completely dissolved After, stand 6min, be subsequently adding after transferrins is completely dissolved, stand 4min, then use Sodium Pyruvate Regulation pH value is to 7.38, and the membrane filtration then using aperture to be 0.1 μm obtains T cell culture medium.
Embodiment 2
A kind of T cell culture medium that the present invention proposes, its component includes: RPMI-1640 culture medium, Bai Jie Element-2, endothelial cell growth factor (ECGF), cholesterol, triglyceride and transferrins；Wherein the end of interleukin-2 is dense Degree is 10 μ g/L, the final concentration of 0.1 μ g/L of endothelial cell growth factor (ECGF), cholesterol final concentration of 100mg/L, the final concentration of 80mg/L of triglyceride, the final concentration of 100mg/L of transferrins.
The preparation method of the above-mentioned T cell culture medium that the present invention also proposes, adds in RPMI-1640 culture medium Enter after interleukin-2 is completely dissolved, stand 6min, add after endothelial cell growth factor (ECGF) is completely dissolved, quiet Put 4min, be subsequently added into after cholesterol is completely dissolved, stand 6min, continuously add triglyceride and be completely dissolved After, stand 4min, be subsequently adding after transferrins is completely dissolved, stand 6min, then use Sodium Pyruvate Regulation pH value is to 7.35, and then membrane filtration obtains T cell culture medium.
Embodiment 3
A kind of T cell culture medium that the present invention proposes, its component includes: RPMI-1640 culture medium, Bai Jie Element-2, endothelial cell growth factor (ECGF), cholesterol, triglyceride and transferrins；Wherein the end of interleukin-2 is dense Degree is 5 μ g/L, final concentration of 0.2 μ g/L, the final concentration of 50mg/L of cholesterol of endothelial cell growth factor (ECGF), The final concentration of 100mg/L of triglyceride, the final concentration of 50mg/L of transferrins.
The preparation method of the above-mentioned T cell culture medium that the present invention also proposes, is 20～25 DEG C, humidity in room temperature Be 40～60%, be in the two stage biological safety cabinet of atmospheric pressure state, to 500mLRPMI-1640 culture medium After middle addition 2.5 μ g interleukin-2 is completely dissolved, stand 5min, add 0.1 μ g endothelial cell growth because of After son is completely dissolved, stand 5min, be subsequently added into after 25mg cholesterol is completely dissolved, stand 5min, continue After continuous addition 50mg triglyceride is completely dissolved, stands 5min, be subsequently adding 25mg transferrins the most molten Xie Hou, stands 5min, then uses Sodium Pyruvate regulation pH value to 7.40, and then using aperture is 0.1 μm Membrane filtration remove all microorganisms obtain T cell culture medium.
Embodiment 3 gained T cell culture medium is phenol red liquid, and clarification is without muddy.
Example 3 gained T cell culture medium is at 37 DEG C, 5%CO2Under the conditions of empty training 96h, outward appearance is without bright Aobvious change, has no muddy thing under microscope.
Example 3 gained T cell culture medium is at 37 DEG C, 5%CO2Under the conditions of cultivate T cell, 48h adopts Detecting cytoactive by cell counting and MTS method, cell culture efficiency reaches more than 50%, has proliferation activity T cell > 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 T cell culture medium, it is characterised in that its component includes: RPMI-1640 culture medium, white Interleukin-2, endothelial cell growth factor (ECGF), cholesterol, triglyceride and transferrins.
T cell culture medium the most according to claim 1, it is characterised in that interleukin-2 final concentration of 2～10 μ g/L, final concentration of 0.1～0.5 μ g/L of endothelial cell growth factor (ECGF), cholesterol final concentration of 30～ 100mg/L, final concentration of the 80 of triglyceride～200mg/L, transferrins final concentration of 30～ 100mg/L。
T cell culture medium the most according to claim 1 or claim 2, it is characterised in that described T cell is trained The pH value supporting base is 7.35～7.40.
4. according to T cell culture medium described in any one of claim 1-3, it is characterised in that interleukin-2 Final concentration of 5 μ g/L, the final concentration of 0.2 μ g/L of endothelial cell growth factor (ECGF), cholesterol final concentration of 50mg/L, the final concentration of 100mg/L of triglyceride, the final concentration of 50mg/L of transferrins.
5. the preparation method of T cell culture medium as described in any one of claim 1-4, it is characterised in that After addition interleukin-2 is completely dissolved in RPMI-1640 culture medium, stands, add endothelial cell growth After the factor is completely dissolved, stand, be subsequently added into after cholesterol is completely dissolved, stand, continuously add glycerol three After ester is completely dissolved, stand, be subsequently adding after transferrins is completely dissolved, stand, then regulate pH value, Then membrane filtration obtains T cell culture medium.
The preparation method of T cell culture medium the most according to claim 5, it is characterised in that time of repose It is 4～6min.
7. according to the preparation method of T cell culture medium described in claim 5 or 6, it is characterised in that use Sodium Pyruvate regulation pH.
8. according to the preparation method of T 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.
CN201610482947.XA 2016-06-24 2016-06-24 T cell culture medium and preparation method thereof Pending CN105969729A (en)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1566331A (en) * 2003-06-27 2005-01-19 北京天润善达生物科技有限责任公司 Serum-free culture medium for lymphocyte
CN102816735A (en) * 2012-09-12 2012-12-12 山东省齐鲁细胞治疗工程技术有限公司 Method for culturing autologous peripheral blood lymphocytes
CN103013914A (en) * 2012-12-13 2013-04-03 吉林省拓华生物科技有限公司 Method for in-vitro culture of killer T cells
CN103146648A (en) * 2013-03-14 2013-06-12 北京京蒙高科干细胞技术有限公司 Animal source-free and serum-free culture medium of lymphocyte
CN104818248A (en) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 Immunocyte culture medium, and culture method and application of immunocytes
CN104946588A (en) * 2015-07-07 2015-09-30 英普乐孚生物技术(上海)有限公司 Separation and efficient amplification culture method for antigen specific T lymphocyte

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1566331A (en) * 2003-06-27 2005-01-19 北京天润善达生物科技有限责任公司 Serum-free culture medium for lymphocyte
CN102816735A (en) * 2012-09-12 2012-12-12 山东省齐鲁细胞治疗工程技术有限公司 Method for culturing autologous peripheral blood lymphocytes
CN103013914A (en) * 2012-12-13 2013-04-03 吉林省拓华生物科技有限公司 Method for in-vitro culture of killer T cells
CN103146648A (en) * 2013-03-14 2013-06-12 北京京蒙高科干细胞技术有限公司 Animal source-free and serum-free culture medium of lymphocyte
CN104818248A (en) * 2015-03-25 2015-08-05 苏州佰通生物科技有限公司 Immunocyte culture medium, and culture method and application of immunocytes
CN104946588A (en) * 2015-07-07 2015-09-30 英普乐孚生物技术(上海)有限公司 Separation and efficient amplification culture method for antigen specific T lymphocyte

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Application publication date: 20160928