CN107043748A - The serum free medium of peripheral blood cells - Google Patents

The serum free medium of peripheral blood cells Download PDF

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Publication number
CN107043748A
CN107043748A CN201710216879.7A CN201710216879A CN107043748A CN 107043748 A CN107043748 A CN 107043748A CN 201710216879 A CN201710216879 A CN 201710216879A CN 107043748 A CN107043748 A CN 107043748A
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concentration
serum free
peripheral blood
free medium
blood cells
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CN201710216879.7A
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Inventor
颜学恒
苏恩筠
张智培
薛宜青
熊延狮
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Shanghai Xp Biomed Ltd
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Shanghai Xp Biomed Ltd
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Priority to CN201710216879.7A priority Critical patent/CN107043748A/en
Publication of CN107043748A publication Critical patent/CN107043748A/en
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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
    • 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
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • 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
    • 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
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced 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
    • 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)
    • 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
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/59—Lectins
    • 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
    • C12N2501/90—Polysaccharides
    • C12N2501/91—Heparin

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Hematology (AREA)
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  • Immunology (AREA)
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  • General Engineering & Computer Science (AREA)
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Abstract

The present invention relates to a kind of serum free medium of peripheral blood cells, it includes basal medium and addO-on therapy, wherein, the basal medium is the culture mediums of RPMI 1640；The addO-on therapy and its concentration are as follows：Interleukin 2,35 50mg/L；Heparin, 1 2g/L；Transferrins, 25 30mg/L；Biosynthetic human insulin, 20 30mg/L；M type phytohemagglutinins, 10 20mg/L；L glutamine, 7 10g/L；Sodium acid carbonate, 2 4g/L；The concentration of each addO-on therapy is on the basis of the cumulative volume of the serum free medium of the peripheral blood cells.Compared with prior art, the serum free medium of peripheral blood cells of the invention disclosure satisfy that leucocyte in peripheral blood cells is quick, substantial amounts of propagation demand, be particularly suitable for the culture of peripheral blood cells, with preferable cultivation effect and cell survival rate.

Description

The serum free medium of peripheral blood cells
Technical field
The present invention relates to a kind of serum free medium of peripheral blood cells, belong to technical field of cell culture.
Background technology
Cell culture has been widely used in the every field such as biology, medical science, new drug development, as most important basis One of science.
Human peripheral blood cell culture is widely used in medical inspection, especially applies in science of heredity, is important charge-sheet model skill One of art.
The culture medium of the peripheral blood cells generally used at present uses traditional culture medium containing cow's serum, but ox blood is aloof from politics and material pursuits Cost, and there is viral pollution risk, the research of cow's serum composition interference experiment, may all it cause inspection data inaccurate, therefore Serum free medium and free serum culture are a main trend of cell culture.
However, after employing serum free medium, which being added in the medium and is adapted to the blood that peripheral blood cells grow It is clear to substitute composition, it is those skilled in the art's technical issues that need to address.
The content of the invention
In view of the above mentioned problem and/or other problemses of correlation technique, one aspect of the present invention provide a kind of peripheral blood cells Serum free medium, it includes basal medium and addO-on therapy, wherein, the basal medium be RPMI 1640 cultivate Base；The addO-on therapy and its concentration are as follows：Interleukin 2,35-50mg/L；Heparin, 1-2g/L；Transferrins, 25- 30mg/L；Biosynthetic human insulin, 20-30mg/L；M type phytohemagglutinins, 10-20mg/L；Glu, 7-10g/L； Sodium acid carbonate, 2-4g/L；The concentration of each addO-on therapy is on the basis of the cumulative volume of the serum free medium of the peripheral blood cells.
It is preferred that, on the basis of the cumulative volume of the peripheral blood cells culture medium, the dry powder of the culture mediums of RPMI 1640 Measure as 16-18g/L.
It is preferred that, on the basis of the cumulative volume of the peripheral blood cells culture medium, the dry powder of the culture mediums of RPMI 1640 Measure as 16.8g/L.
It is preferred that, in the addO-on therapy, the concentration of the interleukin 2 is 42mg/L, the M types plant blood cell The concentration of agglutinin is 15.5mg/L.
It is preferred that, in the addO-on therapy, the concentration of the heparin is 1.26g/L, and the concentration of the transferrins is 28mg/L, the concentration of the biosynthetic human insulin is 27mg/L, and the concentration of the Glu is 8.5g/L, the bicarbonate The concentration of sodium is 3.2g/L.
It is preferred that, the addO-on therapy also includes gentamicin and streptomysin, and the concentration of the gentamicin is 2 × 105– 3.5×105IU/L, the concentration of the streptomysin is 2 × 105-4×105IU/L。
It is preferred that, the addO-on therapy also includes phenol red sodium, and the concentration of the phenol red sodium is 6-8mg/L.
It is preferred that, the pH value of the serum free medium is 7.2~7.6.
The serum free medium of the peripheral blood cells of the present invention, use based on the culture mediums of RPMI 1640 culture medium with And the combination of specific addO-on therapy, the interleukin 2 and M type plants blood cell that especially with the addition of certain concentration range coagulate Collect the combination of element, disclosure satisfy that the leucocyte in peripheral blood cells is quick, the demand of substantial amounts of propagation, be particularly suitable for periphery The culture of haemocyte, with preferable cultivation effect and cell survival rate.
Brief description of the drawings
Fig. 1 mutually detects picture for the cell division of application examples 1；
Fig. 2 mutually detects picture for the cell division of comparative example 1.
Embodiment
The present invention is further illustrated by the following examples, but the present invention is not limited to these specific embodiment parties Formula.
In one embodiment of the invention, a kind of serum free medium of peripheral blood cells, it includes basis Culture medium and addO-on therapy, wherein, the basal medium is the culture mediums of RPMI 1640；The addO-on therapy and its concentration are such as Under：Interleukin 2,35-50mg/L；Heparin, 1-2g/L；Transferrins, 25-30mg/L；Biosynthetic human insulin, 20- 30mg/L；M type phytohemagglutinins, 10-20mg/L；Glu, 7-10g/L；Sodium acid carbonate, 2-4g/L；Each addition The concentration of component is on the basis of the cumulative volume of the serum free medium of the peripheral blood cells.
The present inventor has found by substantial amounts of development test, when the serum free medium of peripheral blood cells is used The combination of culture medium and above-mentioned specific addO-on therapy based on the culture mediums of RPMI 1640, especially with the addition of specific dense The interleukin 2 of scope and the combination of M type phytohemagglutinins are spent, disclosure satisfy that the leucocyte in peripheral blood cells is fast The demand of fast, substantial amounts of propagation, is particularly suitable for the culture of peripheral blood cells, with preferable cultivation effect and cell survival Rate.
In a preferred embodiment of the invention, in the addO-on therapy, the concentration of the interleukin 2 is 42mg/L, the concentration of the M types phytohemagglutinin is 15.5mg/L.In another further preferred embodiment of the present invention In, in the addO-on therapy, the concentration of the heparin is 1.26g/L, and the concentration of the transferrins is 28mg/L, people's weight The concentration of group insulin is 27mg/L, and the concentration of the Glu is 8.5g/L, and the concentration of the sodium acid carbonate is 3.2g/ L；When these materials all meet above-mentioned concentration simultaneously, the culture effect of peripheral blood cells is more preferably.
Embodiment 1
The process for preparation of the serum free medium of the peripheral blood cells of embodiment 1 is as follows：
Step 1)：Aseptically the dry powder of RPMI 1640 of synthesis is weighed to be placed in after 168g on electronic balance and disappeared Stir acquisition basal medium in malicious container, plus after the dilution of 9L purified waters.
Step 2)：By addO-on therapy：32g sodium acid carbonates, 85g Glus, 420mg interleukin 2s, 12.6g Heparin, 280mg transferrins, 270mg biosynthetic human insulins, 155mg M types phytohemagglutinin, 2.5g gentamicin (phases When in 2.5 × 105) and 3g streptomysins are (equivalent to 3 × 10 IU/L5IU/L) and the phenol red sodium salts of 70mg sequentially add step 1) obtain In the basal medium obtained, fully mix.
Step 3)：Filtration sterilization, then pH value is adjusted to 7.4 ± 0.2, it is eventually adding purified water and adjusts volume to 10L；
Step 4)：After the completion of culture medium is prepared, filtration sterilization, packing, sealing finally refrigerate (- 20 DEG C of environment) standby.
Application examples 1
Human peripheral is gathered first, is then added to 0.25mL human peripherals in aseptic manipulation work table real equipped with 5mL In the blake bottle (Tissue Culture Flask of commercially available Corning brand T25 models) for applying the peripheral blood cells serum free medium of example 1 Mix, put 37 DEG C, 5%CO2Cultivated in incubator.
Effect data
Comparative example 1：Using the serum free medium of commercially available peripheral blood cells, incubation is identical with application examples 1, specifically Repeat no more.
The cell proliferation rate and cell survival rate of application examples 1 and comparative example 1 are detected respectively.
Application examples 1 and comparative example 1, in culture 1, after 2 and 3 days, separately sampled nutrient solution carries out the detection of cell concentration (every group detection three times, average), testing result is referring to table 1 below.
Table 1
On the other hand, the cell culture fluid of application examples 1 and comparative example 1 (after culture 3 days) is taken, cell division phase, inspection is detected Result is surveyed respectively referring to Fig. 1 and Fig. 2.
To sum up result can be seen that the cell proliferation rate of the serum free medium of the peripheral blood cells of the embodiment of the present invention All it is higher than the serum free medium of commercially available peripheral blood cells with survival rate, the split coil method of generation is also significantly more than commercially available periphery The serum free medium of haemocyte.
It should be understood that, although the present specification is described in terms of embodiments, but not each embodiment only includes one Individual independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art will should say Bright book is as an entirety, and the technical scheme in each embodiment may also be suitably combined to form those skilled in the art can With the other embodiment of understanding.
Those listed above is a series of to be described in detail only for feasibility embodiment of the invention specifically Bright, they simultaneously are not used to limit the scope of the invention, all equivalent implementations made without departing from skill spirit of the present invention Or change should be included in the scope of the protection.

Claims (8)

1. a kind of serum free medium of peripheral blood cells, it includes basal medium and addO-on therapy, wherein,
The basal medium is the culture mediums of RPMI 1640；
The addO-on therapy and its concentration are as follows：
Interleukin 2,35-50mg/L；
Heparin, 1-2g/L；
Transferrins, 25-30mg/L；
Biosynthetic human insulin, 20-30mg/L；
M type phytohemagglutinins, 10-20mg/L；
Glu, 7-10g/L；
Sodium acid carbonate, 2-4g/L；
The concentration of each addO-on therapy is on the basis of the cumulative volume of the serum free medium of the peripheral blood cells.
2. serum free medium as claimed in claim 1, it is characterised in that：
On the basis of the cumulative volume of the peripheral blood cells culture medium, the dry powder amount of the culture mediums of RPMI 1640 is 16-18g/ L。
3. serum free medium as claimed in claim 2, it is characterised in that：
On the basis of the cumulative volume of the peripheral blood cells culture medium, the dry powder amount of the culture mediums of RPMI 1640 is 16.8g/ L。
4. serum free medium as claimed in claim 2, it is characterised in that：
In the addO-on therapy, the concentration of the interleukin 2 is 42mg/L, the concentration of the M types phytohemagglutinin For 15.5mg/L.
5. serum free medium as claimed in claim 4, it is characterised in that：
In the addO-on therapy, the concentration of the heparin is 1.26g/L, and the concentration of the transferrins is 28mg/L, the people The concentration of Recombulin is 27mg/L, and the concentration of the Glu is 8.5g/L, and the concentration of the sodium acid carbonate is 3.2g/L。
6. the serum free medium as described in any one in claim 1 to 5, it is characterised in that：
The addO-on therapy also includes gentamicin and streptomysin, and the concentration of the gentamicin is 2 × 105–3.5×105IU/ L, the concentration of the streptomysin is 2 × 105-4×105IU/L。
7. the serum free medium as described in any one in claim 1 to 5, it is characterised in that：
The addO-on therapy also includes phenol red sodium, and the concentration of the phenol red sodium is 6-8mg/L.
8. serum free medium as claimed in claim 7, it is characterised in that：
The pH value of the serum free medium is 7.2~7.6.
CN201710216879.7A 2017-04-05 2017-04-05 The serum free medium of peripheral blood cells Pending CN107043748A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112795537A (en) * 2019-11-13 2021-05-14 苏州易迈吉生物医药科技有限公司 Serum-free medium for culturing human peripheral blood dendritic cells

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CN104830766A (en) * 2015-05-22 2015-08-12 广州和能生物科技有限公司 Serum-free human peripheral blood lymphocyte culture medium
CN105713873A (en) * 2016-04-20 2016-06-29 广东艾时代生物科技有限责任公司 Serum-free medium for vitro amplification culture of immune cells and application thereof
CN106047807A (en) * 2016-08-19 2016-10-26 上海逍鹏生物科技有限公司 Serum-free culture medium for high-density cell culture system of immune cells
CN106047806A (en) * 2016-08-19 2016-10-26 上海逍鹏生物科技有限公司 Peripheral blood cell culture medium for high-density cell culture system

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Publication number Priority date Publication date Assignee Title
CN104830766A (en) * 2015-05-22 2015-08-12 广州和能生物科技有限公司 Serum-free human peripheral blood lymphocyte culture medium
CN105713873A (en) * 2016-04-20 2016-06-29 广东艾时代生物科技有限责任公司 Serum-free medium for vitro amplification culture of immune cells and application thereof
CN106047807A (en) * 2016-08-19 2016-10-26 上海逍鹏生物科技有限公司 Serum-free culture medium for high-density cell culture system of immune cells
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112795537A (en) * 2019-11-13 2021-05-14 苏州易迈吉生物医药科技有限公司 Serum-free medium for culturing human peripheral blood dendritic cells

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Application publication date: 20170815