CN106047806B - A kind of peripheral blood cells culture medium suitable for concentration cultivation system - Google Patents

A kind of peripheral blood cells culture medium suitable for concentration cultivation system Download PDF

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CN106047806B
CN106047806B CN201610693937.0A CN201610693937A CN106047806B CN 106047806 B CN106047806 B CN 106047806B CN 201610693937 A CN201610693937 A CN 201610693937A CN 106047806 B CN106047806 B CN 106047806B
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peripheral blood
blood cells
culture medium
addo
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CN106047806A (en
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颜学恒
周艳
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Shanghai Xp Biomed Ltd
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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
    • C12N2500/30—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
    • C12N2500/30—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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2501/998—Proteins not provided for elsewhere

Abstract

The present invention relates to a kind of peripheral blood cells culture mediums suitable for concentration cultivation system comprising basal medium and addO-on therapy, wherein basal medium is 1640 culture medium of RMPI, and addO-on therapy and its concentration are as follows: cow's serum, 160-220ml/L；PHA- phytohemagglutinin, 16-30g/L；L-Glutamine, 8-12g/L；Glutathione, 8-14g/L；Linoleic acid, 0.1-0.3g/L；Lipoic acid, 0.1-0.3g/L；The concentration of each addO-on therapy is on the basis of the total volume of peripheral blood cells culture medium.Compared with prior art, peripheral blood cells culture medium of the invention enables to peripheral blood cells quickly, to be largely proliferated, and cultivation effect is good, and incubation time is short, is particularly suitable for the concentration cultivation system of peripheral blood cells.

Description

A kind of peripheral blood cells culture medium suitable for concentration cultivation system
Technical field
The present invention relates to a kind of peripheral blood cells culture mediums suitable for concentration cultivation system, belong to cell culture Technical field.
Background technique
Cell culture has been widely used in the every field such as biology, medicine, new drug development, becomes most important basis One of science.
About Cell culture invitro system, in the prior art in addition to conventional culture plate, culture dish, culture bottle etc. are two-dimensional Cell culture system has been developed that dimensional culture system, such as presently commercially available concentration cultivation bucket now, be it is a kind of both It can be adapted for attached cell, and can be adapted for the dimensional culture system of suspension cell, can rapidly cultivate a large amount of cells, And long term cell culture more than half a year by a definite date can be carried out, it does not need often to replace consumptive material, with traditional culture plate, culture dish Compared with culture bottle, be more convenient, cost it is lower, therefore the extensive concern by this field research staff.
However, traditional peripheral blood cells culture medium in the prior art is suitable for medium scale cell culture body mostly System, in general can not be suitable for large-scale, highdensity cell culture；On the other hand, existing serum free medium is only Suitable for the two-dimensional cell culture system such as culture plate, culture dish, culture bottle, concentration cultivation bucket can not be suitable in this way Dimensional culture system.
The peripheral blood cells culture medium for being exclusively used in concentration cultivation system is urgently developed now.
Summary of the invention
In view of the above problem and/or other problems of the relevant technologies, one aspect of the present invention provides a kind of suitable for highly dense Spend the peripheral blood cells culture medium of cell culture system comprising basal medium and addO-on therapy, wherein the basis culture Base is 1640 culture medium of RMPI；The addO-on therapy and its concentration are as follows: cow's serum, 160-220ml/L；PHA- plant blood cell Agglutinin, 16-30g/L；L-Glutamine, 8-12g/L；Glutathione, 8-14g/L；Linoleic acid, 0.1-0.3g/L；Lipoic acid, 0.1-0.3g/L；The concentration of each addO-on therapy is on the basis of the total volume of the peripheral blood cells culture medium.
Preferably, on the basis of the total volume of the peripheral blood cells culture medium, the dry powder of 1640 culture medium of RMPI Amount is 10-15g/L.It is furthermore preferred that the RMPI 1640 is cultivated on the basis of the total volume of the peripheral blood cells culture medium The dry powder amount of base is 13.6g/L.
Preferably, in the addO-on therapy, the concentration of the cow's serum is 200ml/L, the PHA- plant hemagglutination The concentration of element is 24g/L, and the concentration of the L-Glutamine is 11g/L, and the concentration of the glutathione is 12g/L.
Preferably, in the addO-on therapy, the linoleic concentration is 0.2g/L, and the concentration of the lipoic acid is 0.2g/L。
Preferably, the addO-on therapy further includes penicillin and streptomysin, and the concentration of the penicillin is 1 × 105-4× 105IU/L, the concentration of the streptomysin are 1 × 105-4×105IU/L。
Preferably, the addO-on therapy further includes phenol red sodium, and the concentration of the phenol red sodium is 5-7mg/L.
Preferably, the pH value of the peripheral blood cells culture medium is 7.3~7.5.
Another aspect of the present invention provides above-mentioned peripheral blood cells culture medium answering in concentration cultivation system With.
Preferably, the concentration cultivation system is concentration cultivation bucket.
Peripheral blood cells culture medium suitable for concentration cultivation system of the invention, using RMPI1640 culture medium As the combination of basic culture medium and specific addO-on therapy, especially cow's serum, PHA- phytohemagglutinin, L- paddy ammonia Amide and glutathione, four when meeting above-mentioned concentration range simultaneously, enable to peripheral blood cells quickly, a large amount of increase It grows, is particularly suitable for the concentration cultivation system of peripheral blood cells.
Specific 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 peripheral blood cells suitable for concentration cultivation system Culture medium comprising basal medium and addO-on therapy, wherein the basal medium is 1640 culture medium of RMPI；It is described to add Add component and its concentration as follows: cow's serum, 160-220ml/L；PHA- phytohemagglutinin, 16-30g/L；L-Glutamine, 8-12g/L；Glutathione, 8-14g/L；Linoleic acid, 0.1-0.3g/L；Lipoic acid, 0.1-0.3g/L；The concentration of each addO-on therapy On the basis of the total volume of the peripheral blood cells culture medium.
The present inventor has found by a large amount of development test, when peripheral blood cells culture medium uses RMPI 1640 Combination of the culture medium as basic culture medium and above-mentioned specific addO-on therapy, especially cow's serum, PHA- plant blood cell are solidifying Collection element, L-Glutamine and glutathione, four when meeting above-mentioned concentration range simultaneously, enables to peripheral blood cells fast Speed, a large amount of proliferation, are particularly suitable for the concentration cultivation system of peripheral blood cells.
In a preferred embodiment of the invention, on the basis of the total volume of the peripheral blood cells culture medium, institute The dry powder amount for stating 1640 culture medium of RMPI is 10-15g/L.
In a further preferred embodiment of the invention, the dry powder amount of 1640 culture medium of RMPI is 13.6g/ L。
In another further preferred embodiment of the invention, in the addO-on therapy, the concentration of the cow's serum is 200ml/L, the concentration of the PHA- phytohemagglutinin are 24g/L, and the concentration of the L-Glutamine is 11g/L, and institute The concentration for stating glutathione is 12g/L.In peripheral blood cells culture medium of the invention, the concentration of 1640 culture medium of RMPI, with And cow's serum, PHA- phytohemagglutinin, L-Glutamine and glutathione simultaneously using above-mentioned specific concentration when, meet The nutritional need of the High Density Cultivation of peripheral blood cells is particularly suitable for the concentration cultivation system of peripheral blood cells, especially It is the dimensional culture system of such as concentration cultivation bucket etc, there is preferable cultivation effect.
In a preferred embodiment in accordance with this invention, in the addO-on therapy, the linoleic concentration is 0.2g/ L, the concentration of the lipoic acid are 0.2g/L.The combination of both substances and certain concentration plays the growth of peripheral blood cells The survival rate of peripheral blood cells can be increased particularly with the High Density Cultivation system of peripheral blood cells to protective effect.
In a preferred embodiment of the invention, the addO-on therapy can be with antibiotic, for example including penicillin And streptomysin；The concentration of the penicillin is 1 × 105-4×105IU/L, the streptomysin concentration are 1 × 105-4× 105IU/L.It is furthermore preferred that the concentration of penicillin is 2 × 105IU/L, streptomysin concentration are 2 × 105IU/L.Both antibiotic Combination and its specific concentration are particularly suitable for the concentration cultivation system of peripheral blood cells, and antibacterial effect is excellent, and cell is deposited Motility rate is high.
In a preferred embodiment in accordance with this invention, the addO-on therapy further includes phenol red sodium, the phenol red sodium Concentration is 5-7mg/L.
In a preferred embodiment of the invention, the pH value of the serum free medium is controlled 7.3~7.5.
In a preferred embodiment of the invention, peripheral cells culture medium of the invention is to be exclusively used in high-density cells Cultivate the peripheral cells culture medium of bucket.
Embodiment 1
The process for preparation of the peripheral blood cells culture medium suitable for concentration cultivation system of embodiment 1 is as follows:
Step 1): aseptically the RPMI1640 dry powder of synthesis is weighed into 136g on an electronic balance and is placed on disinfection In container, acquisition basal medium is stirred evenly after adding 7.5L redistilled water to dilute；
Step 2): by addO-on therapy: 2L cow's serum, 240g PHA- phytohemagglutinin, 110g L-Glutamine, 120g glutathione, 1.2g penicillin (are equivalent to 2 × 106IU), 2g streptomysin (is equivalent to 2 × 106IU), 2g linoleic acid, 2g sulphur Octanoic acid and the phenol red sodium of 60mg sequentially add in the basal medium of step 1) acquisition, mix well；
Step 3): filtration sterilization, then pH value is adjusted to 7.4 ± 0.1, redistilled water, which is eventually adding, by volume is adjusted to 10L；
Step 4): after the completion of culture medium is prepared, filtration sterilization, it is spare finally to refrigerate (- 20 DEG C of environment) for packing, sealing.
Embodiment 2
The process for preparation of the peripheral blood cells culture medium suitable for concentration cultivation system of embodiment 2 is as follows:
Step 1): aseptically the RPMI1640 dry powder of synthesis is weighed into 140g on an electronic balance and is placed on disinfection In container, acquisition basal medium is stirred evenly after adding 7.5L redistilled water to dilute；
Step 2): by addO-on therapy: 1.7L cow's serum, 260g PHA- phytohemagglutinin, 100g L-Glutamine, 110g glutathione, 2g penicillin (are equivalent to 3.3 × 106IU), 3g streptomysin (is equivalent to 3 × 106IU), 1.8g linoleic acid, 1.8g lipoic acid and the phenol red sodium of 66mg sequentially add in the basal medium of step 1) acquisition, mix well；
Step 3): filtration sterilization, then pH value is adjusted to 7.4 ± 0.1, redistilled water, which is eventually adding, by volume is adjusted to 10L；
Step 4): after the completion of culture medium is prepared, filtration sterilization, it is spare finally to refrigerate (- 20 DEG C of environment) for packing, sealing.
Application examples 1
It disinfects finger skin in alcohol, with blood-taking needle broken skin skin, acquires peripheral blood with the hollow heparin tube containing heparin sodium, Mixing is jiggled, 5ml peripheral blood is added in the peripheral blood cells culture medium obtained to above-described embodiment 1, and by peripheral blood Cell concentration is diluted to 1.2 × 106A/ml.
Using the concentration cultivation bucket of commercially available FiberCell brand C2025 model (referring to FiberCell cell Culture systems product sell webpage) and the embodiment of the present invention 1 obtain peripheral blood cells culture medium cultivated.
It is 1.2 × 10 by 5ml peripheral blood cells concentration6It is thin that the culture medium of a/ml with syringe is loaded into above-mentioned high density Born of the same parents cultivate in bucket, then 25ml Peripheral blood culture base are added to (peripheral blood cells in 50ml concentration cultivation bucket liquid storage bottle Inoculum density be 2 × 105A/ml)；Specifically, the culture medium in liquid storage bottle is connect by silicone tube with cell culture bucket, Under the action of peristaltic pump, persistent loop flowing, culture medium is swapped by the doughnut of cell culture bucket, gives cell culture Cell in bucket constantly provides nutriment；Entire concentration cultivation bucket is put into 5%CO2, in 37 DEG C of incubator Culture.
Application examples 2: detailed process is identical as application examples 1, and difference is the peripheral blood cells culture obtained using embodiment 2 The culture of base progress peripheral blood cells.
Effect data
Comparative example 1: using the concentration cultivation bucket of commercially available FiberCell brand C2025 model and commercially available The 1640 peripheral blood cells culture medium of RPMI of Qingdao Lay Buddhist brand, incubation is identical as application examples 1, specifically repeats no more.
Comparative example A: using the Tissue Culture Flask and commercially available Qingdao Lay Buddhist product of commercially available Corning brand T25 model The 1640 peripheral blood cells culture medium of RPMI of board, the peripheral blood cells culture medium of 5ml Lay Buddhist brand is transferred to pipette In T25 culture bottle, adding 1ml peripheral blood cells concentration is 1.2 × 106(inoculation of peripheral blood cells is close for the culture medium of a/ml Degree is 2 × 105A/ml) suspension culture is carried out, culture bottle is put into 5%CO2, cultivate 72 hours in 37 DEG C of incubator.
The cell proliferation rate and cell survival rate of application examples 1, application examples 2, comparative example 1 and comparative example A are detected respectively.
Detect cell proliferation rate
Application examples 1 and application examples 2 and comparative example 1 and comparative example A, after culture 1,3 and 6 day, separately sampled culture Liquid carries out the detection of cell concentration, and testing result is referring to the following table 1.
Table 1
Detect cell survival rate
After culture 6 days, the culture solution of each group is collected, carries out flow cytometer detection.Testing result is as follows:
The cell survival rate of application examples 1: 98.4%；The cell survival rate of application examples 2: 96.7%；The cell of comparative example 1 is deposited Motility rate: 84.2%；The cell survival rate of comparative example A: 88.6%.
Result in table 1, which is compared, can be seen that, on the one hand, the cell proliferation rate and cell of comparative example 1 are deposited The result of motility rate (cell culture effect) be slightly inferior to comparative example A's as a result, this illustrate: conventional medium is more suitable for such as cell The small-scale cultivating system of tradition of bottle etc, is not suitable for concentration cultivation system；On the other hand, application examples 1 of the present invention Cell proliferation rate and cell survival rate are much higher than comparative example 1, this explanation: the peripheral blood cells culture medium of the embodiment of the present invention Compared to conventional RMPI peripheral blood cells culture medium more suitable for concentration cultivation system.
It should be appreciated that although this specification is described in terms of embodiments, but not each embodiment only includes one A independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should will say As a whole, the technical solution in each embodiment may also be suitably combined to form those skilled in the art can for bright book With the other embodiments of understanding.
The series of detailed descriptions listed above only for feasible embodiment of the invention specifically Protection scope bright, that they are not intended to limit the invention, it is all without departing from equivalent implementations made by technical spirit of the present invention Or change should all be included in the protection scope of the present invention.

Claims (8)

1. a kind of peripheral blood cells culture medium suitable for concentration cultivation system comprising basal medium and addition group Point, wherein
The basal medium is 1640 culture medium of RMPI；
The addO-on therapy and its concentration are as follows:
Cow's serum, 170-200ml/L；
PHA- phytohemagglutinin, 24-26g/L；
L-Glutamine, 10-11g/L；
Glutathione, 11-12g/L；
Linoleic acid, 0.18-0.2g/L；
Lipoic acid, 0.18-0.2g/L；
The concentration of each addO-on therapy is on the basis of the total volume of the peripheral blood cells culture medium；
On the basis of the total volume of the peripheral blood cells culture medium, the dry powder amount of 1640 culture medium of RMPI is 10-15g/ L；
The pH value of the peripheral blood cells culture medium is 7.3~7.5.
2. peripheral blood cells culture medium as described in claim 1, it is characterised in that:
On the basis of the total volume of the peripheral blood cells culture medium, the dry powder amount of 1640 culture medium of RMPI is 13.6g/ L。
3. peripheral blood cells culture medium as described in claim 1, it is characterised in that:
In the addO-on therapy, the concentration of the cow's serum is 200ml/L, and the concentration of the PHA- phytohemagglutinin is 24g/L, the concentration of the L-Glutamine is 11g/L, and the concentration of the glutathione is 12g/L.
4. peripheral blood cells culture medium as described in claim 1, it is characterised in that:
In the addO-on therapy, the linoleic concentration is 0.2g/L, and the concentration of the lipoic acid is 0.2g/L.
5. the peripheral blood cells culture medium as described in any one of Claims 1-4, it is characterised in that:
The addO-on therapy further includes penicillin and streptomysin, and the concentration of the penicillin is 1 × 105-4×105IU/L, it is described The concentration of streptomysin is 1 × 105-4×105IU/L。
6. the peripheral blood cells culture medium as described in any one of Claims 1-4, it is characterised in that:
The addO-on therapy further includes phenol red sodium, and the concentration of the phenol red sodium is 5-7mg/L.
7. the peripheral blood cells culture medium as described in any one of claim 1 to 6 is in the high density for peripheral blood cells Application in cell culture system.
8. application as claimed in claim 7, it is characterised in that: the concentration cultivation system is concentration cultivation Bucket.
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