CN110904025A - Serum-free suspension domestication method of Vero cells - Google Patents

Serum-free suspension domestication method of Vero cells Download PDF

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Publication number
CN110904025A
CN110904025A CN201910950096.0A CN201910950096A CN110904025A CN 110904025 A CN110904025 A CN 110904025A CN 201910950096 A CN201910950096 A CN 201910950096A CN 110904025 A CN110904025 A CN 110904025A
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serum
suspension
free
vero
cells
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李智力
方芳
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Yuan Pei Biotech Inc Shanghai
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Yuan Pei Biotech Inc Shanghai
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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
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • 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

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Abstract

The present invention relates to the field of biology. A serum-free suspension domestication method of Vero cells comprises the following steps of firstly, culturing the Vero cells in an adherent manner until the confluence rate reaches 80%, discarding supernatant, and adding D-PBS (phosphate buffer solution) without calcium and magnesium ions for washing; step two, removing the D-PBS, adding pancreatin, removing pancreatin digestive juice, leaving a little pancreatin enough to cover the cells, incubating until the cells become round or incubating for a certain time, and then tapping the bottom of the bottle to dissociate the cells; step three, resuspending by using a Vero-S suspension serum-free culture medium; step four, discarding the supernatant, and resuspending the supernatant by using a Vero-S suspension serum-free culture medium; step five, diluting the cell density, and inoculating the diluted cell density into a disposable shake flask for culture; step six, when the cell density reaches 3 multiplied by 106cells/ml for cell passage; step seven, once the cell density of more than 10 continuous generations can reach 3 multiplied by 10 within 48 hours6cell/ml, cell survival rate is not less than 95%, and the cell is considered to be suitable for suspension culture.

Description

Serum-free suspension domestication method of Vero cells
Technical Field
The invention relates to the field of biology, in particular to cell culture.
Background
At present, adherent culture is mostly adopted for culturing Vero cells, but because the attached area provided by a culture system is limited, the proliferation and amplification quantity is limited, and the culture process is difficult to amplify.
Disclosure of Invention
The invention aims to provide a serum-free suspension domestication method of Vero cells so as to solve the technical problems.
The serum-free suspension domestication method of the Vero cells is characterized by comprising the following steps:
step one, culturing Vero cells in an adherent manner until the confluence rate reaches 80%, discarding supernatant, and adding D-PBS without calcium and magnesium ions for washing;
step two, removing D-PBS, adding pancreatin, shaking for a moment at room temperature, removing pancreatin digestive juice, leaving a little pancreatin enough to cover cells, incubating at 36-38 ℃ until the cells become round or incubating for a certain time, and then tapping the bottom of the bottle to dissociate the cells;
step three, resuspending by using a Vero-S suspension serum-free culture medium, and centrifuging for 3-6min at 1000 rpm;
step four, discarding the supernatant, and resuspending the supernatant by using a Vero-S suspension serum-free culture medium;
step five, diluting the cell density to 1 × 106cells/ml, inoculated into a disposable shake flask with a gas permeable lid, and then placed at 37 ℃ and 130rpm in 5% CO2Suspension culture of the shaking table;
step six, when the cell density reaches 3 multiplied by 10 in 48h6cells/ml, passage by dilution, cell density diluted to 1X 10 with fresh medium6cells/ml；
When the cell density is lower than 3X 10 in 48h6cells/ml, passaging by centrifugation, suspending the cells with fresh medium after centrifugation, at 1X 106cells/ml were inoculated at the density and the culture was continued.
Step seven, once the cell density of more than 10 continuous generations can reach 3 multiplied by 10 within 48 hours6cell/ml, cell survival rate is not less than 95%, and the cell is considered to be suitable for suspension culture.
Has the advantages that: by the method, the Vero cells capable of being cultured in a suspension manner can be obtained, the Vero cells cultured in the suspension manner are large in proliferation and amplification quantity, and the culture process is convenient to amplify.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below.
A serum-free suspension domestication method of Vero cells,
step one, culturing Vero cells in an adherent manner until the confluence rate reaches 80%, discarding supernatant, and adding D-PBS without calcium and magnesium ions for washing. In the first step, the adherent culture is carried out by adopting a disposable cell culture square bottle with the product number of T-75 produced by Shanghai culture Biotechnology GmbH. After discarding the supernatant, 5ml of D-PBS free of calcium and magnesium ions was added to wash 2 times. The D-PBS free of calcium and magnesium ions is preferably a D-PBS free of calcium and magnesium ions, having a product number of B210KJ, manufactured by Shanghai-derived Baek Biotech Co., Ltd.
And step two, removing the D-PBS, adding pancreatin, shaking for a moment at room temperature, removing pancreatin digestive juice, leaving a little pancreatin enough to cover the cells, incubating at 36-38 ℃ until the cells become round or incubating for a certain time, and then tapping the bottom of the bottle to dissociate the cells. The moment in the second step is preferably 15s, and the certain time is preferably 12-20 min. The incubation temperature is preferably 37 ℃. After discarding PBS, 5ml of pancreatin is preferably added. The pancreatin is preferably a 0.25% pancreatin-EDTA solution, and further preferably pancreatin having a product number of S310KJ, manufactured by Shanghai-culture Biotech, Inc.
And step three, resuspending the suspension by using a Vero-S suspension serum-free culture medium, and centrifuging the suspension for 3 to 6min at 1000 rpm. In the third step, the Vero-S suspension serum-free culture medium is preferably Vero-S suspension serum-free culture medium with the product number of H570KJ, which is produced by Shanghai culture Biotech Co. The amount of Vero-S suspension serum-free medium is preferably 10 ml. The centrifugation time is preferably 5 min.
Step four, discarding the supernatant, and resuspending the supernatant by using a Vero-S suspension serum-free culture medium; in the fourth step, the Vero-S suspension serum-free culture medium is preferably Vero-S suspension serum-free culture medium with the product number of H570KJ, which is produced by Shanghai culture Biotech Co. The amount of Vero-S suspension serum-free medium is preferably 5 ml. In the fourth step, the resuspended solution can be sampled, preferably 100 microliters, and the cell density and the survival rate can be counted.
Step five, diluting the cell density to 1 × 106cells/ml, inoculated into a disposable shake flask with a vented lid, and then placed at 37 ℃ with 130rpm 5% CO2The shaking table of (4) was cultured. In the fifth step, the disposable shake flask is preferably a disposable shake flask with a product number of Corning 125, manufactured by Shanghai-culture Biotechnology GmbH, and the culture volume is 25 ml. In the fifth step, 100 microliters can be sampled every day during the culture process, and the cell density and the survival rate are counted.
Step six, when the cell density reaches 3 multiplied by 10 in 48h6cells/ml, passage by dilution, cell density diluted to 1X 10 with fresh medium6cells/ml; when the cell density is lower than 3X 10 in 48h6cells/ml, passaging by centrifugation, suspending the cells with fresh medium after centrifugation, at 1X 106cells/ml were inoculated at the density and the culture was continued. In the sixth step, the fresh medium is preferably Vero-S suspension serum-free medium with the product number of H570KJ, manufactured by Shanghai culture Biotech Co.
Step seven, once the cell density of more than 10 continuous generations can reach 3 multiplied by 10 within 48 hours6cell/ml, cell survival rate is not less than 95%, and the cell is considered to be suitable for suspension culture.
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made without departing from the spirit and scope of the invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (10)

  1. A serum-free suspension domestication method of Vero cells is characterized in that:
    step one, culturing Vero cells in an adherent manner until the confluence rate reaches 80%, discarding supernatant, and adding D-PBS without calcium and magnesium ions for washing;
    step two, removing D-PBS, adding pancreatin, shaking for a moment at room temperature, removing pancreatin digestive juice, leaving a little pancreatin enough to cover cells, incubating at 36-38 ℃ until the cells become round or incubating for a certain time, and then tapping the bottom of the bottle to dissociate the cells;
    step three, resuspending by using a Vero-S suspension serum-free culture medium, and centrifuging for 3-6min at 1000 rpm;
    step four, discarding the supernatant, and resuspending the supernatant by using a Vero-S suspension serum-free culture medium;
    step five, diluting the cell density to 1 × 106cells/ml, inoculated into a disposable shaker with vented coverThe flask was then charged with 5% CO at 37 ℃ and 130rpm2Suspension culture in a shaking table;
    step six, when the cell density reaches 3 multiplied by 10 in 48h6cells/ml, passage by dilution, cell density diluted to 1X 10 with fresh medium6cells/ml; when the cell density is lower than 3X 10 in 48h6cells/ml, passaging by centrifugation, suspending the cells with fresh medium after centrifugation, at 1X 106Inoculating cells/ml, and continuously culturing;
    step seven, once the cell density of more than 10 continuous generations can reach 3 multiplied by 10 within 48 hours6cell/ml, cell survival rate is not less than 95%, and the cell is considered to be suitable for suspension culture.
  2. 2. The method for serum-free suspension acclimatization of Vero cells according to claim 1, which comprises: in the first step, the adherent culture is carried out by adopting a disposable cell culture square bottle with the product number of T-75 produced by Shanghai culture Biotechnology GmbH.
  3. 3. The method for serum-free suspension acclimatization of Vero cells according to claim 1, which comprises: in the first step, the D-PBS free of calcium and magnesium ions is a D-PBS free of calcium and magnesium ions, which is manufactured by Shanghai-source Baekey Biotech Co., Ltd and has a product number of B210 KJ.
  4. 4. The method for serum-free suspension acclimatization of Vero cells according to claim 1, which comprises: the moment in step two is 15 s.
  5. 5. The method for serum-free suspension acclimatization of Vero cells according to claim 1, which comprises: in the second step, pancreatin is pancreatin manufactured by Shanghai-source culture Biotechnology GmbH with a product number of S310 KJ.
  6. 6. The method for serum-free suspension acclimatization of Vero cells according to claim 1, which comprises: in the third step and the fourth step, the Vero-S suspension serum-free culture medium is a Vero-S suspension serum-free culture medium with the product number of H570KJ, which is produced by Shanghai culture Biotechnology GmbH.
  7. 7. The method for serum-free suspension acclimatization of Vero cells according to claim 6, which comprises: in the third step, the dosage of the Vero-S suspension serum-free medium is 10ml, and in the fourth step, the dosage of the Vero-S suspension serum-free medium is 5 ml.
  8. 8. The method for serum-free suspension acclimatization of Vero cells according to claim 1, which comprises: in the fifth step, the disposable shake flask is a product number Corning 125 disposable shake flask produced by Shanghai-source culture Biotechnology GmbH, and the culture volume is 25 ml.
  9. 9. The method for serum-free suspension acclimatization of Vero cells according to claim 1, which comprises: in the fourth step, the resuspended solution can be sampled, preferably 100 microliters, and the cell density and the survival rate can be counted.
  10. 10. The method for serum-free suspension acclimatization of Vero cells according to claim 1, which comprises: in the fifth step, 100 microliters can be sampled every day during the culture process, and the cell density and the survival rate are counted.
CN201910950096.0A 2019-10-08 2019-10-08 Serum-free suspension domestication method of Vero cells Pending CN110904025A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111718889A (en) * 2020-07-27 2020-09-29 华农（肇庆）生物产业技术研究院有限公司 Serum-free full-suspension domestication method of Sf9 cells

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111718889A (en) * 2020-07-27 2020-09-29 华农（肇庆）生物产业技术研究院有限公司 Serum-free full-suspension domestication method of Sf9 cells
CN111718889B (en) * 2020-07-27 2021-10-22 华农（肇庆）生物产业技术研究院有限公司 Serum-free full-suspension domestication method of Sf9 cells

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Application publication date: 20200324