CN105176916A - Low-serum protein-free culture medium applicable to Vero cell growth and preparation method thereof - Google Patents
Low-serum protein-free culture medium applicable to Vero cell growth and preparation method thereof Download PDFInfo
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- CN105176916A CN105176916A CN201510664916.1A CN201510664916A CN105176916A CN 105176916 A CN105176916 A CN 105176916A CN 201510664916 A CN201510664916 A CN 201510664916A CN 105176916 A CN105176916 A CN 105176916A
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Abstract
The invention belongs to the field of cell culture and particularly relates to a low-serum protein-free culture medium applicable to Vero cell growth and a preparation method thereof. The culture medium comprises amino acid, vitamin, inorganic salt, micro elements and other ingredients. The low-serum protein-free culture medium applicable to Vero cell growth and the preparation method of the low-serum protein-free culture medium have the advantages of being free of protein and animal source ingredients and definite in chemical ingredient, the interference to cell culture of serum is industrially reduced, the production cost is lowered, and great convenience is brought to industrial production of cells.
Description
Technical field
The invention belongs to field of cell culture, be specifically related to a kind of low serum protein-free medium being suitable for Vero Growth of Cells and preparation method thereof.
Background technology
Vero cell is African green monkey kidney cell, is a kind of heteroploid cell, produces after the derivation of RhMK cell cultures.This clone is isolated and cultured in 1962 by scholar Y.Yasumura and Y.Kawakita of Chiba, Japan university from the kidney epithelia cell of cercopithecus aethiops.Vero cell is the clone of the production vaccine of the World Health Organization and China's biological products code accreditation, convenient sources, biological safety is high, can continuous passage in vitro, to multiple virus is responsive and virus multiplication titre is high, and be the desirable cell matrix of producing vaccine for man by world health organisation recommendations.
The current mainly adherent culture mode of production technique of Vero cell, needs the bovine serum adding about 10% in traditional substratum, with the support albumen providing the nutritive ingredient needed for Growth of Cells and cell to keep normal morphology.But the chemical composition contained by serum is indefinite, also mass discrepancy is there is between each batch, the stdn of experiment and serialization are restricted, also easily bring the biological pollutions such as bacterium, fungi, virus and mycoplasma, the protein of a large amount of complicated component is had in serum, very large difficulty can be brought to the separation and purification of the biological products such as vaccine, cytokine and monoclonal antibody, the more important thing is the expensive of serum, source difficulty.Therefore become the development trend of Vero cell cultures less with serum, the research and apply of low serum cell culture technology has been put into 12 national science and technology supporting plan main projects simultaneously as far as possible.
Summary of the invention
Invention broadly provides a kind of low serum protein-free medium being suitable for Vero Growth of Cells and preparation method thereof, have without albumen, non-animal derived composition, clearly several large advantage of Chemical Composition, industrially decrease the interference of serum to cell cultivation process, reduce production cost, industrialization for cell is produced and is brought great convenience, use substratum of the present invention, make to be carry out under 2% low-serum-concentration condition in the culturing process of Vero cell, decrease the consumption of serum.Its technical scheme is as follows: a kind of low serum protein-free medium being suitable for VERO Growth of Cells, and it comprises following component:
Amino acid moiety comprises following component:
Vitamin moieties comprises following component:
Inorganic salt part comprises following component:
Trace element part comprises following component:
Also comprise other components following:
Preferably, a kind of component of low serum protein-free medium of the Vero of being suitable for Growth of Cells is as follows: amino acid moiety comprises following component:
Vitamin moieties comprises following component:
Inorganic salt part comprises following component:
Trace element part comprises following component:
Also comprise other components following:
Be suitable for a preparation method for the low serum protein-free medium of Vero Growth of Cells, comprise the steps:
(1) getting dried each component is respectively charged in ball grinder, and ball milling 1.5-3 hour, obtains culture medium dry powder;
(2) culture medium dry powder that step (1) is obtained is dissolved in ultrapure water, and carries out constant volume, with the sterilizing of bacteriological filtration membrane filtration, the low serum of VERO Growth of Cells must be suitable for without protein liquid substratum.
Preferably, in step (1), Ball-milling Time is 2 hours, and in step (2), the aperture of bacteriological filtration film is 0.22 μm.
Adopt above-mentioned substratum and preparation method thereof, the present invention has the following advantages:
(1) substratum of the present invention, have without albumen, non-animal derived composition, clearly several large advantage of Chemical Composition, scientific research can reduce the interference of serum to experimentation and result, industrial production can be enhanced productivity, ensure quality product, on the basis remaining traditional process equipment, production cost is reduced by the usage quantity reducing serum, the most important thing is simultaneously, it does not change the adherent growth state of Vero cell, substratum provided by the invention is applicable to the adherent culture of cell, with other combine with technique such as microcarriers, can in shaking flask, bio-reactors etc. carry out pilot scale culture, thus be widely used in suitability for industrialized production,
(2) the low serum protein-free medium of Vero cell 2% of the present invention and basic medium add 10% calf serum and have carried out cell cultures and compare, easier decreasing pollution, be conducive to the separation of derived product, security is high, it is close that the growth curve of its cell cultures and virus connect malicious multiplication capacity, and without marked difference, substratum of the present invention does not change traditional processing technology, do not need the production unit more renewed, can be used for existing domestic production;
(3) substratum of the present invention overcomes during Vero cell routine is cultivated the shortcoming that must use high-content calf serum ability growth promoting effects, poor stability, the process of culture medium culturing Vero cell of the present invention is carried out under 2% low-serum-concentration, decreases the consumption of serum.
Accompanying drawing explanation
Fig. 1 is state graph under the BHK21 microcytoscope cultivated under the 10% serum-concentration condition that provides of ATCC (American Type Culture Collection);
Fig. 2 adds state graph under the bimestrial Vero microcytoscope of 2% calf serum cultured continuously for using low blood serum medium of the present invention;
Fig. 3 uses low blood serum medium of the present invention to add the growth curve of the Vero cell that 2% calf serum is cultivated and the growth curve comparison diagram of the Vero cell using the high sugar of existing 10% calf serum DMEM to cultivate.
Embodiment
Embodiment 1
The present embodiment is for the preparation of being suitable for the low serum of Vero Growth of Cells without albuminous cell substratum.
1. the formula of substratum
Table 1 culture medium prescription
2. preparation method
(1) getting dried each component in formula is respectively charged in ball grinder, and ball milling 2 hours, obtains culture medium dry powder;
(2) the culture medium dry powder 16.73g that step (1) is obtained is dissolved in 900ml ultrapure water, then 1L is settled to ultrapure water, with the bacteriological filtration membrane filtration sterilizing that aperture is 0.22 μm, the low serum that must be suitable for Vero Growth of Cells, without protein liquid substratum, refrigerates for subsequent use at 6 DEG C.
Embodiment 2
The present embodiment is for the preparation of being suitable for the low serum of Vero Growth of Cells without albuminous cell substratum.
1. the formula of substratum
Table 2 culture medium prescription
2. preparation method
(1) getting dried each component in formula is respectively charged in ball grinder, and ball milling 1.5 hours, obtains culture medium dry powder;
(2) culture medium dry powder that step (1) is obtained is dissolved in 900ml ultrapure water, then 1L is settled to ultrapure water, with the bacteriological filtration membrane filtration sterilizing that aperture is 0.22 μm, the low serum that must be suitable for Vero Growth of Cells, without protein liquid substratum, refrigerates for subsequent use at 8 DEG C.
Embodiment 3
The present embodiment is for the preparation of being suitable for the low serum of Vero Growth of Cells without albuminous cell substratum.
1. the formula of substratum
Table 3 culture medium prescription
2. preparation method
(1) getting dried each component in formula is respectively charged in ball grinder, and ball milling 3 hours, obtains culture medium dry powder;
(2) culture medium dry powder that step (1) is obtained is dissolved in 900ml ultrapure water, then 1L is settled to ultrapure water, with the bacteriological filtration membrane filtration sterilizing that aperture is 0.22 μm, the low serum that must be suitable for Vero Growth of Cells, without protein liquid substratum, refrigerates for subsequent use at 4 DEG C.
Embodiment 4
The present embodiment is for illustration of the laboratory using method of low serum without albuminous cell medium liquid being applicable to Vero Growth of Cells of the present invention.
1.Vero cell cultures
A) in existing DMEM high glucose medium (DMEM high glucose medium is known and the substratum containing each seed amino acid and glucose determined of composition), add the Vero cell that 10% concentration calf serum is cultivated, grow to and to be paved with bottom T25 square vase after about 80-90%, remove nutrient solution, use the aseptic PBS liquid (0.2ml/cm of not calcium-magnesium-containing
2) rinsing cell;
B) the aseptic Digestive system (0.2ml/cm containing 0.25% (w/v) trypsinase and 0.02% (w/v) EDTA is added
2) after, be placed in 5%CO
2, saturated humidity incubator, hatch for 37 DEG C and become circle, sucking-off Digestive system to cellular contraction, go down to posterity after adding substratum piping and druming evenly, inoculum density is 1 ~ 2 × 10
5cells/ml.
2. with substratum of the present invention, low serum domestication is carried out to Vero cell
Adopt direct method or progressively fall adaptation and the domestication that serum adjustment procedure carries out the low serum free culture system of Vero cell.
A) direct method: the cell that aforementioned stable grows directly is inoculated in low serum protein-free medium prepared by embodiment 1,2 or 3 and cultivates, substratum adds the calf serum of 2% when cell cultures, stablize and namely represent after energy continuous passage tame successfully until its growth conditions.
B) serum adjustment procedure progressively falling: is inoculated in by the cell that aforementioned stable grows in the substratum of the present invention containing 10% calf serum concentration and cultivates, progressively reduce the serum content in substratum, namely serum content is respectively from 10%, 5% is finally down to 2%, carry out the 2% low serum free culture system that cell continues, under each serum-concentration, cell carries out continuing to go down to posterity, cell is transferred in the substratum containing lower concentration serum after in stable condition, until adapt to low serum protein-free medium completely, the substratum of the present invention of above-mentioned use is embodiment 1, the substratum prepared in 2 or 3.
Interpretation of result: containing the Vero cell cultivated in 10% calf serum DMEM high glucose medium, cultivates in adopting direct method and progressively falling 2% low blood serum medium that Vero cell all can successfully adapt to from 10% calf serum cultivation the present invention by serum adjustment procedure.
Direct method domestication process time is short, but adaptive process incipient cell there will be obvious inadaptability, and does not occur too significantly incompatibility phenomenon progressively falling cell in serum adaptive process.No matter be direct or serum free culture system domestication falls in round-about way, the low serum protein-free medium in the present invention can well for Vero cell provides a suitable growing environment under interpolation 2% calf serum concentration conditions.
The Vero cell picture cultivated under the 10% serum-concentration condition that Fig. 1 provides for ATCC, Fig. 2 is for using low blood serum medium of the present invention cultured continuously bimestrial Vero cell picture under interpolation 2% calf serum concentration conditions.Contrasted from Fig. 1 and Fig. 2, adopt substratum of the present invention under 2% calf serum condition, be more suitable for the growth of Vero cell.
Embodiment 5
The present embodiment is more better than conventional use 10% calf serum concentration cultures culturing cell for illustration of the low serum of the Vero of being suitable for Growth of Cells of the present invention without albuminous cell culture medium culturing cell.
Cell growth curve measures: the DMEM high glucose medium cultured cells adding 10% calf serum by existing, add 2% calf serum embodiment 1 in the cell of culture medium culturing be inoculated in 12 orifice plates respectively, 1 × 10
5cells/ hole, and use corresponding culture medium culturing, survey a cell density every 24h, often kind of cell gets 3 holes at every turn, after trysinization, counts with Trypan Blue.
Interpretation of result: two kinds of culture system cultured cells growth power curves, as that shown in fig. 3, the Vero cellular form that the speed of growth of the Vero cell of 2% low serum free culture system and 10% calf serum DMEM high sugar is cultivated does not have notable difference, and the Vero cell of 2% low serum free culture system does not have notable difference compared to the Vero vitro growth rates of the high sugar cultivation of 10% calf serum DMEM and cell quantity yet.It can thus be appreciated that the substratum adopting the present invention to prepare is applicable to 2% low serum free culture system environment, can reduce amount of serum, cost-saving.
To one skilled in the art, according to technical scheme described above and design, other various corresponding change and deformation can be made, and all these change and deformation all should belong within the protection domain of the claims in the present invention.
Claims (4)
1. be suitable for a low serum protein-free medium for Vero Growth of Cells, it is characterized in that: it comprises following component:
Amino acid moiety comprises following component:
Vitamin moieties comprises following component:
Inorganic salt part:
Trace element part comprises following component:
Also comprise other components following:
Thymidine 0.1-1mg/L xanthoglobulin 1-10mg/L
Linolic acid 0.01-1mg/LD-glucose 3000-5000mg/L
Phenol red 1-20mg/LHEPES1000-4000mg/L.
2. a kind of low serum protein-free medium being suitable for Vero Growth of Cells according to claim 1, is characterized in that: it comprises following component:
Amino acid moiety comprises following component:
Vitamin moieties comprises following component:
Inorganic salt part comprises following component:
Trace element part comprises following component:
Also comprise other components following:
Thymidine 0.47mg/L xanthoglobulin 1.42mg/L
Linolic acid 0.042mg/LD-glucose 4500mg/L
Phenol red 8.57mg/LHEPES2979mg/L.
3. be suitable for a preparation method for the low serum protein-free medium of Vero Growth of Cells as claimed in claim 1, it is characterized in that: comprise the steps:
(1) getting dried each component is respectively charged in ball grinder, and ball milling 1.5-3 hour, obtains culture medium dry powder;
(2) culture medium dry powder that step (1) is obtained is dissolved in ultrapure water, and carries out constant volume, with the sterilizing of bacteriological filtration membrane filtration, the low serum of Vero Growth of Cells must be suitable for without protein liquid substratum.
4. the preparation method being suitable for the low serum protein-free medium of Vero Growth of Cells according to claim 3, is characterized in that: in step (1), Ball-milling Time is 2 hours, and in step (2), the aperture of bacteriological filtration film is 0.22 μm.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034172A (en) * | 2017-05-17 | 2017-08-11 | 四川农业大学 | Suitable for the method for building up of low serum Vero cell line |
| CN110938586A (en) * | 2019-12-24 | 2020-03-31 | 依科赛生物科技（太仓）有限公司 | Vero cell low serum culture medium and application thereof |
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| CN101974481A (en) * | 2010-10-29 | 2011-02-16 | 国家兽用生物制品工程技术研究中心 | Serum free culture medium for growing various cells derived from kidney tissue |
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Non-Patent Citations (3)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107034172A (en) * | 2017-05-17 | 2017-08-11 | 四川农业大学 | Suitable for the method for building up of low serum Vero cell line |
| CN110938586A (en) * | 2019-12-24 | 2020-03-31 | 依科赛生物科技（太仓）有限公司 | Vero cell low serum culture medium and application thereof |
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Application publication date: 20151223 |