Patent Application
20170369836
This application claims priority to Chinese Patent Application No. 201610486303.8 with a filing date of Jun. 24, 2016. The content of the aforementioned application, including any intervening amendments thereto, are incorporated herein by reference.
The present invention relates to the field of the preparation of a serum-free medium, and in particular relates to a serum-free medium for full suspension culture of MDCK cells and a preparation method of the serum-free medium.
Madin-Darby canine kidney (MDCK) cells are regarded as one of cell lines most suitable for producing influenza A and B virus vaccines and may be used to substitute chick embryo to culture the influenza virus. At present, a micro-carrier adherent culture production method utilizing an MDCK cell line to substitute the chick embryo to culture the influenza virus has already been developed. Although the technological method can reach certain production scale, the method still has some defects; 1. micro-carriers are difficult to reuse repeatedly, resulting in high production cost; 2. the culture density of adherent cells is limited by an adherent area, resulting in low yield of the viruses; and 3. the adherent culture generally needs to add the serum to help the attachment and growth of the cells and needs the medium change, so the process is complicated, and mycoplasma, chlamydia or animal protein may be introduced to cause the pollution, thereby posing a potential threat to the safety of vaccine products. People pay more attention to the large-scale serum-free full suspension culture technology of the MDCK cells. Reports about the full suspension culture of the MDCK cells in a serum-free medium are rare. At present, commercial serum-free media suitable for the large-scale full suspension culture of the MDCK cells are extremely rare in China. In the prior art, the serum-free medium contains transferrin and other expensive animal-based-proteins, which is not favorable for the development of veterinary biological products. Therefore, it is necessary to develop a serum-free medium for the suspension culture of MDCK single cells, which is definite in components, convenient to prepare and use, lower in cost and suitable for producing the veterinary biological products in a large scale, so that the MDCK cells can be simply and rapidly educated from a serum adherent growth state to a serum-free suspension growth state, and a more advanced serum-free high-density suspension culture process for the MDCK cells is established.
In view of the deficiencies of the prior art, one of objectives of the present invention is to provide a serum-free medium for full suspension culture of MDCK cells. The medium supports the high-density full suspension culture of the MDCK single cells, greatly shortens the time for educating the MDCK cells from the serum adherent cultured cells to the serum-free full suspension cultured cells, and is applicable to the mass production of biological products and particularly the veterinary biological products.
In order to realize the above objective, the present invention adopts a technical solution as follows: a serum-free medium for full suspension culture of MDCK cells comprises components of the following concentrations:
basic metabolic nutrients:
nucleotide:
vitamins:
inorganic salts:
shear force protective agent: 500 to 2500 mg/L;
cell clustering resisting agent: 20 to 150 mg/L;
pH buffer agent:
pH indicator:
influenza virus proliferation
accelerant:
other additives:
As a preferred embodiment of the present invention: in the serum-free medium for the full suspension culture of the MDCK cells, concentrations of various components are:
basic metabolic nutrients:
nucleotide:
vitamins:
inorganic salts:
shear force protective agent: 1600 mg/L;
cell clustering resisting agent: 50 mg/L;
pH buffer agent:
pH indicator:
influenza virus proliferation
accelerant:
other additives:
As a preferred embodiment of the present invention, the shear force protective agent is segmented polyether F68,
As a preferred embodiment of the present invention, the cell clustering resisting agent is dextran sulfate.
Another objective of the present invention is to provide a preparation method of a serum-free medium for full suspension culture of MDCK cells. The method is simple, rapid and high in efficiency and facilitates the mass production.
In order to realize the above objective, the present invention adopts a technical solution as follows: a preparation method of a serum-free medium for full suspension culture of MDCK cells comprises the following steps:
1) preparing a mixed solution: dissolving and mixing raw materials in one of the following methods:
I) mixing the raw materials and grinding them into fine powder, and then dissolving the obtained fine powder in a solvent at 10 to 30° C. to obtain a mixed solution;
II) respectively dissolving raw materials in the solvent to obtain a raw material solution; and mixing the obtained raw material solutions at a temperature of 10 to 30° C. to obtain a mixed solution; and
2) regulating pH: regulating the pH of the mixed solution to 6.3 to 6.7, and setting a constant volume to obtain the serum-free medium for the full suspension culture of the MDCK cells.
As a preferred embodiment of the present invention, in step 1 the solvent, is pyrogen-free ultra-pure water.
As a preferred embodiment of the present invention, in step 2), sodium hydroxide is added to regulate the pH value of the obtained mixed solution.
The present invention has the beneficial effects as follows:
1 The serum-free medium for the full suspension culture of the MDCK cells contains no animal serum, is low in cost, supports the high-density full suspension culture of the MDCK single cells, and is definite in components, easy to prepare and convenient to use;
2. the medium of the present invention effectively, shortens the time for educating the MDCK cells from the serum adherent cultured cells to the serum-free full-suspension cultured cells, increases the production efficiency and obtains high-quality full suspension cells; and
3 the preparation method of the present invention is simple, fast and high in efficiency and facilitates the mass production.
The present invention is further described below in combination with specific implementation manners:
Specific implementation manners:
A serum-free medium for full suspension culture of MDCK cells comprises components of the following concentrations:
basic metabolic nutrients:
nucleotide:
vitamins:
inorganic salts:
shear force protective agent:
cell clustering resisting agent:
pH buffer agent:
pH indicator:
influenza virus proliferation accelerant:
other additives:
The hypoxanthine and the thymidine are selected, in the nucleotide, so that the nucleotide synthesis of the MDCK cells can be promoted, and the, growth of the cells can be ensured; and if the proportion of the hypoxanthine and the thymidine is excessively high, the growth of the cells may be inhibited.
Ammonium ferric citrate is selected in other additives and used to substitute transferrin to play the original effect, the growth of the cells and the iron metabolism are not affected, animal protein components in the serum-free medium can be reduced, and the cost of the medium and the uncertainty and unsafety in production can be reduced; the ammonium ferric citrate absorbs iron through a divalent metal ion channel DMT1, while the transferrin absorbs the iron through a transferrin receptor, so that compared with the transferrin, the ammonium ferric citrate increases the absorption rate of the iron in the MDCK cells; if the concentration of the ammonium ferric citrate is excessively high, the growth of the MDCK cells may be inhibited; and if the concentration is excessively low, the MDCK cells are insufficient to absorb the iron.
The concentration of insulin in the other additives is 2 to 15 mg/L, so that the metabolism of the glucose can be promoted, the growth of the MDCK cells can be ensured and the activity of the MDCK cells can be maintained.
The concentration of the soybean hydrolysate in the other additives is 1000 to 5000 mg/mL, so that the supply of other auxiliary factors such as the vitamins, metal ions, amino acid and the like can be ensured, and the absorption of the amino acid in the MDCK cells can be improved.
A preparation method of a serum-free medium for full suspension culture of MDCK cells comprises the following steps:
1) a mixed solution is prepared: raw materials are dissolved end mixed in one of the following methods:
I) raw materials are mixed and then ground into fine powder, and the obtained fine powder is dissolved in pyrogen-free ultra-pure water at 10 to 30° C. to obtain a mixed solution;
II) raw materials are respectively dissolved in the pyrogen-free ultra-pure water to obtain, a raw material solution; and then the obtained raw material solutions are mixed at the temperature of 10 to 30° C. to obtain a mixed solution; and
2) pH is regulated: the sodium hydroxide is added to regulate the pH of the mixed solution to 6.3 to 6.7, and after a constant volume is set, the serum-free medium for the full suspension culture of the MDCK cells is obtained.
Specific embodiments
The present embodiment discloses a serum-free medium for full suspension culture of MDCK cells. The serum-free medium for the full suspension culture of the MDCK cells comprises components of the following concentrations: basic metabolic nutrients:
nucleotide:
vitamins:
inorganic salts:
shear force protective agent:
cell clustering resisting agent:
pH buffer agent:
pH indicator:
influenza virus proliferation
accelerant:
other additives:
The serum-free medium for the full suspension culture of MDCK cells is prepared according to the following steps:
1) the raw materials are mixed and then ground into fine powder, the obtained fine powder is dissolved in pyrogen-free ultra-pure water at 10 to 30° C., the concentration of each raw material is as described above, and the mixed solution is obtained;
2) the sodium hydroxide is added to regulate the pH of the mixed solution to 6.4, and after the constant volume is set, the serum-free medium DHN-1 for the full suspension culture of the MDCK cells is obtained.
According to the present embodiment, the serum-free medium for the full suspension culture of the MDCK cells comprises components of the following concentrations
basic metabolic nutrients:
nucleotide:
vitamins:
inorganic salts:
shear force protective agent:
cell clustering resisting agent:
pH buffer agent:
pH indicator:
influenza virus proliferation accelerant:
other additives:
The serum-free medium for the full suspensor culture of the MDCK cells is prepared according to the following steps:
1 ) the raw materials are mixed and then ground into fine powder, the obtained fine powder is dissolved in pyrogen-free ultra-pure water at 10 to 30° C., the concentration of each raw material is as described above, and the mixed solution is obtained: and
2) the sodium hydroxide is added to regulate the pH of the mixed solution to 6.5, and after the constant volume is set, the serum-free medium DHN-2 for the full suspension culture of the MDCK cells is obtained.
According to the present embodiment, the serum-free medium for the full suspension culture of the MDCK cells comprises components of the following concentrations:
basic metabolic nutrients:
nucleotide:
vitamins:
inorganic salts:
shear force protective agent:
cell clustering resisting agent:
pH buffer agent:
pH indicator:
influenza virus proliferation accelerant:
other additives:
The serum-free medium for the full suspension culture of the MDCK cells is prepared according to the following, steps:
1) the raw materials are mixed and then ground into fine powder, the obtained fine powder is dissolved in pyrogen-free ultra-pure water at 10 to 30° C., the concentration of each raw material is as described above, and the mixed solution is obtained;
2) the sodium hydroxide is added to regulate the pH of the mixed solution to 6.7, and after the constant'volume is set, the serum-free medium DHN-3 for the full suspension culture of the MDCK cells is obtained.
A characteristic test is carried out for the medium obtained in embodiments 1 to 3:
1. Instrument: bio-reactor Bio-Bundle purchased from Holland Applikon Biotechnology Company), and a volume of a tank body is 3L;
2. Cells: MDCK cell lines suitable for the serum-free full suspension culture, provided by East China University of Science and Technology;
3. Serum-free medium for reference: commercial serum-free medium SFM4 Mega Vir (purchased from Hyclone Company);
4. Culture method: the cells are inoculated into the bio-reactor at a cell density of 0.5×106 cells/mL and subjected to mass culture under the conditions of 37° C. and 5% CO2, the cells are sampled every 24 h for counting the living cells, and the growth rate of the cells is calculated. Results are shown in Table 1 and Table 2:
Compared with the commercial serum-free medium SFM4 Mega Vir of a control group for the suspension culture of the MDCK cells, by adopting the serum-free medium provided by the present invention, the supported living cell density in the culture process is greatly increased; and furthermore, the specific, growth rate of the cells at the non-exponential growth period is increased from the maximum 0.57 d-1 of the control group to 0.91 d-1 in DHN-2 of the embodiment 2, and the doubling time of the cells is shortened from the maximum 0.79 d of the control group to 0.32 d in DHN-2 of the embodiment 2. It can be seen that by adopting the serum-free medium of the present invention to culture the MDCK cells, both the cell growth rate and the cell activity are greatly improved.
The serum-free medium DHN-2 prepared in embodiment 2 is used to perform the serum-free full suspension culture education for the serum adherent cultured MDCK cells. The cell education process is as follows:
1) when the adherent MDCK cells cultured by DMEM containing 10% of new-born calf serum are cultured to the cell confluence of 80% to 90%, the original medium is abandoned, the cell layers are washed twice with pancreatin so as to neutralize the residual serum, and liquid is abandoned; a pancreatin solution is continuously added to cover the MDCK cells to perform the digestion for 5 to 15 min; after all cells become round, the digestion was terminated by adding medium containing 10% of fetal bovine serum with the volume four times of the volume of a digestive solution; and the cells are blown and beaten by using a pipette, the cells are suspended, a cell suspension solution is collected and centrifuged at 1000 rpm for 5 min, then supernatant is abandoned, and cell clusters are obtained;
2) the cell clusters are re-suspended by using the serum-free medium DHN-2 until the cell density is about 1.5*106 cells/mL, and a cell re-suspension solution is obtained;
3) the cell re-suspension solution is added into a square vase and cultured in an incubator at :a rotation speed of 30 rpm, a temperature of 37° C., and 5% of CO2; after two-generation culture, the cultured cell re-suspension solution is transferred into a 125 mL of shake flask, and the rotation speed is increased to 120 rpm. The cells are sampled every 24 h, the sampled cells are counted and subjected to the activity analysis, the cell density is diluted with fresh medium DHN-2 to about 1.5*106 cells/mL every 48 h, subculture is coontinued on a shaking table, and the MDCK cells suitable for the serum-free full suspension culture are obtained, and
4) the living cell density and the cell activity are shown in
The morphology of the serum-free full suspension cultured MDCK cells educated with the medium of the present invention is compared with the morphology of the adherent culture cells and the cells cultured with other serum-free media, and results are shown in
In
Therefore, the adherent cultured MDCK cells are educated to the suspension culture state in the serum-free medium of the present invention, the cells grow in an individually scattering manner, the cell morphology is full and the size is uniform; and the cell quality is high.
It will be apparent to those skilled in the art that various other corresponding changes and variations may be made in accordance with the technical solutions and concepts described above, and all of the changes and variations shall belong to the protection scope of the claims of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201610486303.8 | Jun 2016 | CN | national |
