CN107841482B - H9 subtype influenza vaccine produced by MDCK cell serum-free suspension culture technology - Google Patents

H9 subtype influenza vaccine produced by MDCK cell serum-free suspension culture technology Download PDF

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CN107841482B
CN107841482B CN201610840523.6A CN201610840523A CN107841482B CN 107841482 B CN107841482 B CN 107841482B CN 201610840523 A CN201610840523 A CN 201610840523A CN 107841482 B CN107841482 B CN 107841482B
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reactor
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CN107841482A (en
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谭文松
刘旭平
黄锭
贾涵婧
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Shanghai Bioengine Biotechnology Co 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
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
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    • 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
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material

Abstract

The invention discloses an H9 subtype influenza vaccine produced by an MDCK cell serum-free suspension culture technology, wherein the culture medium comprises amino acid, vitamin, salt, lipid, trace elements, a buffering agent, protein hydrolysate and an active additive; the method for producing the H9 subtype influenza vaccine comprises the following steps: (1) resuscitating the suspension cells; (2) carrying out cell subculture and seed cell amplification; (3) culturing in a cell reactor; (4) inoculating H9 subtype influenza virus seed venom and harvesting the venom. The method has the advantages of simplified process, enlarged scale, increased yield, no need of digestion, washing, centrifugation and liquid exchange, and remarkable benefit.

Description

H9 subtype influenza vaccine produced by MDCK cell serum-free suspension culture technology
Technical Field
The invention belongs to the field of biological pharmacy, and particularly relates to an H9 subtype influenza vaccine produced by adopting a MDCK cell serum-free single cell suspension culture technology.
Background
In recent years, animal cell culture techniques have been rapidly developed and increasingly used for the production of influenza vaccines, gradually replacing the traditional chick embryo culture method.
In the prior art, cells selected in the influenza vaccine production process are usually adherent cells and grow in a monolayer adhesion manner, so a culture process with a serum microcarrier is mostly adopted. However, serum has the disadvantages of high price, large batch-to-batch difference, exogenous pathogen pollution and the like. Moreover, serum contains a large amount of unknown protein components, which makes the separation and purification of downstream products difficult. Therefore, there is a need to develop serum-free media for efficient production of influenza vaccines. However, in most serum-free production processes, cells still grow in an adherent manner, washing and centrifugation are needed in the cell passage and seed cell amplification processes, and microcarriers are needed to provide an attachment matrix when the cells are cultured in large scale and influenza virus is amplified, so that the production cost is increased, the operation is extremely complex, time and labor are wasted, and the expansion of the production scale is not facilitated.
At present, various suspension cells such as MDCK, PER.C6, AGE.CR, EB14/EB66, CAP and the like are applied to influenza vaccine production processes, and particularly the MDCK suspension cells are most widely applied. However, the conventional batch culture method has low virus amplification efficiency, and can obtain a sufficiently high virus titer only by a complicated feeding and perfusion culture mode. However, influenza viruses are lytic and expand rapidly, killing host cells rapidly, and complex time-consuming fedbatch and perfusion culture modes often waste large amounts of culture medium despite the high viral titer achieved.
In view of the above, there is an urgent need in the art to develop a simple and efficient culture method for serum-free single cell suspension, which does not require microcarrier and can be used for batch production.
Disclosure of Invention
The invention aims to provide a serum-free culture medium and a method for producing an H9 subtype influenza vaccine by using a MDCK cell serum-free suspension culture technology.
In a first aspect of the invention, a serum-free medium suitable for suspension culture of MDCK cells is provided, the medium comprising amino acids, vitamins, salts, lipids, trace elements, buffers, protein hydrolysates, and active additives; wherein the content of the first and second substances,
the amino acids include 16 to 19 amino acids selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cystine, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;
the vitamins are selected from the group consisting of: biotin, folic acid, nicotinamide, pyridoxine, thiamine, lipoic acid, or a combination thereof;
the lipid is selected from the group consisting of: cholesterol, tocopherol acetate, myristic acid, palmitic acid, palmitoleic acid, stearic acid, tween 80, segmented polyether F68, or a combination thereof;
the trace elements are selected from the group consisting of: copper sulfate, ferric nitrate, ferrous sulfate, sodium selenite, or a combination thereof;
the protein hydrolysate is trypsin hydrolysate;
the active additive is selected from the group consisting of: glucose, insulin, soy hydrolysate, hypoxanthine, thymidine, ferric ammonium citrate, albumin, or a combination thereof.
In another preferred embodiment, the glucose content is in the range of 6000-8500mg/L medium (preferably 7000-8000mg/L medium) based on the volume of the medium.
In another preferred embodiment, the insulin is present in an amount ranging from 1 to 5mg/L of the culture medium (preferably 3 to 4.6mg/L of the culture medium) by volume of the culture medium.
In another preferred embodiment, the amount of the soybean hydrolysate is in the range of 1000-3000mg/L of the culture medium (preferably 2000-2800mg/L of the culture medium), based on the volume of the culture medium.
In another preferred embodiment, the content of the ammonium ferric citrate is in the range of 5-45mg/L of the culture medium (preferably 25-40mg/L of the culture medium), based on the volume of the culture medium.
In another preferred embodiment, the hypoxanthine is contained in the range of 2 to 15mg/L of the culture medium (preferably 3 to 11mg/L of the culture medium) by volume of the culture medium.
In another preferred embodiment, thymidine is present in the range of 0.1-0.8mg/L of medium (preferably 0.15-0.5mg/L of medium), based on the volume of medium.
In another preferred embodiment, the buffer is a buffer with a pH of 6.0-8.0 (preferably 6.8-7.2).
In another preferred embodiment, the buffer is selected from the group consisting of: sodium bicarbonate, hydroxyethylpiperazine ethanesulfonic acid, or a combination thereof.
In another preferred embodiment, the culture medium is in liquid form or, when reconstituted in liquid form, has a pH of 6.0 to 8.0, preferably 6.8 to 7.2.
In another preferred embodiment, the salt is selected from the group consisting of: magnesium chloride, magnesium sulfate, calcium chloride, potassium chloride, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium pyruvate, or combinations thereof.
In another preferred embodiment, the medium comprises one or more components selected from the group consisting of: 3-6mg/L alanine, 50-200mg/L arginine, 5-15mg/L asparagine, 2-15mg/L aspartic acid, 5-30mg/L cystine, 10-40mg/L cysteine, 7-20mg/L glutamic acid, 100-1000mg/L glutamine, 5-30mg/L glycine, 10-50mg/L histidine, 20-60mg/L isoleucine, 30-70mg/L leucine, 50-150mg/L lysine, 10-30mg/L methionine, 20-50mg/L phenylalanine, 2-30mg/L proline, 10-40mg/L serine, 10-60mg/L threonine, 5-20mg/L tryptophan, 30-70mg/L tyrosine and 30-70mg/L valine.
In another preferred embodiment, the medium comprises one or more components selected from the group consisting of: 0.003-0.1mg/L of biotin, 2-5mg/L of folic acid, 2-10mg/L of nicotinamide, 1-5mg/L of pyridoxine, 1-5mg/L of thiamine and 0.1-0.5mg/L of lipoic acid.
In another preferred embodiment, the medium comprises one or more components selected from the group consisting of: 20-100mg/L magnesium chloride, 20-100mg/L magnesium sulfate, 150-200mg/L calcium chloride, 500mg/L potassium chloride, 1000-7000mg/L sodium chloride, 50-100mg/L disodium hydrogen phosphate, 50-100mg/L sodium dihydrogen phosphate and 50-150mg/L sodium pyruvate.
In another preferred embodiment, the medium comprises one or more components selected from the group consisting of: 1-5mg/L of cholesterol, 0.005-0.1mg/L of tocopherol acetate, 0.005-0.1mg/L of myristic acid, 0.005-0.1mg/L of palmitic acid, 0.005-0.1mg/L of palmitoleic acid, 0.005-0.1mg/L of stearic acid, 1-3.1mg/L of Tween 80 and 0-2000 mg/L of block polyether F680.1.
In another preferred embodiment, the medium comprises one or more components selected from the group consisting of: 5-20mg/L of copper sulfate, 20-100mg/L of ferric nitrate, 0.1-0.6mg/L of ferrous sulfate and 50-150mg/L of sodium selenite.
In another preferred embodiment, the culture medium comprises 3000mg/L of trypsin hydrolysate 1500-.
In another preferred example, the culture medium comprises 6000mg/L of glucose 1000-.
In a second aspect of the present invention, there is provided a method for producing an H9 subtype influenza vaccine in the MDCK medium according to the first aspect of the present invention, comprising the steps of:
(1) providing a serum-free medium according to the first aspect of the invention;
(2) recovering the suspension MDCK cells;
(3) subculturing suspension cells: taking MDCK cell suspension, subculturing, inoculating density range of 1 × 105cell/ml-1X 107Cells/ml (preferably 2X 10)5cell/ml-1X 106Cells/ml, more preferably 4X 105cell/ml-9X 105Cells/ml);
(4) suspension cell reactor culture: adding the MDCK cell suspension obtained in the step (3) into a reactor for culturing;
(5) inoculation of H9 subtype influenza virus and venom harvest: inoculating the virus liquid with virus infection Multiplicity (MOI) of 0.01-1 (preferably 0.01-0.1) into a reactor to obtain the H9 subtype influenza virus.
In another preferred embodiment, the obtained HA titer of the influenza virus of H9 subtype is in the range of 211.5-212(hemagglutination unit/50. mu.l).
In another preferred example, in the steps (1) to (5), centrifugal liquid changing operation is not required.
In another preferred embodiment, the MDCK cells are fully suspended non-adherent.
In another preferred example, the step (2) includes: placing a freezing tube of MDCK single cell suspension culture type cells frozen by liquid nitrogen in a water bath at 37 ℃ for rapid dissolution, inoculating the tube into a 125 ml shake flask containing 30 ml of serum-free culture medium, placing the shake flask at 37 ℃ and 5% CO2The shaking culture of (1) was performed at a stirring speed of 120 rpm for 48 hours, and then the cells were passaged at an inoculation density of 5X 105Cells/ml.
In another preferred example, the step (3) includes: taking MDCK cells cultured for 48 hours in a single cell suspension mode, sucking cell suspension into a stirring bottle, adding a fresh culture medium, wherein the stirring speed of the shaking bottle is 120 revolutions per minute, and the stirring speed of the stirring bottle is 150 revolutionsPer minute, at 37 deg.C and 5% CO2The incubator of (2) and repeated subculture.
In another preferred embodiment, in the step (4), the stirring speed in the reactor is 100-.
In another preferred example, in the step (4), the set temperature in the reactor is 32-37 ℃.
In another preferred embodiment, in the step (4), the pH in the reactor is 6.8 to 7.2 (preferably 6.9 to 7.1).
In another preferred embodiment, in the step (4), the culture time in the reactor is 24-96h (preferably 30-85h, more preferably 50-80h, and most preferably 60-75 h).
In another preferred example, the step (4) includes: inoculating the MDCK cell suspension obtained in the step (3) into a reactor, and setting parameters of the reactor to three paths of gases including air and O2And CO2。
In another preferred example, the step (4) includes: inoculating the cell suspension obtained by culture into a 3L Aplikon stirred tank reactor with the inoculation density of 5 × 105Setting three paths of gases of air and O respectively according to the parameters of the cell/ml reactor2And CO2Stirring at 150 rpm, 37 deg.C and pH 7.0, and sampling every 24 hr to count viable cells and calculate cell viability by means of a bench blue staining method.
In another preferred example, in the step (5), the method further includes the steps of: pancreatin is added to the reactor at a final concentration of 0-18mg/L, preferably 1-16mg/L, more preferably 5-10 mg/L.
In another preferred embodiment, in the step (5), the virus inoculation time is 24-96h (preferably 30-85h, more preferably 50-80h, and most preferably 60-75 h).
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 shows the batch cell growth curve and cell viability profile of MDCK cells in example 1 of the present invention
Figure BDA0001118066260000051
Is a viable cell density curve;
Figure BDA0001118066260000052
the cell activity curve is shown.
FIG. 2 shows the batch culture growth and H9 subtype influenza virus propagation curves of MDCK cells in example 1 of the present invention
Figure BDA0001118066260000053
Is a viable cell density curve;
Figure BDA0001118066260000054
is H9 subtype influenza virus hemagglutination value curve.
FIG. 3 shows the batch cell growth curve and cell viability profile of MDCK cells in example 2 of the present invention
Figure BDA0001118066260000055
Is a viable cell density curve;
Figure BDA0001118066260000056
the cell activity curve is shown.
FIG. 4 shows the batch culture growth and H9 subtype influenza virus propagation curves of MDCK cells in example 2 of the present invention
Figure BDA0001118066260000057
Is a viable cell density curve;
Figure BDA0001118066260000058
is H9 subtype influenza virus hemagglutination value curve.
FIG. 5 shows a batch of MDCK cells in example 3 of the present inventionGrowth curve and cell activity curve, in which
Figure BDA0001118066260000059
Is a viable cell density curve;
Figure BDA00011180662600000510
the cell activity curve is shown.
FIG. 6 shows the batch culture growth and H9 subtype influenza virus propagation curves of MDCK cells in example 3 of the present invention
Figure BDA00011180662600000511
Is a viable cell density curve;
Figure BDA00011180662600000512
is H9 subtype influenza virus hemagglutination value curve.
FIG. 7 shows the batch cell growth curve and cell activity change curve of MDCK cells in comparative example 1 of the present invention
Figure BDA00011180662600000513
Is a viable cell density curve;
Figure BDA00011180662600000514
the cell activity curve is shown.
FIG. 8 is a graph showing the batch culture growth and H9 subtype influenza virus propagation curves of MDCK cells in comparative example 1 of the present invention
Figure BDA00011180662600000515
Is a viable cell density curve;
Figure BDA00011180662600000516
is H9 subtype influenza virus hemagglutination value curve.
FIG. 9 shows the batch cell growth curve and cell activity change curve of MDCK cells in comparative example 2 of the present invention
Figure BDA00011180662600000517
Is a viable cell density curve;
Figure BDA00011180662600000518
the cell activity curve is shown.
FIG. 10 is a graph showing the batch culture growth and H9 subtype influenza virus propagation curves of MDCK cells in comparative example 2 of the present invention
Figure BDA0001118066260000061
Is a viable cell density curve;
Figure BDA0001118066260000062
is H9 subtype influenza virus hemagglutination value curve.
FIG. 11 shows the batch cell growth curves of MDCK cells in comparative example 3 of the present invention in commercial medium and serum-free medium, respectively
Figure BDA0001118066260000063
Is a cell density curve in the serum-free culture medium;
Figure BDA0001118066260000064
cell density curves in commercial media.
FIG. 12 shows histograms of proliferation of influenza virus subtype H9 in commercial and serum-free media, respectively, for MDCK cells of comparative example 3 of the invention, black in the figure is proliferation of influenza virus subtype H9 in serum-free media; white is the proliferation of influenza virus subtype H9 in commercial medium.
Detailed Description
The inventor of the invention, through extensive and intensive research, unexpectedly discovers for the first time that the method for producing the H9 subtype influenza vaccine by using the MDCK single cell suspension mode only relates to a single animal cell, but not to animal tissues and embryos, has stable, rich and reliable sources and does not have batch difference. The present invention has been completed based on this finding.
Description of the terms
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the term "about" when used in reference to a specifically recited value means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).
As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….
Method for producing influenza vaccine subtype H9
The method for producing the H9 subtype influenza vaccine comprises the following steps:
(1) providing a serum-free medium;
(2) resuscitating the suspension cells;
(3) subculturing suspension cells and expanding seed cells;
(4) culturing in a suspension cell reactor;
(5) inoculating H9 subtype influenza virus seed venom and harvesting the venom.
The method of the invention does not need the steps of digestion, washing and centrifugal liquid exchange in the whole process.
The method reduces the using amount of the culture medium, the amount of the harvested virus liquid is the consumed culture medium amount, and the amount of the culture medium consumed in the process of producing the influenza vaccine by using serum adherent culture technologies such as spinner bottles, microcarriers and the like is more than 2 times of the amount of the harvested virus liquid, and bovine serum with the culture medium amount of about 10% is required. In addition, in the traditional influenza vaccine production process, which needs serum adherent culture technologies such as spinner bottles, microcarriers and the like, pancreatin digestion is needed for cell passage and amplification, and before virus inoculation, a phosphate buffer solution is needed for washing cells and a fresh serum-free culture medium is completely replaced to serve as a maintenance culture medium in the virus production process.
Titer of the product
As used herein, TCID50 titer refers to the half tissue culture infectious dose, also known as 50% tissue cell infectious dose, i.e., the amount of virus that can cause cytopathic effect (CPE) in half of the wells or tubes of a cell culture plate.
As used herein, HA titer refers to hemagglutinin of influenza virus, which causes agglutination of chicken erythrocytes, a phenomenon known as Hemagglutination (HA), also known as direct hemagglutination, and an assay designed to exploit this property is known as Hemagglutination Assay (HA).
The main advantages of the invention are:
(1) the method for producing the H9 subtype influenza vaccine by the serum-free culture medium and amplifying the cells step by step does not need the steps of digestion, washing, centrifugation, liquid exchange and the like, can greatly reduce the cost of raw materials, simplify the process, avoid the pollution of exogenous pathogens, improve the stability and the uniformity of the product quality, and reduce the burden of downstream separation and purification.
(2) The process method does not relate to wall attachment and digestion, needs a pancreatin digestion step, does not need microcarrier, roller bottles and other attaching matrixes, makes the amplification process easier, and can greatly reduce the time, space and labor cost.
(3) The process method reduces the using amount of the culture medium by a batch culture virus inoculation mode, the amount of the harvested virus liquid is the consumed culture medium amount, the influenza vaccine produced by culture processes such as spinner bottles, microcarriers with serum adherence and the like in the prior art has serious consumption of the culture medium, 10 percent of fetal calf serum is also needed, and pancreatin digestion and phosphate buffer solution are needed for washing in the culture process.
(4) The process method is verified by scale amplification from a 3L Applikon reactor to a 30L reactor to a 650L reactor, and finally 2 or more is obtained on the 650L reactor12The hemagglutination unit/50 microliter virus yield, higher virus titer can be obtained by batch culture, the yield and the quality are stable, and the further optimization and the amplification of the process are easy.
(5) In the process method, the virus is directly inoculated when the cells grow to the required density, only one culture medium is involved in the process, and the process is different from the traditional two-stage culture process which needs two culture media, namely a cell growth culture medium and a maintenance culture medium, so that the production efficiency of the culture medium in unit volume is improved.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are percentages and parts by weight.
The test materials and reagents used in the following examples are commercially available without specific reference.
The experimental methods in the following examples are conventional methods unless otherwise specified; the reagents used were purchased from Sigma-Aldrich, USA, unless otherwise specified.
Universal feedstock and process
1. Cell line
MDCK cells were purchased from ATCC (CCL-34) and acclimatized to a serum-free single cell suspension culture by animal cells and tissue engineering laboratories of university of eastern science and technology.
2. Serum-free medium
In the embodiment of the invention, the preparation method of the serum-free culture medium comprises the following steps: dissolving the culture medium dry powder prepared according to the table 1 in 80% of deionized ultrapure water in the final volume, and stirring for 30 minutes to completely dissolve the culture medium dry powder; adding 2.2 g/L of sodium bicarbonate, and stirring for 30 minutes; adding 1.0 mol/L hydrochloric acid or 1.0 mol/L sodium hydroxide to adjust pH to 6.8-7.0; sterilizing with 0.22 μm filter membrane, and storing at 0-4 deg.C.
Table 1 serum-free medium composition is as follows:
Figure BDA0001118066260000081
Figure BDA0001118066260000091
influenza H9 strain having a TCID50 titer of 108TCID50/mL。
TPCK pancreatin was purchased from Sigma-Aldrich, USA.
5. A bioreactor: aplikon stirred tank reactor, 3L (Aplikon Biotechnology, Holland).
6. Shaking table: from Adolf Kuhner, under the model Lab-Therm LT-X (Adolf Kuhner, Sweden).
7.CO2A constant-temperature incubator: available from Thermo Corporation, model number Forma 3951Reach-In (Thermo Fisher Scientific Corporation, USA).
8. Hemagglutination test: hemagglutinin of influenza virus selectively agglutinates erythrocytes of one or more animals, and this phenomenon of agglutinating erythrocytes is called Hemagglutination (HA), also called direct hemagglutination.
9. Commercial culture medium: the commercial medium used in the present invention was Ex-Cell MDCK, purchased from Sigma-Aldrich.
Example 1:
(1) preparation of serum-free medium
Taking 25g/L of culture medium dry powder, dissolving the culture medium dry powder in deionized ultrapure water with a final volume of 90%, and stirring for 30 minutes to completely dissolve the culture medium dry powder. Adding 2.2 g/L of sodium bicarbonate, and stirring for 30 minutes; adjusting pH to 7.0 with 1 mol/L hydrochloric acid solution or 1 mol/L sodium hydroxide, diluting to desired volume, sterilizing with 0.22 μm filter membrane (Millipore, USA), and storing at 0-4 deg.C.
(2) Resuscitating suspension cells
Freezing tube for MDCK single cell suspension culture type cell frozen by liquid nitrogen (the amount of MDCK cell is 2 × 10)7Individual cells/ml), rapidly dissolved in a 37 ℃ water bath, inoculated into a 125 ml shake flask containing 30 ml of serum-free medium, placed at 37 ℃ in 5% CO2The shaking culture was performed at a stirring speed of 120 rpm. After culturing for 48 hours, counting dead and live cells and calculating cell viability by a tray blue staining method, carrying out cell passage, and inoculating with the density of 5 multiplied by 105Cells/ml.
(3) Subculturing suspension cells and expanding seed cells
30 ml of MDCK cells cultured in a single cell suspension mode for 48 hours are taken, and the counted viable cell density is 5.3 multiplied by 106Cells/ml. The desired cell suspension was pipetted into a 150 ml stirred flask (seeding density about 1X 106Cells/ml) a volume of 120 ml fresh medium was added. The shaking flask stirring speed was 120 rpm. Placing at 37 ℃ and 5% CO2Until reaching the number of cells needed by the reactor, i.e. 5X 108And (4) cells.
The batch cell growth curve and cell activity change curve of MDCK cells in example 1 are shown in FIG. 1, and the cell density reaches the highest value of 7.5X 10 at 96h6The activity of the cells/ml is kept above 95%, and the serum-free single-cell suspension culture technology can support the normal growth of the cells.
(4) Suspension cell reactor culture
Viable cell density of 5.6X 10 was sampled and counted from the cell suspension obtained by the above culture6Cells/ml, the volume of 90 ml was inoculated into a 3.0 l Aplikon stirred tank reactor, and fresh serum-free medium was added to the reactor to a working volume of 1 l.
Setting three paths of gases as air and O according to reactor parameters2And CO2Stirring at 150 rpm, 37 deg.C and pH 7.0, and sampling every 24 hr to count viable cells and calculate cell viability by means of a bench blue staining method.
(5) Inoculation of H9 subtype influenza virus seed venom and venom harvest
MDCK cells are cultured in single cell suspension for 72 hours, and samples are taken to count the viable cell density by a bench-top blue staining method, and the cell viability is calculated. The seed venom was inoculated at a virus multiplicity of infection (MOI) of 0.01 into a stirred tank bioreactor, at which time 5 ml (concentration of 1 mg/ml) of TPCK pancreatin solution was added so that the final concentration was 5mg/l, the reactor temperature was set at 37 ℃, the dissolved oxygen at 50%, and the pH at 7.0, and samples were taken every 24 hours to count viable cells and calculate cell viability by the Takara blue staining method, while sampling and centrifuging the supernatant to detect the total virus number by the hemagglutination test.
Inoculation of virusesAfter 72 hours all the culture broth was removed aseptically and used for downstream virus concentration purification preparations. Viral titer 212The hemagglutination unit/50. mu.l, the results are shown in FIG. 2. As can be seen from fig. 2, the MDCK cell serum-free single cell suspension culture technology of this example can support normal growth of cells and proliferation of influenza virus subtype H9, and both the highest cell density and influenza virus hemagglutination value titer exceed those of known commercial culture media.
Example 2:
the difference from example 1 is that in step (4), the working volume is 20.0L, and inoculation is carried out at 1X 1010And (4) cells. In the step (4), the volume of the Aplikon stirred tank reactor is 30.0L, and a fresh serum-free culture medium is supplemented until the working volume of the reactor is 20.0L.
The batch cell growth curve and cell activity change curve of MDCK cells in example 2 are shown in FIG. 3, and the cell density reaches the highest value of 7.0X 10 at 96h6The activity of the cells/ml is kept above 90 percent, and the serum-free single-cell suspension culture technology can support the normal growth of the cells.
All culture fluids were aseptically taken out 72 hours after virus inoculation, and the virus titer was 211.5Hemagglutination units/50. mu.l, the results are shown in FIG. 4. As can be seen from fig. 4, the MDCK cell serum-free single cell suspension culture technique of example 2 can support normal growth of cells and proliferation of influenza virus subtype H9, with the highest cell density and influenza virus hemagglutination value titer exceeding those of known commercial media.
Example 3:
the difference from example 1 is that in step (4), the working volume is 400.0L, and 2X 10 inoculations are required11And (4) cells. In the step (4), the volume of the Aplikon stirred tank reactor is 650 liters, and fresh serum-free culture medium is supplemented until the working volume of the reactor is 400.0 liters.
The batch cell growth curve and cell activity change curve of MDCK cells in example 3 are shown in FIG. 5, and the cell density reaches the highest value of 7.0X 10 at 96h6The activity of the cells/ml is kept above 80 percent, which proves that the serum-free single-cell suspension culture of the inventionThe technology is capable of supporting normal growth of cells.
All culture fluids were aseptically taken out 72 hours after virus inoculation, and the virus titer was 211.5Hemagglutination unit/50 microliter, as shown in fig. 6, the MDCK cell serum-free single cell suspension culture technique of example 3 can support normal growth of cells and proliferation of influenza virus subtype H9, with the highest cell density and hemagglutination titer of influenza virus exceeding those of known commercial culture media.
Comparative example 1:
the same as example 1, except that in the step (5), the seed venom was inoculated into the stirred tank bioreactor at a multiplicity of viral infection (MOI) of 0.001.
The results are shown in FIG. 7, where the cell density reached a maximum of 7.8X 10 in 96h6The activity of the cells/ml is kept above 85%, the late death of the cells is slow, and the serum-free single-cell suspension culture technology can support the normal growth of the cells.
All culture fluids were aseptically removed 72 hours after virus inoculation, and the virus titer was only 28Hemagglutination unit/50 microliter, the result is shown in fig. 8, the MDCK cell serum-free single cell suspension culture technique of comparative example 1 can support normal cell growth but the H9 subtype influenza virus yield is low due to too low MOI.
Comparative example 2:
the same as example 1, except that in the step (5), 25 ml of TPCK pancreatin solution (concentration: 1 mg/ml) was added so that the final concentration was 25 mg/l, the reactor temperature was set at 37 ℃, the dissolved oxygen at 50% and the pH at 7.0, and samples were taken every 24 hours to count live cells and calculate cell viability by the trayed blue staining method, while sampling and centrifuging the retained supernatant to detect the total virus number by the hemagglutination test.
As shown in FIG. 9, the cell density reached a maximum of about 6.9X 10 at 96h6The activity of the cells/ml is kept above 75%, the damage to the cells is serious, and the activity is low.
All culture fluids were aseptically removed 72 hours after virus inoculation, and the virus titer was only 28Hemagglutination units/50. mu.l, the results are shown in FIG. 10. As can be seen from FIG. 10, the present pairThe serum-free single-cell suspension culture technology of the MDCK cells can support normal growth of the cells, but the yield of the H9 subtype influenza virus is low due to the excessively high addition amount of TPCK-pancreatin.
Comparative example 3:
the same as example 1, except that, in the step (1), a commercial medium was prepared according to the preparation method of the commercial medium, the volume was fixed, and the medium was sterilized with a 0.22 μm-pore filter (Millipore, USA) and stored at 0 to 4 ℃ for use.
As shown in FIG. 11, the highest cell density of the serum-free medium of the present invention was about 7X 10 at 96 hours6Cells/ml, whereas the commercial medium of comparative example 3 is only 5X 106Cells/ml, it can be seen that the serum-free medium of the present invention is better able to support cell growth.
All culture fluids were aseptically taken out 72 hours after virus inoculation, and the virus titer was 210Hemagglutination units/50. mu.l, the results are shown in FIG. 12. As can be seen from fig. 12, the MDCK cells of comparative example 3 had slow cell growth, low peak density, and low production of influenza virus subtype H9 in commercial culture medium.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (12)

1. A serum-free medium suitable for suspension culture of MDCK cells, wherein the medium comprises amino acids, vitamins, salts, lipids, trace elements, buffers, protein hydrolysates, and active additives; wherein the content of the first and second substances,
the amino acids include: alanine 22.3 mg/L, arginine 273 mg/L, asparagine 33 mg/L, aspartic acid 33.3 mg/L, cystine 42 mg/L, cysteine 68.0 mg/L, glutamic acid 36.8 mg/L, glutamine 876 mg/L, glycine 25 mg/L, histidine 73 mg/L, isoleucine 90 mg/L, leucine 161 mg/L, lysine 107.3 mg/L, methionine 89 mg/L, phenylalanine 100.2 mg/L, proline 95 mg/L, serine 78 mg/L, threonine 136 mg/L, tryptophan 57 mg/L, tyrosine 58 mg/L and valine 99 mg/L;
the vitamins are 0.072-0.1mg/L biotin, 2-5mg/L folic acid, 2-10mg/L nicotinamide, 1-5mg/L pyridoxine and 1-5mg/L thiamine, calculated by the volume of the culture medium;
the lipid is: 1-5mg/L cholesterol, 1.39 mg/L tocopherol acetate, 0.2284 mg/L myristic acid and 0.256 mg/L palmitic acid;
the trace elements are: 5-20mg/L of copper sulfate, 20-100mg/L of ferric nitrate, 0.1-0.6mg/L of ferrous sulfate and 50-150mg/L of sodium selenite, calculated by the volume of the culture medium;
the protein hydrolysate is trypsin hydrolysate, and the content of the trypsin hydrolysate is 1500-3000mg/L in terms of the volume of the culture medium;
the active additive is as follows: glucose, insulin, soy hydrolysate, hypoxanthine, thymidine, and ferric ammonium citrate;
wherein the content range of the glucose is 7000-8000mg/L culture medium, the content range of the insulin is 5.34mg/L culture medium, the content range of the soybean hydrolysate is 2000-2800mg/L culture medium, the content range of the hypoxanthine is 3-11mg/L culture medium, the content range of the thymidine is 0.15-0.5mg/L culture medium, and the content range of the ammonium ferric citrate is 25-40mg/L culture medium, calculated by the volume of the culture medium.
2. The serum-free medium of claim 1, wherein the medium is in a liquid state or, when reconstituted in a liquid state, has a pH of 6.0 to 8.0.
3. The serum-free medium according to claim 1, wherein the salt is: magnesium chloride, magnesium sulfate, calcium chloride, potassium chloride, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium pyruvate;
and the content of the salts is as follows: 2856mg/L of magnesium chloride, 20-100mg/L of magnesium sulfate, 150mg/L of calcium chloride, 500mg/L of potassium chloride, 1000mg/L of sodium chloride, 50-100mg/L of disodium hydrogen phosphate and 50-100mg/L of sodium dihydrogen phosphate.
4. The serum-free medium of claim 1, wherein the buffer is sodium bicarbonate.
5. A method for producing an H9 subtype influenza vaccine using the MDCK medium of claim 1, comprising the steps of:
(1) providing a serum-free medium according to claim 1;
(2) recovering the suspension MDCK cells;
(3) subculturing suspension cells: taking MDCK cell suspension, subculturing, inoculating density range of 1 × 105cell/ml-1X 107Cells/ml;
(4) suspension cell reactor culture: adding the MDCK cell suspension obtained in the step (3) into a reactor for culturing;
(5) inoculation of H9 subtype influenza virus and venom harvest: inoculating the seed venom into a reactor within the range of 0.01-1 of the virus multiplicity of infection to obtain the H9 subtype influenza virus.
6. The method of claim 5, wherein the obtained HA titer of the influenza virus subtype H9 ranges from 211.5Hemagglutination unit/50 microliter-212Hemagglutination unit/50 microliter.
7. The method of claim 5, wherein in steps (1) to (5), no centrifuge exchange is required and/or the MDCK cells are fully suspended non-adherent.
8. The method as claimed in claim 5, wherein in the step (4), the stirring speed in the reactor is 100-200 rpm.
9. The method of claim 5, wherein in step (4), the set temperature in the reactor is 32-37 ℃; and/or the presence of a gas in the gas,
in the step (4), the pH value in the reactor is 6.8-7.2; and/or the presence of a gas in the gas,
in the step (4), the culture time in the reactor is 24-96 h.
10. The method of claim 5, wherein step (3) comprises: taking MDCK cells cultured for 48 hours in a single cell suspension mode, sucking cell suspension into a stirring bottle, adding a fresh culture medium, placing the mixture at 37 ℃ and 5% CO, wherein the stirring speed of the shaking bottle is 120 r/min and the stirring speed of the stirring bottle is 150 r/min2The incubator of (2) and repeated subculture.
11. The method of claim 5, wherein the step (5) further comprises the steps of: adding pancreatin into the reactor, wherein the final concentration of the pancreatin is 0-18 mg/L.
12. The method of claim 5, wherein in step (5), the virus is inoculated for 24-96 h.
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