CN105368794A - Method of utilizing stirred bioreactor to produce infectious Bursal disease virus - Google Patents
Method of utilizing stirred bioreactor to produce infectious Bursal disease virus Download PDFInfo
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Abstract
The invention provides a method of utilizing a stirred bioreactor to produce an infectious Bursal disease virus. Bioreactor microcarrier cell culture technology is used to replace conventional roller bottle culture, so that the problems of low production efficiency, unstable product quality and low virus titer can be solved. On this basis, biological characteristics of the infectious Bursal disease virus and DF1 cells are combined, and proper conditions are matched from the perspectives of microcarrier adding amount, cell inoculation density, virus inoculation amount, cell density during virus inoculation, virus collection time and reactor operation parameters, so that virus culture efficiency is improved remarkably, and unit culture titer is improved by 10-100 times. In addition, compared with roller bottle culture, the method utilizing the bioreactor has the advantages that culture scale is large, and parameter control is comprehensive, so that systematic risk of being polluted is lowered, quality stability is improved, and the method has a wide application prospect.
Description
Technical field
The present invention relates to veterinary biologics technical field, be specifically related to a kind of method utilizing stirring type bioreactor to produce infectious bursa of Fabricius virus.
Background technology
Infectious bursal disease (InfectiousBursalDisease, IBD) be by birnavirus section, a kind of high degree in contact sexually transmitted disease that the infectious bursa of Fabricius virus (InfectiousBursalDiseaseVirus, IBDV) of Avibirnavirus causes.This disease mainly encroaches on the Lymphoid tissues such as the humoral immunization maincenter organ fabricius bursa of chicken, mainly comprises two aspects to the harm of aviculture.On the one hand that disease itself can cause 3 week age or larger chick clinical onset or death; Long-term immunosuppression can be produced on the other hand to chicken body, cause secondary Other diseases (mainly chicken horse Garrick, newcastle disease, colibacillosis, salmonellosis etc.), cause mortality ratio obviously to raise, can reach more than 80%, provisions fowl industrial belt carrys out serious financial loss.
Prevent this disease to realize mainly through vaccination, wherein inactivated vaccine account for very large ratio.The method of current China vitro culture infectious bursa of Fabricius virus mainly utilizes chicken embryo to breed or cell lies in spinner culture two kinds of methods.This method labour intensity is large, length consuming time, and efficiency is low, and production cost is high; Difference between different production batch or between the different rolling bottle of same production batch is large, and unstable product quality, is difficult to expanding production; Easily by environmental pollution, often there is the pollution of bacterium or other virus thus cause vaccine quality to there is hidden danger, relating to Biosafety and public health problem.The bio-reactor of mass-producing is adopted to be expected to improve above-mentioned defect; but there is diverse biological characteristics due to different cell strain, strain; therefore in the process utilizing bio-reactor execution infectious bursa of Fabricius virus to prepare, lack a kind of working method of ubiquity; that is, need to design specific culture condition for different cell strain, strain.In addition, for spinner culture, bioreactor culture infectious bursa of Fabricius virus larger, controllable parameter is more, on this scale basis, therefore how to ensure tiring of the multiplication rate of cell, virus, not yet form effective ways in prior art.
The kind of current zooblast bio-reactor is more, and for the cultivation of DF1 cell, current domestic existing unit adopts rip current type bio-reactor to complete scale operation.But due to the special construction of rip current type bio-reactor, it can only utilize scraps of paper carrier to cultivate, employing be disposable biological consumptive material, easily cause waste, the more important thing is in cell cultivation process, can not the state of observation of cell at any time, be difficult to control the best flexibly and connect the poison time.Compared with rip current type bio-reactor, the power that stirring type bioreactor provides liquid phase to stir by stirring rake, has larger operating restraint, good mixing and even concentration, is easy to cleaning and sterilization, can repeats and life-time service.It adopts most widely used microcarrier as culture carrier, can sample at any time, observation of cell state, draws growth curve, accurately grasps the best and connects poison and receive the poison time, obtain the virus liquid of the highest titre.Therefore, in mass cell is cultivated, occupy critical positions, have a lot of correlative studys and application both at home and abroad at present.
Summary of the invention
The present invention is intended to the technological deficiency for prior art, provides a kind of method utilizing stirring type bioreactor to produce infectious bursa of Fabricius virus, to solve the technical problem that in prior art, methods involving level of automation is low, quality is unstable.
Another technical problem that the present invention will solve is that the infectious bursa of Fabricius virus that the methods involving of prior art is produced is tired lower.
The technical problem again that the present invention will solve is that the methods involving security of prior art is lower.
How to ensure the quality of products when the another technical problem that the present invention will solve is and utilizes bio-reactor to produce infectious bursa of Fabricius virus stable.
How the another technical problem that the present invention will solve promotes vaccine valence when being and utilizing bio-reactor to produce infectious bursa of Fabricius virus.
For realizing above technical purpose, the present invention by the following technical solutions:
Utilize stirring type bioreactor to produce a method for infectious bursa of Fabricius virus, comprise the following steps:
1) in stirring type bioreactor, mixed by the microcarrier after sterilizing, obtain mixed culture medium with the consumption of 3 ~ 6g/L with steril cell growth media, the density inoculating DF1 cell is wherein 0.6 ~ 1.2 × 10
6individual/mL, makes DF1 cell adsorb cultivation on microcarrier, culture temperature 36 ~ 37 DEG C, pH7.2 ~ 7.3, dissolved oxygen 40 ~ 60%, stirring velocity 30 ~ 80rpm;
2) after inoculating DF1 cell 48 ~ 72h, discard the cell growth medium in mixed culture medium, add cell maintenance medium, the infectious bursa of Fabricius virus of access 0.3 ~ 1.2MOI, continue to cultivate;
3) after virus inoculation 24 ~ 48h, results virus liquid.
As preferably, step 1) in mixed culture medium cumulative volume be 40 ~ 60% of stirring type bioreactor cubic capacity.
As preferably, step 1) in before inoculating cell reactor first stir 20 ~ 60min.
As preferably, step 1) to plant described microcarrier be sterilizing by the following method:
A) with PBS damping fluid, more than 4h is soaked to microcarrier;
B) by steps A) soak after microcarrier PBS buffer solution for cleaning;
C) by step B) cleaning after microcarrier 115 ~ 125 DEG C of sterilizing 25 ~ 35min under PBS damping fluid soaking conditions.
As preferably, step 1) in for the DF1 cell inoculated first through the digestion of EDTA-trysinization liquid, then inoculate; Preferred further on this basis, described EDTA-trysinization liquid is the PBS damping fluid of pancreatin and 0.02% (w/v) EDTA containing 0.03 ~ 0.05% (w/v).
As preferably, step 1) new-born calf serum of DMEM/F12 substratum and 10% (v/v) containing 90% (v/v) in cell growth medium; Preferred further on this basis, before described cell growth medium inoculation DF1 cell, first in cell growth medium, add benzylpenicillin sodium and Vetstrep, to the whole content 100IU/mL of benzylpenicillin sodium, the whole content 100IU/mL of Vetstrep.
As preferably, step 2) in DF1 cell is to the rate that occupies of micro-carrier surface higher than 85% when discarding in mixed culture medium cell growth medium, the empty ball rate of microcarrier is lower than 5%.
As preferably, step 3) in results virus liquid time virus titer reach the highest.
As preferably, described microcarrier is Cytodex series microcarrier.
In above technical scheme, viral seed culture of viruses for inoculating can be prepared by the following method: be that 0.3-1.2 be inoculated in individual layer passage cell cultivate by infectious bursa of Fabricius virus kind poison by MOI amount with virus-culturing fluid, to meeting the rear 24-48h of poison, when malicious valency is the highest, results virus liquid; Again using this virus liquid as kind of a poison, cell carries out cell adaptation continuous passage rejuvenation, and the product that at every turn goes down to posterity carries out viral TCID
50and Sterility testing, reach viral TCID
50stablize and reach 10
8.5tCID
50rejuvenation is stopped, as production kind of a poison during/more than mL.Wherein, the screening formulation of described virus-culturing fluid is: 98%DMEM/F12,2% new-born calf serum, final concentration are 100U/ml benzylpenicillin sodium and Vetstrep, and pH value is 7.2-7.3.
In above technical scheme, described empty ball rate refers to ratio microcarrier not being accounted for microcarrier total surface area by the surface-area of cell attachment, and this index is for evaluating the attaching upgrowth situation of cell on microcarrier.Described DF1 cell is a kind of chick embryo fibroblast system of going down to posterity, and can buy from market.Described Cytodex series microcarrier, be only defined in by GE Company, model is a series of microcarrier products of Cytodex.
Present invention utilizes that stirring type bioreactor operating restraint is comparatively large, Combination good and the advantage such as even concentration, promote nutrient solution matter by stirring action and pass effect, guarantee the nutrient of nutrient solution and being uniformly distributed of oxyty, reach the object of large scale culturing cell.Compare with other biological reactor, its structure is simple, cost is lower.
The present invention has following beneficial effect:
(1) substitute traditional rolling bottle cell culture technology with bio-reactor Microcarrier Cell Culture Techniques and produce infectious bursa of Fabricius virus vaccine, the problem that production efficiency is low, unstable product quality, virus titer are low can be solved, by the change of production technology and production technique, improve viral units cultivation and tire 10-100 doubly, greatly reduce production cost, General Promotion vaccine quality and output, improve the security of vaccine.
(2) compared with rip current type bio-reactor, stirring type bioreactor mixes, structure is simple, easy to operate, have good transmission effect and turndown ratio, cell growth state can be grasped in real time, more easily grasp the best harvest time of virus liquid, improve vaccine valence.
(3) low stain: the present invention's clone replaces primary cell or chicken embryo tissue to cultivate infectious bursa of Fabricius virus, the problem that can solve chicken embryo self and pollute by exogenous virus, by the strict control of starting material and culture condition, ensure that the vaccine produced is pure.
(4) production technique is controlled: high-accuracy cultivation, Real-Time Monitoring dissolved oxygen, aerogenesis, residual sugar, PH, cell state etc., and curve is cultivated in comprehensive parameters setting, directly ensures the state of carrier cell; Production lot is large, differences between batches are little; Save space, man power and material.
Embodiment
Below will be described in detail the specific embodiment of the present invention.In order to avoid too much unnecessary details, in the examples below to belonging to known structure or function will not be described in detail.
The approximating language used in following examples can be used for quantitative expression, shows to allow quantity to have certain variation when not changing basic function.Therefore, this exact value itself is not limited to the numerical value that the language such as " approximately ", " left and right " is revised.In certain embodiments, " approximately " represents and allows its numerical value revised to change in the positive and negative scope of 10 (10%), such as, and any numerical value that what " about 100 " represented can be between 90 to 110.In addition, in the statement of " about first numerical value is to second value ", revise the first and second numerical value two numerical value approximately simultaneously.In some cases, approximating language may be relevant with the precision of surveying instrument.
Apart from outside definition, technology used in following examples and scientific terminology have the identical meanings generally understood with those skilled in the art of the invention.
Test reagent consumptive material used in following examples, if no special instructions, is routine biochemistry reagent; Described experimental technique, if no special instructions, is ordinary method; Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged; % in following examples, if no special instructions, is mass percentage.
Embodiment 1
(1) select bio-reactor as the means of cultivating: 10L Germany sartoriusstedim bio-reactor.
(2) carrier selecting microcarrier to grow as cell attachment: microcarrier Cytodex1.
(3) cleaning of microcarrier, sterilising method: 1) weigh Cytodex1 microcarrier 3g/L, soak microcarrier with 1LPBS ambient temperature overnight; 2) 3 times are cleaned with 1LPBS; 3) microcarrier is soaked with 1LPBS, 121 DEG C of steam sterilizing 30min.
(4) select DF1 cell as seedling cell.
(5) the going down to posterity and cultivation of seedling cell: above-mentioned cell is through EDTA-pancreatin (PBS containing 0.03% pancreatin, 0.02%EDTA) had digestive transfer culture, with containing 90%DMEM/F12 nutrient solution, 10% new-born calf serum, the benzylpenicillin sodium of 100IU/mL and Vetstrep, the cell growth medium that pH value is adjusted to 7.2 continues to cultivate.Culture temperature is 37 DEG C, when forming good individual layer, is inoculated in bio-reactor and carries out microcarrier suspension culture.
(6) breeding of cell seed culture of viruses: (be benzylpenicillin sodium and the Vetstrep of 100IU/mL containing the DMEM/F12 nutrient solution of new-born calf serum 2%, final concentration with virus-culturing fluid, being measured by MOI by the kind of infectious bursa of Fabricius virus HQ strain poison is the well-grown above-mentioned cell monolayer of 0.6 inoculation, continue to cultivate, culture temperature is 37 DEG C.To connect poison after 40h and malicious valency the highest time results virus liquid; Measure the TCID of virus
50, treat that virus titer is stablized and reaches 10
8.5tCID
50rejuvenation is stopped, using this virus as production kind of a poison during/more than mL.
(7) microcarrier suspension culture of DF1 cell in stirring type bioreactor: get well-grown DF1 cell on rolling bottle, clean twice with the PBS that pH value is 7.2, add the digestion of aforesaid EDTA-trysinization liquid, treat that cellular layer starts to occur that full wafer comes off, add described cell growth medium, blow to hang and make cell suspending liquid, cell counting density is by 0.8 × 10
6in individual/mL reaction of inoculation device.Bio-reactor is set as follows parameter: temperature 37 DEG C, pH value 7.2, stirring velocity 40rpm, dissolved oxygen content 40% carries out reactor and automatically control to cultivate.
(8) breeding of seedling venom: cultivating 48h observation cells on microcarriers and substantially cover with (more than 85%), is 0.6 virus inoculation with MOI.The culture parameters such as design temperature 37 DEG C, pH value 7.2, stirring velocity 50rpm, dissolved oxygen content 50%, carry out reactor and automatically control to cultivate.Sample at regular intervals after connecing poison, periodic monitor consumption sugar and residual sugar content, and by microscope observing cell pathology situation, detect the TCID of sample
50.When virus liquid is tired the highest, observe cells on microcarriers and substantially all come off (about 40h), and the obvious ascendant trend of DO value, stopped reaction device stirs, and after microcarrier all sinks to reactor bottom, gathers in the crops supernatant liquor and microcarrier respectively.
(9) process of virus liquid is gathered in the crops: after the packing of results supernatant ,-20 DEG C of freezen protective are for subsequent use.
Embodiment 2
(1) select bio-reactor as the means of cultivating: 10L Germany sartoriusstedim bio-reactor bio-reactor.
(2) carrier selecting microcarrier to grow as cell attachment: microcarrier Cytodex2.
(3) cleaning of microcarrier, sterilising method: 1) weigh Cytodex1 microcarrier 4g/L, soak microcarrier with 1LPBS ambient temperature overnight; 2) 3 times are cleaned with 1LPBS; 3) microcarrier is soaked with 1LPBS, 121 DEG C of steam sterilizing 30min.
(4) select DF1 cell as seedling cell.
(5) the going down to posterity and cultivation of seedling cell: above-mentioned cell is through EDTA-pancreatin (PBS containing 0.03% pancreatin, 0.02%EDTA) had digestive transfer culture, with containing 90%DMEM/F12 nutrient solution, 10% new-born calf serum, the benzylpenicillin sodium of 100IU/mL and Vetstrep, the cell growth medium that pH value is adjusted to 7.2 continues to cultivate.Culture temperature is 37 DEG C, when forming good individual layer, is inoculated in bio-reactor and carries out microcarrier suspension culture.
(6) breeding of cell seed culture of viruses: (be benzylpenicillin sodium and the Vetstrep of 100IU/mL containing the DMEM/F12 nutrient solution of new-born calf serum 2%, final concentration with virus-culturing fluid, being measured by MOI by the kind of infectious bursa of Fabricius virus HQ strain poison is the well-grown above-mentioned cell monolayer of 0.8 inoculation, continue to cultivate, culture temperature is 37 DEG C.To connect poison after 36h and malicious valency the highest time results virus liquid; Measure the TCID of virus
50, treat that virus titer is stablized and reaches 10
8.5tCID
50rejuvenation is stopped, using this virus as production kind of a poison during/more than mL.
(7) microcarrier suspension culture of DF1 cell in stirring type bioreactor: get well-grown DF1 cell on rolling bottle, clean twice with the PBS that pH value is 7.2, add the digestion of aforesaid EDTA-trysinization liquid, treat that cellular layer starts to occur that full wafer comes off, add described cell growth medium, blow to hang and make cell suspending liquid, cell counting density is by 10
6in individual/mL reaction of inoculation device.Bio-reactor is set as follows parameter: temperature 37 DEG C, pH value 7.2, stirring velocity 50rpm, dissolved oxygen content 50% carries out reactor and automatically control to cultivate.
(8) breeding of seedling venom: cultivating 48h observation cells on microcarriers and substantially cover with (more than 85%), is 0.8 virus inoculation with MOI.The culture parameters such as design temperature 37 DEG C, pH value 7.2, stirring velocity 60rpm, dissolved oxygen content 60%, carry out reactor and automatically control to cultivate.Sample at regular intervals after connecing poison, periodic monitor consumption sugar and residual sugar content, and by microscope observing cell pathology situation, detect the TCID of sample
50.When virus liquid is tired the highest, observe cells on microcarriers and substantially all come off (about 36h), and the obvious ascendant trend of DO value, stopped reaction device stirs, and after microcarrier all sinks to reactor bottom, gathers in the crops supernatant liquor and microcarrier respectively.
(9) process of virus liquid is gathered in the crops: after the packing of results supernatant ,-20 DEG C of freezen protective are for subsequent use.
Embodiment 3
Utilize stirring type bioreactor to produce a method for infectious bursa of Fabricius virus, comprise the following steps:
1) in stirring type bioreactor, mixed by the microcarrier after sterilizing, obtain mixed culture medium with the consumption of 3g/L with steril cell growth media, the density inoculating DF1 cell is wherein 0.6 × 10
6individual/mL, makes DF1 cell adsorb cultivation on microcarrier, culture temperature 36 DEG C, pH7.2, dissolved oxygen 40%, stirring velocity 30rpm;
2) after inoculating DF1 cell 48h, discard the cell growth medium in mixed culture medium, add cell maintenance medium, the infectious bursa of Fabricius virus of access 0.3MOI, continue to cultivate;
3) after virus inoculation 48h, results virus liquid.
On the basis of above technical scheme, meet the following conditions:
Step 1) in mixed culture medium cumulative volume be 40% of stirring type bioreactor cubic capacity.
Step 1) in before inoculating cell reactor first stir 20min.
Step 1) to plant described microcarrier be sterilizing by the following method:
A) with PBS damping fluid, more than 4h is soaked to microcarrier;
B) by steps A) soak after microcarrier PBS buffer solution for cleaning;
C) by step B) cleaning after microcarrier 115 DEG C of sterilizing 25min under PBS damping fluid soaking conditions.
Step 1) in for the DF1 cell inoculated first through the digestion of EDTA-trysinization liquid, then inoculate; Described EDTA-trysinization liquid is the PBS damping fluid of pancreatin and 0.02% (w/v) EDTA containing 0.03% (w/v).
Step 1) new-born calf serum of DMEM/F12 substratum and 10% (v/v) containing 90% (v/v) in cell growth medium; Before described cell growth medium inoculation DF1 cell, first in cell growth medium, add benzylpenicillin sodium and Vetstrep, to the whole content 100IU/mL of benzylpenicillin sodium, the whole content 100IU/mL of Vetstrep.
Step 2) in DF1 cell is to the rate that occupies of micro-carrier surface higher than 85% when discarding in mixed culture medium cell growth medium, the empty ball rate of microcarrier is lower than 5%.
Step 3) in results virus liquid time virus titer the highest.
Embodiment 4
Utilize stirring type bioreactor to produce a method for infectious bursa of Fabricius virus, comprise the following steps:
1) in stirring type bioreactor, the microcarrier after sterilizing is mixed with steril cell growth media with the consumption of 6g/L, obtains mixed culture medium, inoculate density to 1.2 × 10 of DF1 cell wherein
6individual/mL, makes DF1 cell adsorb cultivation on microcarrier, culture temperature 37 DEG C, pH7.3, dissolved oxygen 60%, stirring velocity 80rpm;
2) after inoculating DF1 cell 72h, discard the cell growth medium in mixed culture medium, add cell maintenance medium, the infectious bursa of Fabricius virus of access 1.2MOI, continue to cultivate;
3) after virus inoculation 24h, results virus liquid.
On the basis of above technical scheme, meet the following conditions:
Step 1) in mixed culture medium cumulative volume be 60% of stirring type bioreactor cubic capacity.
Step 1) in before inoculating cell reactor first stir 60min.
Step 1) to plant described microcarrier be sterilizing by the following method:
A) with PBS damping fluid, more than 4h is soaked to microcarrier;
B) by steps A) soak after microcarrier PBS buffer solution for cleaning;
C) by step B) cleaning after microcarrier 125 DEG C of sterilizing 35min under PBS damping fluid soaking conditions.
Step 1) in for the DF1 cell inoculated first through the digestion of EDTA-trysinization liquid, then inoculate; Described EDTA-trysinization liquid is the PBS damping fluid of pancreatin and 0.02% (w/v) EDTA containing 0.05% (w/v).
Embodiment 5
Utilize stirring type bioreactor to produce a method for infectious bursa of Fabricius virus, comprise the following steps:
1) in stirring type bioreactor, mixed by the microcarrier after sterilizing, obtain mixed culture medium with the consumption of 5g/L with steril cell growth media, the density inoculating DF1 cell is wherein 0.9 × 10
6individual/mL, makes DF1 cell adsorb cultivation on microcarrier, culture temperature 36.5 DEG C, pH7.2, dissolved oxygen 50%, stirring velocity 60rpm;
2) after inoculating DF1 cell 60h, discard the cell growth medium in mixed culture medium, add cell maintenance medium, the infectious bursa of Fabricius virus of access 0.8MOI, continue to cultivate;
3) after virus inoculation 36h, results virus liquid.
Above embodiments of the invention have been described in detail, but described content is only preferred embodiment of the present invention, not in order to limit the present invention.All make in application range of the present invention any amendment, equivalent to replace and improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. utilize stirring type bioreactor to produce a method for infectious bursa of Fabricius virus, it is characterized in that comprising the following steps:
1) in stirring type bioreactor, mixed by the microcarrier after sterilizing, obtain mixed culture medium with the consumption of 3 ~ 6g/L with steril cell growth media, the density inoculating DF1 cell is wherein 0.6 ~ 1.2 × 10
6individual/mL, makes DF1 cell adsorb cultivation on microcarrier, culture temperature 36 ~ 37 DEG C, pH7.2 ~ 7.3, dissolved oxygen 40 ~ 60%, stirring velocity 30 ~ 80rpm;
2) after inoculating DF1 cell 48 ~ 72h, discard the cell growth medium in mixed culture medium, add cell maintenance medium, the infectious bursa of Fabricius virus of access 0.3 ~ 1.2MOI, continue to cultivate;
3) after virus inoculation 24 ~ 48h, results virus liquid.
2. method according to claim 1, is characterized in that step 1) in mixed culture medium cumulative volume be 40 ~ 60% of stirring type bioreactor cubic capacity.
3. method according to claim 1, is characterized in that step 1) in before inoculating cell reactor first stir 20 ~ 60min.
4. method according to claim 1, is characterized in that step 1) to plant described microcarrier be sterilizing by the following method:
A) with PBS damping fluid, more than 4h is soaked to microcarrier;
B) by steps A) soak after microcarrier PBS buffer solution for cleaning;
C) by step B) cleaning after microcarrier 115 ~ 125 DEG C of sterilizing 25 ~ 35min under PBS damping fluid soaking conditions.
5. method according to claim 1, is characterized in that step 1) in for the DF1 cell inoculated first through the digestion of EDTA-trysinization liquid, then inoculate.
6. method according to claim 5, is characterized in that described EDTA-trysinization liquid is the PBS damping fluid of pancreatin and 0.02% (w/v) EDTA containing 0.03 ~ 0.05% (w/v).
7. method according to claim 1, is characterized in that step 1) new-born calf serum of DMEM/F12 substratum and 10% (v/v) containing 90% (v/v) in cell growth medium.
8. method according to claim 7, it is characterized in that before described cell growth medium inoculation DF1 cell, first in cell growth medium, add benzylpenicillin sodium and Vetstrep, to the whole content 100IU/mL of benzylpenicillin sodium, the whole content 100IU/mL of Vetstrep.
9. method according to claim 1, is characterized in that step 2) in DF1 cell is to the rate that occupies of micro-carrier surface higher than 85% when discarding in mixed culture medium cell growth medium, the empty ball rate of microcarrier is lower than 5%.
10. method according to claim 1, is characterized in that step 3) in results virus liquid time virus titer reach the highest.
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