CN107043740A - A kind of MDBK cell culture fluids and preparation method thereof, application method - Google Patents
A kind of MDBK cell culture fluids and preparation method thereof, application method Download PDFInfo
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Abstract
A kind of MDBK cell culture fluids and preparation method thereof, application method, belong to veterinary biologicses technical field.Contain following components in the every 1000ml deionized waters of the cell culture fluid：DMEM powder, transferrins liquid storage, bFGF liquid storages, EGF liquid storages, IL 3, propolis flavone, astragaloside, penicillin, streptomysin.The invention has the advantages that：MDBK cell culture fluids used in the present invention, greatly reduce the usage amount of serum, reduce the cost of cell culture；Improve MDBK vitro growth rates；Strengthen BVDV to the sensitiveness of MDBK cells, improve viral level；The immunogenicity of BVD MD inactivated vaccines can also be increased substantially by MDBK cell culture fluids used in the present invention.
Description
Technical field
The invention belongs to veterinary biologicses technical field, and in particular to a kind of MDBK cell culture fluids and its preparation side
Method, application method.
Background technology
MDBK cells are tire bovine kidney cells, can continuous passage culture, this cell is mainly used in the culture of virus, particularly ox
Virus diarrhea/mucosal virus（BVDV）It is particularly sensitive to this cell.
Bovine viral diarrhoea/mucosal disease viral disease（BVD-MD）It is by bovine viral diarrhoea/mucosal virus（BVDV）It is caused
A kind of acute, the hot infectious disease of ox, it, which faces to examine, is characterized as mucosa irritation, erosion, necrosis, diarrhoea and Pregnant cows miscarriage,
The disease is classified as three class epidemic diseases by current China.Control BVD-MD main method is to carry out immune prevention and control using vaccine at present,
The quality and security of vaccine depend greatly on the mode of production and process of vaccine, and BVD-MD vaccines are typically big rule
Mould culture cell harvesting virus liquid, but it is at present more use the nutrient solution culture cells containing serum, the complexity of serum component and not
Certainty, and wherein there is the potential risk of the microbiological contaminations such as virus, affect viral vaccine production technology and
Vaccine quality.Consider from viral vaccine production technology angle, the mass discrepancy between the complexity and batch of serum component is added
The unstability of viral vaccine production and the difficulty of control of product quality；Consider from the safety perspective of viral vaccine, in serum
Potential pathogenic microorganism, the use to vaccine brings the potential safety hazard that can not be ignored.
As increased using the cell cultivation process and the uncertain factor of cell expression product security caused by serum
Realistic problem, the development reason as the research of animal cell non-serum culture technique and application.Serum-free medium has many
Advantage：Simplify the isolating and purifying of downstream purpose product, it is cost-effective；The otherness between serum batch is avoided, cell culture is improved
Stability and result reliability；Serum is avoided to the toxic action and serum source contact scar of cell；Serum-free medium by
In the relative determination of adding ingredient, available for researchs such as research cell growth, propagation, differentiation, metabolism and gene regulations.
The content of the invention
The problem of existing for prior art, a kind of MDBK cell culture fluids and its system are provided present invention aims at design
The technical scheme of Preparation Method, application method.
Described a kind of MDBK cell culture fluids, it is characterised in that contain following components in per 1000ml deionized waters：
DMEM powder 9-12 g, transferrins liquid storage 1-3 ml, bFGF liquid storage 0.8-4 ml, EGF liquid storages 0.8-4 ml, IL-3 1-10
Ug, propolis flavone 2-30 mg, astragaloside 20-60 mg, the units of penicillin 5-10 ten thousand, streptomysin 5-10mg.
Described a kind of MDBK cell culture fluids, it is characterised in that contain following components in per 1000ml deionized waters：
DMEM powder 10-12 g, transferrins liquid storage 1.2-2.6 ml, bFGF liquid storage 1.2-2 ml, EGF liquid storage 1.2-2 ml, IL-3
2-8 ug, propolis flavone 6-25 mg, astragaloside 30-50 mg, the units of penicillin 6-8 ten thousand, streptomysin 6-8mg.
Described a kind of MDBK cell culture fluids, it is characterised in that described transferrins liquid storage passes through following steps system
：Using volume ratio as 1:1 physiological saline and 90% glycerine dissolving transferrins, compound concentration is 15mg/ml liquid storage, is put
It is standby in -20 DEG C.
Described a kind of MDBK cell culture fluids, it is characterised in that described bFGF, EGF liquid storage passes through following steps system
：Using volume ratio as 1:1 physiological saline and 90% glycerine dissolve bFGF, EGF respectively, and concentration is made into respectively for 50 ug/
Ml, 30 ug/ml liquid storage, be placed in -20 DEG C it is standby.
The preparation method of described a kind of MDBK cell culture fluids, it is characterised in that comprise the following steps：
1）Using volume ratio as 1:1 physiological saline and 90% glycerine dissolving transferrins, compound concentration is 15mg/ml storage
Liquid, be placed in -20 DEG C it is standby；
2）Using volume ratio as 1:1 physiological saline and 90% glycerine dissolve bFGF, EGF respectively, and it is 50 that concentration is made into respectively
Ug/ml, 30 ug/ml liquid storage, be placed in -20 DEG C it is standby；
3）Take the component of the formula ratio, be added in deionized water, it is stirring while adding, until be completely dissolved, then mend go from
The quantitative 1000ml of sub- water, and filtration sterilization is carried out by 0.22um bacterial filters, it is standby that packing is carried out afterwards.
The application method of described a kind of MDBK cell culture fluids, it is characterised in that comprise the following steps：It is biological in 2.5L
200-500ml MDBK cell culture fluids are added in reactor, the PH of the sodium acid carbonate for being 8.8% with concentration adjustment nutrient solution is
7.0-7.4, by 2 × 105-5×105Individual/ml initial cell densities inoculation, is stirred with 800-1200r/min mixing speed
Mix, be placed at 37 DEG C and cultivate 72-80h, by 1:3-1:5 carry out Secondary Cultures, and method Secondary Culture is to 10-15 generations according to this, you can obtain
The MDBK cells of stable suspersion culture can be carried out by obtaining.
The application method of described a kind of MDBK cell culture fluids, it is characterised in that be linked into BVDV virus liquids stable outstanding
In the MDBK cells of floating culture, suspension culture is carried out with 800-1200r/min mixing speed, is placed at 37 DEG C and cultivates 48-56h
Afterwards, treat that cytopathy reaches more than 75% progress harvest virus liquid.
Each component is existing product in the present invention, wherein DMEM powder, transferrins, bFGF, EGF, penicillin, strepto-
Element and IL-3 are bought in Sigma；Propolis flavone is bought in Shaanxi Sen Fu natural products Co., Ltd；Astragaloside is bought in Shanghai
The triumphant chemical Science and Technology Ltd. of one hundred generation.
The invention has the advantages that：
1st, the MDBK cell culture fluids prepared by the present invention, are no added serum free culture system liquid, can both be greatly lowered and be produced into
This, in turn ensure that the quality and security of vaccine.
2nd, the MDBK cell culture fluids prepared by the present invention, can also greatly improve the speed of growth of cell.
3rd, the MDBK cell culture fluids prepared by the present invention, can improve infection sensibilities of the BVDV to MDBK cells, carry
Its high viral level, so as to also improve the immunogenicity of BVD-MD inactivated vaccines.
4th, the MDBK cell culture fluids prepared by the present invention, improve the adaptability of MDBK cells, BVDV to the culture that suspends,
So as to improve the reproductive efficiency of virus.
Embodiment
In order that the present invention is easier to understand, with reference to specific embodiment, the present invention is expanded on further.It should be understood that
These embodiments are only illustrative of the invention and is not intended to limit the scope of the invention, NM specific reality in the following example
Proved recipe method, generally routinely experimental method is carried out.
Embodiment 1：The preparation of nutrient solution
DMEM powder：9 g (buy in：Sigma )
Transferrins liquid storage：1ml (transferrins buy in：Sigma )
BFGF liquid storages:0.8 ml (bFGF buy in：Sigma )
EGF liquid storages:0.8 ml (EGF buy in：Sigma )
IL-3：1ug (buy in：Sigma )
Propolis flavone：2 mg （Buy in：Shaanxi Sen Fu natural products Co., Ltd）
Astragaloside：20 mg （Buy in：The triumphant chemical Science and Technology Ltd. of the generation of Shanghai one hundred）
Penicillin：50000 units (buy in：Sigma)
Streptomysin：5 mg (buy in：Sigma)
Above-mentioned transferrins liquid storage is made by following steps：Using volume ratio as 1:1 physiological saline and 90% glycerine are molten
Solve transferrins, compound concentration be 15mg/ml liquid storage, be placed in -20 DEG C it is standby.
Above-mentioned bFGF, EGF liquid storage is made by following steps：Using volume ratio as 1:1 physiological saline and 90% sweet
Oil does not dissolve bFGF, EGF, and concentration is made into respectively for 50 ug/ml, 30 ug/ml liquid storage, be placed in -20 DEG C it is standby.
Any of the above composition is sufficiently mixed, is added in deionized water, it is stirring while adding, until being completely dissolved, then
The quantitative 1000ml of deionized water is mended, and filtration sterilization is carried out by 0.22um bacterial filters, packing is carried out afterwards standby.
Embodiment 2：The preparation of nutrient solution
DMEM powder：10 g (buy in：Sigma )
Transferrins liquid storage：1.2 ml (buy in：Sigma )
BFGF liquid storages:1.4 ml (bFGF buy in：Sigma )
EGF liquid storages:1.6 ml (EGF buy in：Sigma )
IL-3：5 ug (buy in：Sigma )
Propolis flavone：9mg （Buy in：Shaanxi Sen Fu natural products Co., Ltd）
Astragaloside：35mg （Buy in：The triumphant chemical Science and Technology Ltd. of the generation of Shanghai one hundred）
Penicillin：80000 units (buy in：Sigma)
Streptomysin：8 mg (buy in：Sigma)
Above-mentioned transferrins liquid storage is made by following steps：Using volume ratio as 1:1 physiological saline and 90% glycerine are molten
Solve transferrins, compound concentration be 15mg/ml liquid storage, be placed in -20 DEG C it is standby.
Above-mentioned bFGF, EGF liquid storage is made by following steps：Using volume ratio as 1:1 physiological saline and 90% sweet
Oil does not dissolve bFGF, EGF, and concentration is made into respectively for 50 ug/ml, 30 ug/ml liquid storage, be placed in -20 DEG C it is standby.
Any of the above composition is sufficiently mixed, is added in deionized water, it is stirring while adding, until being completely dissolved, then
The quantitative 1000ml of deionized water is mended, and filtration sterilization is carried out by 0.22um bacterial filters, packing is carried out afterwards standby.
Embodiment 3：The preparation of nutrient solution
DMEM powder：12 g (buy in：Sigma )
Transferrins liquid storage：3 ml (buy in：Sigma )
BFGF liquid storages:4 ml (bFGF buy in：Sigma )
EGF liquid storages:4 ml (EGF buy in：Sigma )
IL-3：10 ug (buy in：Sigma )
Propolis flavone： 30 mg （Buy in：Shaanxi Sen Fu natural products Co., Ltd）
Astragaloside： 60 mg （Buy in：The triumphant chemical Science and Technology Ltd. of the generation of Shanghai one hundred）
Penicillin：100000 units (buy in：Sigma)
Streptomysin：10mg (buy in：Sigma)
Above-mentioned transferrins liquid storage is made by following steps：Using volume ratio as 1:1 physiological saline and 90% glycerine are molten
Solve transferrins, compound concentration be 15mg/ml liquid storage, be placed in -20 DEG C it is standby.
Above-mentioned bFGF, EGF liquid storage is made by following steps：Using volume ratio as 1:1 physiological saline and 90% sweet
Oil does not dissolve bFGF, EGF, and concentration is made into respectively for 50 ug/ml, 30 ug/ml liquid storage, be placed in -20 DEG C it is standby.
Any of the above composition is sufficiently mixed, is added in deionized water, it is stirring while adding, until being completely dissolved, then
The quantitative 1000ml of deionized water is mended, and filtration sterilization is carried out by 0.22um bacterial filters, packing is carried out afterwards standby.
The application of test example nutrient solution of the present invention
1 material
1.1 MDBK cell culture fluids
It is made by 1-3 of the embodiment of the present invention
1.2 DMEM basic culture solutions, hyclone are purchased from GIBCO companies,
THIOGLYCOLLIC ACID salt nutrient solution (TG), pancreas casein peptone nutrient solution（TSB）It is purchased from Beijing Zhonghai Biotech Co., Ltd.
1.3 MDBK cells are purchased from ATCC, are preserved by Zhejiang Mei Baolong Bioisystech Co., Ltd
1.4 bovine viral diarrhoeas/mucosal virus（BVDV）, it is purchased from China Veterinery Drug Inspection Office
The age in days cleaning grade Kunming white mouse of 1.5 experimental animal 15, is purchased from Beijing magnificent experimental animal technology of dimension tonneau limited
Company
2 methods
2.1 nutrient solutions are examined
2.1.1 steriling test
MDBK cell culture fluids prepared by embodiment 1-3, respectively take 3 different batches to carry out steriling test, each batch difference
Take 1ml to be inoculated in 2 big pipes of 50ml TG, 1 is placed in 35-37 DEG C of culture, 1 be placed in 23-25 DEG C of culture, 3d after respectively draw
Culture is inoculated in 10 TG tubules respectively, then is respectively placed in 35-37 DEG C and 23-25 DEG C culture, and each batch takes respectively in addition
0.2ml is inoculated in TSB tubules, is placed in 23-25 DEG C of culture, while doing negative control, is cultivated 7, is observed result.
2.1.2 endotoxin measurement
MDBK cell culture fluids prepared by embodiment 1-3, respectively take 3 different batches to carry out endotoxin inspection, each batch point
Do not take 1.7ml to be detected respectively with tachypleus amebocyte lysate box, its OD value is surveyed with ELIASA selection wavelength 405nm, while doing negative control.
The domestication of 2.2 cells and the measure of the speed of growth
First, the culture domestication that suspends is carried out to MDBK cell applications bioreactor, be separately added into 2.5L bioreactors
The MDBK cell culture fluids that 300ml is prepared by embodiment 1-3, use sodium acid carbonate（8.8%）The PH for adjusting nutrient solution is 7.0-7.4,
By 2 × 105-5×105Individual/ml initial cell densities inoculation, is stirred with 800-1200r/min mixing speed, is placed in 37
72-80h is cultivated at DEG C, by 1:3-1:5 carry out Secondary Culture, and method Secondary Culture is to 10-15 generations according to this, and acquisition can carry out stabilization
Suspend the MDBK cells cultivated.
Be separately added into 2.5L bioreactors MDBK cell culture fluids that 300ml prepared by embodiment 1-3, containing 8%,
10%FBS complete culture solution, respectively by 3 × 105Individual/ml initial cell densities inoculation, with 800-1200r/min stirring speed
Degree is stirred, and is placed at 37 DEG C and is cultivated 72-80h.
The cell culture samples of each culture that suspends uniformly are drawn, is removed by 1000r/min centrifugations after culture supernatant, uses PBS
Cleaning 3 times, free cell is obtained after digesting 10 min in 37 DEG C through 0. 25% trypsin solution.With trypan blue staining, blood is used
Ball count plate count respectively extremely, viable count, calculate cell density and survival rate.
2.3 Virus culture
BVDV is diluted to 1500 TCID with DMEM basic culture solutions50/ ml, is inoculated into thin by 5 kinds of MDBK of 2.2 cultures respectively
In born of the same parents' microcarrier culture, it is stirred, is placed at 37 DEG C with 800-1200r/min mixing speed, after culture culture 50h
Carry out receiving poison.
2.4 TCID50 are determined
The DMEM nutrient solutions by the cell of the embodiment 1-3 MDBK cell culture fluid cultures prepared, containing 8%, 10%FBS are trained respectively
Foster cell, is diluted to 105~106Individual/m L, are transferred in 96 well culture plates by 0. 2 m L/hole, are placed in 37 DEG C, 5%
2d is cultivated in incubator, nutrient solution supernatant is discarded, by 2.3 harvest BVDV respectively with DMEM basic culture solutions make continuous 10 times it is dilute
Release, be inoculated with by 0.2 mL/hole, be placed in 37 DEG C, 5% incubator and cultivate 4d, cytopathy is observed daily and is recorded, is pressed
Reed-Muench methods calculate TCID50。
2.5 immunogenicity determining
Five kinds of virus liquids of 2.3 harvests are qualified through examining, the immunogenicity that vaccine determines vaccine is made after inactivation.By 60
15 age in days cleaning grade small white mouses are divided into 6 groups, and every group 10, the Ith group uses the nutrient solution prepared through the embodiment of the present invention 1, uses
In culture MDBK cell proliferation BVDV, the vaccine being made after inactivating emulsification treatment, carries out hypodermic injection and exempted from according to 0.5ml/ only
Epidemic disease；IIth group uses the nutrient solution prepared through the embodiment of the present invention 2, for cultivating MDBK cell proliferation BVDV, is emulsified through inactivation
The vaccine being made after processing, carries out hypodermic injection immune according to 0.5ml/ only；IIIth group through the embodiment of the present invention 3 using preparing
Nutrient solution, for cultivating MDBK cell proliferation BVDV, the vaccine that is made after inactivating emulsification treatment, enters according to 0.5ml/ only
Row is subcutaneously injected immune；IVth group uses nutrient solution containing 10%FBS, for cultivating MDBK cell proliferation BVDV, through inactivating at emulsification
The vaccine being made after reason, carries out hypodermic injection immune according to 0.5ml/ only；Vth group uses nutrient solution containing 8%FBS, for cultivating
MDBK cell proliferation BVDV, the vaccine being made after inactivating emulsification treatment, carries out hypodermic injection immune according to 0.5ml/ only.The
IV group, vaccine control group, without immune.Each group is before immune and immune 7th d, 14d, 21d, 28d, 35d takes a blood sample
And serum is separated, the antibody level of each group is measured with ELISA method.
3 results
3.1 assay
The steriling test result of table 1
The endotoxin assay of table 2（405nm）
It can be seen from table 1, table 2 by embodiment 1,2,3 prepare different batches nutrient solution not only asepsis growth again without in
Toxin, nutrient solution prepared by this method is safe and reliable.
3.2 cell densities and vitality test result
The cell density measurement result of table 3
As can be seen from Table 3, the MDBK cell densities of the nutrient solution culture prepared by embodiment 1,2,3 are significantly greater than containing FBS trainings
The MDBK cells of nutrient solution culture.
The Cell viability measurement result of table 4
As can be seen from Table 4, in identical incubation time, the MDBK cells of the nutrient solution culture prepared by embodiment 1,2,3
Survival rate is significantly greater than the MDBK cells by the culture of nutrient solution containing FBS.
3.3 TCID50 measurement results
The TCID50 measurement results of table 5
As can be seen from Table 5, the MDBK cells ratio of the culture solution additive culture prepared by embodiment 1,2,3 containing FBS by cultivating
The MDBK cells of liquid culture are more beneficial for BVDV breeding, and sensitiveness is stronger.
3.4 immunogenicity determinations
The PRRSV of table 6 antibody level measurement result
As can be seen from Table 6, I, II, III group of antibody level apparently higher than IV, V group, the culture prepared by embodiment 1,2,3
The BVDV of liquid culture immunogenicity is higher than the BVDV of the culture of nutrient solution containing FBS.
4 conclusions
In summary, MDBK cell culture fluids used in the present invention, in the premise for the speed of growth that can greatly improve cell
Under, it on the one hand can strengthen sensitiveness of the BVDV to MDBK cells, improve viral level, on the other hand, BVD-MD can also be strengthened
The immunogenicity of inactivated vaccine.
Claims (7)
1. a kind of MDBK cell culture fluids, it is characterised in that contain following components in per 1000ml deionized waters：DMEM powder 9-
12 g, transferrins liquid storage 1-3 ml, bFGF liquid storage 0.8-4 ml, EGF liquid storages 0.8-4 ml, IL-3 1-10 ug, propolis are yellow
Ketone 2-30 mg, astragaloside 20-60 mg, the units of penicillin 5-10 ten thousand, streptomysin 5-10mg.
2. a kind of MDBK cell culture fluids as claimed in claim 1, it is characterised in that contain in per 1000ml deionized waters with
Lower component：DMEM powder 10-12 g, transferrins liquid storage 1.2-2.6 ml, bFGF liquid storage 1.2-2 ml, EGF liquid storages 1.2-2
Ml, IL-3 2-8 ug, propolis flavone 6-25 mg, astragaloside 30-50 mg, the units of penicillin 6-8 ten thousand, streptomysin 6-8mg.
3. a kind of MDBK cell culture fluids as claimed in claim 1 or 2, it is characterised in that described transferrins liquid storage passes through
Following steps are made：Using volume ratio as 1:1 physiological saline and 90% glycerine dissolving transferrins, compound concentration is 15mg/
Ml liquid storage, be placed in -20 DEG C it is standby.
4. a kind of MDBK cell culture fluids as claimed in claim 1 or 2, it is characterised in that described bFGF, EGF liquid storage passes through
Following steps are made：Using volume ratio as 1:1 physiological saline and 90% glycerine dissolve bFGF, EGF respectively, and concentration is made into respectively
For 50 ug/ml, 30 ug/ml liquid storage, be placed in -20 DEG C it is standby.
5. a kind of preparation method of MDBK cell culture fluids as claimed in claim 1 or 2, it is characterised in that including following step
Suddenly：
1）Using volume ratio as 1:1 physiological saline and 90% glycerine dissolving transferrins, compound concentration is 15mg/ml storage
Liquid, be placed in -20 DEG C it is standby；
2）Using volume ratio as 1:1 physiological saline and 90% glycerine dissolve bFGF, EGF respectively, and it is 50 that concentration is made into respectively
Ug/ml, 30 ug/ml liquid storage, be placed in -20 DEG C it is standby；
3）Take the component of the formula ratio, be added in deionized water, it is stirring while adding, until be completely dissolved, then mend go from
The quantitative 1000ml of sub- water, and filtration sterilization is carried out by 0.22um bacterial filters, it is standby that packing is carried out afterwards.
6. a kind of application method of MDBK cell culture fluids as claimed in claim 1 or 2, it is characterised in that including following step
Suddenly：200-500ml MDBK cell culture fluids are added in 2.5L bioreactors, are adjusted with concentration for 8.8% sodium acid carbonate
The PH of nutrient solution is 7.0-7.4, by 2 × 105-5×105Individual/ml initial cell densities inoculation, with 800-1200r/min stirring
Speed is stirred, and is placed at 37 DEG C and is cultivated 72-80h, by 1:3-1:5 carry out Secondary Cultures, and method Secondary Culture is to 10-15 according to this
Generation, you can acquisition can carry out the MDBK cells of stable suspersion culture.
7. a kind of application method of MDBK cell culture fluids as claimed in claim 6, it is characterised in that connect BVDV virus liquids
Enter into the MDBK cells of stable suspersion culture, suspension culture is carried out with 800-1200r/min mixing speed, is placed at 37 DEG C
Cultivate after 48-56h, treat that cytopathy reaches more than 75% progress harvest virus liquid.
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Application publication date: 20170815 |