CN105505853B - A low-serum medium for high-density suspension culture of BHK-21 cells and its application in the proliferation of foot-and-mouth disease virus - Google Patents
A low-serum medium for high-density suspension culture of BHK-21 cells and its application in the proliferation of foot-and-mouth disease virus Download PDFInfo
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- CN105505853B CN105505853B CN201510980603.7A CN201510980603A CN105505853B CN 105505853 B CN105505853 B CN 105505853B CN 201510980603 A CN201510980603 A CN 201510980603A CN 105505853 B CN105505853 B CN 105505853B
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Abstract
The invention discloses a kind of low blood serum medium for the suspension culture of BHK-21 high cell densities and its applications in foot and mouth disease virus Multiplying culture.The present invention is adjusted by metaboilic levels of nutriments such as glucose in research BHK-21 cell growth process and mutually, the low blood serum medium of personalized BHK-21 cell is proposed, amino acid moiety, vitamin moieties, balance salt part and other additive parts are included.It is used for the high density suspension culture of BHK-21 cell using culture medium of the present invention, cell can be greatly improved to the effective rate of utilization of nutriment, metabolic by-product concentration is reduced, to improve cell culture efficiency；Furthermore, the BHK-21 cell obtained using culture medium culture of the present invention, FMDV different serotypes sensibility is increased, viral Effective Antigens 146S content is greatly improved, it can reach the high efficiency, the controllability of cell growth and the purpose of metabolic by-product concentration minimumization of cell growth, keep the production efficiency of biological products higher, production cost is lower, has significant economic benefit.
Description
Technical field
The present invention relates to a kind of developments of the low serum personalization culture medium of suspension high density and its in mouth entirely of BHK-21 cell
Application in aphtovirus proliferation, belongs to field of biological pharmacy for animals.
Background technique
With the fast development of biological products technology, the existing inactivated foot-and-mouth disease vaccine production technology in China, from using tradition
Rolling bottle attached cell culture, develop for bioreactor mass cell suspend cultivate.It is advised greatly using bioreactor
Mould cell culture can increase substantially the specific yield of scale animal cell culture, realize High Density Cultivation, the height of cell
Density expression, while can simplify production technology, production cost is reduced, guarantees the product quality of large-scale production.And it commonly trains
Feeding base is unable to satisfy the High Density Cultivation of cell in reactor, can not the unique training method of supporting reactions device.In foreign countries
Personalized culture medium has become the mainstream of culture medium development, has been commonly used and has generated great economic benefit.Suitable training
The culture density and Cell viability of cell can be improved in feeding based formulas, improves viral yield.Therefore how to be trained according to scale cell
The difference of technique is supported, personalized culture medium is researched and developed, improves stability, the output capacity of target product, be the research of current this field
Hot and difficult issue.
The present invention is according to existing cell strain, cell culture mode, purpose product and production technology, cell metabolism and nutrition
The demand of substance establishes culture medium design scheme, establishes the detection method of various nutriments, analyzes cell metabolism stream, design
Culture medium prescription, compared with being commercialized culture medium, serum content reduces by 30%, and cell proposes the effective rate of utilization of nutriment
Height, metabolic by-product concentration reduce, and improve the culture density and Cell viability of BHK-21 cell, and cell production capacity increases by 28%,
It being proliferated through foot and mouth disease virus, cell increases FMDV different serotypes sensibility, and antigenic content is stable and increase rate is up to 43%,
To guarantee to take into account the high density level of culture cell under stable cell cultivation process and produce the highly concentrated of viral antigen
Degree, produces the viral vaccine of high quality, lays a good foundation for the raising and industrialization production of target product quality.
Summary of the invention
An object of the present invention be to provide it is a kind of low cost, ingredient more explicitly be suitable for BHK-21 high cell densities
Suspend the low serum personalization culture medium cultivated.
The second object of the present invention is to provide purposes of the culture medium in the suspension culture of BHK-21 high cell densities.
The third object of the present invention is to provide purposes of the culture medium in virus multiplication.
In order to achieve the above object, present invention employs following technological means:
1, culture medium strategy process is developed to be completed by following steps:
(1), the preparation and preparation of cell base culture solution
It weighs MEM culture medium powder to be dissolved in a certain amount of distilled water (distillation coolant-temperature gage is lower than 40 DEG C), dissolution is sufficiently stirred,
With sodium hydroxide or salt acid for adjusting pH value to 6.95~7.15, last constant volume is up to BHK-21 cell suspension cultures culture medium.
(2), cell recovery and culture
Take one, cell from suspension BHK-21 cell work library, after recovery stationary culture in T100 cell square vase, pass 2~
3 generations, cell use trypsin digestion after covering with single layer, add appropriate cell culture fluid and be transferred in 1000ml triangular flask, and 37 DEG C
Oscillation, which suspends, cultivates, and shaking speed 90r/min, pH value is 7.0~7.2, per daily NaHCO3Adjust pH value.To cell culture body
Product reach specified quantity after, collect cell, be transferred in 5L bioreactor and cultivate, cultivate setup parameter: temperature be 37.0~
37.5 DEG C, pH value is that 7.0~7.2, DO value is 60.0%~80.0%, and speed of agitator is 80~120r/min, culture 50h passage
Culture.
(3), the control of BHK-21 cell cell growth metabolism parameter in basal medium
1. batch experiments are carried out in 5L bioreactor by obtained cell in above-mentioned steps 2, with cell density and carefully
Born of the same parents' motility rate be evaluation index, culture 0h, for 24 hours, 48h, 72h and 96h take cell sample, be centrifuged under the conditions of 4 DEG C, 1000rpm
5min leaves and takes cell supernatant；
2. above-mentioned 1. gained cell supernatant is carried out Major Nutrient substance glucose and glutamine tests and analyzes, grape
Sugar is measured with glutamine concentration and using 100 Biochemical Analyzer of NOVA PLUS；
3. above-mentioned 1. gained cell supernatant to be carried out to the detection and analysis of Major Nutrient substance amino acid, amino acid concentration is adopted
With pre-column derivatization, the analysis of superelevation liquid chromatograph, derivative reagent uses dilution in acetonitrile molten to certain density PITC and TEA
Liquid, it is spare；
4. above-mentioned 1. gained cell supernatant to be carried out to the detection and analysis of main metabolic by-product ammonia and lactic acid, using NOVA
The detection of 100 Biochemical Analyzer of PLUS.
(4), the determination of culture medium prescription
In conjunction with the cell metabolism stream monitoring result of above-mentioned steps (3), the main each battalion for influencing cell growth metabolism is determined
The content of feeding substance, especially concentration of glucose are not less than 16.0mmol/L, glutamine concentration is not less than 4.5mmol/L, and two
Person's concentration ratio about 3.5:1；Amino acid is not less than initial content 5% at the end of cell culture；Cellular process has been determined
In to the tolerable concentration of by-product, i.e. lactic acid concn is not higher than 8mmol/L not higher than 19mmol/L, ammonia density, so that it is determined that
The personalized low serum free culture system based formulas of BHK-21 cell, and it is named as ZN-MEM.
(5) the actual effect verifying of culture medium prescription
5.15L and 500L bioreactor continuous passage culture
The ZN-MEM culture solution that above-mentioned steps 4 are obtained carries out the outstanding of BHK-21 cell in 5L and 500L bioreactor
Floating culture, setup parameter: temperature is 37.0~37.5 DEG C, and pH value is that 7.0~7.2, DO value is 60.0%~80.0%, and stirring turns
Speed is 60~80r/min, culture 0h, for 24 hours, 48h, 72h and 96h cell sample, 800r/min is centrifuged, stays supernatant, adopt
It is analyzed with cytoanalyze, investigates BHK-21 cell under different scales condition of culture, cell density and Cell viability change feelings
Condition.
As a result: passing within culture 48 hours, cell density is 3.8 × 106A/ml or more, and its motility rate is above 94%.
The virus multiplication effect of 5.2 low serum free culture system cells
The cell for taking 500L bioreactor in above-mentioned steps 5.1 to harvest, be inoculated with respectively foot and mouth disease virus A type, it is O-shaped and
1 type of Asia, sets control parameter: for temperature as 36.5~37.0 DEG C, pH value is that 7.4~7.6, DO value is
60.0%~70.0%, speed of agitator is 40~60r/min.When cytopathy variability is up to 90% or more, harvest is viral,
The time of record harvest virus, routinely method measurement harvests the TCID of virus liquid50、LD50With Effective Antigens 146S content.
As a result: being detected, harvest the TCID of virus liquid50、LD50Meet vaccine industrialization production requirements, compared to routine
Method, cytopathy time shorten about 14%, and Effective Antigens 146S content improves 43%.
On the basis of the studies above, the invention proposes a kind of low blood for the suspension culture of BHK-21 high cell densities
Clear culture medium, is named as ZN-MEM, including amino acid moiety, balances salt part, vitamin moieties, other additives, serum with
And distilled water, pH value 7.0~7.2, wherein it is 1% (v/v) that serum, which adds concentration, and other each raw materials are in the medium
It is final concentration of:
1. amino acid moiety, comprising:
2. balancing salt part, comprising:
3. vitamin moieties, comprising:
4. other additives, comprising:
In the present invention, it is preferred to, other each raw materials in the medium final concentration of: 1. amino acid moiety, packet
It includes:
2. balancing salt part, comprising:
3. vitamin moieties, comprising:
4. other additives, comprising:
Further, the use the invention also provides the culture medium in the suspension culture for carrying out BHK-21 cell
On the way.
In the present invention, it is preferred to, the suspension culture, condition of culture are as follows: inoculum density is 0.50 × 106A/
Ml, cultivation temperature are 37.0~37.5 DEG C, and pH value be that 7.0~7.2, DO value is 60.0%~80.0%, speed of agitator for 60~
80r/min, culture 48h are passed on.
Further, the invention also provides the culture mediums, and disease is being carried out using BHK-21 cell as host cell
Purposes in poison proliferation.
In the present invention, it is preferred to, the virus multiplication includes obtaining to through culture medium culture of the present invention
BHK-21 cell inoculation virus, sets control parameter are as follows: temperature is 36.5~37.0 DEG C, and pH value is that 7.4~7.6, DO value is
60.0%~70.0%, speed of agitator is 40~60r/min, carries out virus multiplication culture.
Wherein, the virus is preferred foot and mouth disease virus, more preferably FMD/O/MYA98/BY/2010, FMD/Re-A
WH/09 type or FMD/Asia1JSL/06.
The present invention is by research BHK-21 cell to the metaboilic level of the nutriments such as glucose, glutamine, amino acid
With multifactor mutual adjusting, the type of various composition, quantity and ratio in culture medium is varied or adjusted, optimizes cell culture ring
Border develops and is suitable for the low blood serum medium of the dedicated personalization of BHK-21 suspension cell, guarantees in stable cell culture
The viral vaccine of high quality is produced under journey.Compared with Current commercial culture medium, advantage is as follows:
1. cell quality is promoted
1) cell production capacity improves
Using personalized culture medium ZN-MEM of the invention, cell is substantially increased to the effective rate of utilization of nutriment,
Metabolic by-product concentration is reduced, cell growing environment is optimized, cell growth rate increases, and cultivates the whole density of cell significantly
It improves, by original 2.5 × 106Cells/ml increases to 3.8~5.0 × 106Cells/ml, it is closeer than commercially available culture medium culture cell
Degree increases nearly 1.5~2 times, and cell growth state is stablized, and cell production capacity increases by 28% or more；
2) cell activity improves
The surplus or shortage for avoiding nutriment, reduce the concentration of toxic metabolite by-product in culture solution, make cell
Nutrient environment optimizes, and efficient resource is recycled, while reducing apoptosis rate in scale cell cultivation process,
Cell activity improves；
3) cell amplification ratio improves
High cell growth speed, commercially available BH21 cell culture basal cell passage magnification ratio 1:4 or so, and optimizing application
Culture basal cell expand ratio 1:6~1:10, improve 40%, unit time inner cell output increased.Meanwhile cell expands
The raising of ratio, reduces power-equipment and pressurized equipment, substantially reduces cost, generates indirect economic effect.
2, vaccine antigen increased quality
Using BHK-21 cell as matrix, using personalized culture medium of the invention, cell culture environment is optimized, is improved
Cell density and Cell viability are cultivated, cell production capacity increases, sensibility and expression of the cell to FMD different shaped virus infection
It improves, so that the content of aftosa complete virus particle 146S is greatly enhanced, 146S content reaches the 2 of commercialization culture
Times or more, it lays a good foundation for the raising of aftosa product quality.
3, culture medium economic cost reduces
Currently, aftosa oil adjuvant killed vaccine used in animal aftosa immunization campaign, cell production accounts for its production
80% or more of direct cost.Using in routine culture basal cell's production process, average every liter of nutrient solution direct cost is 5.8
Member, using personalized culture medium culture cell of the invention, every liter of nutrient solution cost is reduced to 4.9 yuan, every liter of nutrient solution cell
Cost decline about 18%.To produce 1,500,000,000 milliliters of aftosa vaccine calculating year, about 7.5 hundred million milliliters of cell nutrient solution are needed, is produced per year
Raw about 600,000 yuan of direct economy or more.
In addition, serum-concentration reduces 30% than commercialization culture medium, production cost is reduced again indirectly, after alleviating
The continuous pressure for concentrating and purifying technique, reduces potential source biomolecule security risk that may be present in vaccine production process.
4, culture medium process for preparation simplification
Personalized culture medium process for preparation of the invention is simple, and the enhancing of reagent solubility does not need to put into a large amount of time
And labour, the investment of personnel has been saved to a certain degree.
To sum up, using personalized culture medium culture cell of the invention, cell culture characteristic is improved, and cell yield increases,
The Effective Antigens content and immune effect of vaccine are improved；Mouth is produced using the cell of personalized culture medium culture of the invention
Aphtovirus, production of vaccine direct cost substantially reduce, currently, the culture medium is applied successfully on my company's pilot production line,
And significant economic benefit is produced, it is with a wide range of applications.
Detailed description of the invention
Fig. 1 is required amino acid classes and content distribution figure in BHK-21 proliferation process；
Fig. 2 is amount of amino acid needed for cell Proliferation；
Fig. 3 is glucose amount needed for cell Proliferation；
Fig. 4 is the variation of cell metabolism by-product concentration；
Fig. 5 is culture cell stability passage figure under different scales；
Fig. 6 is BHK-21 cell cellular morphology in four kinds of culture mediums；
Fig. 7 is the growth curve of BHK-21 cell batch experiments in four kinds of culture mediums；
Fig. 8 is the variation of glucose and lactic acid concn during BHK-21 batch experiments；
Fig. 9 is the variation of BHK-21 batch experiments process glutamine and ammonia density.
Specific embodiment
The present invention is further described with attached drawing combined with specific embodiments below, the advantages and features of the present invention will be with
Description and it is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications and replacement are fallen within the protection scope of the present invention.
One BHK-21 cytotrophy of embodiment metabolism research and the determination of culture medium prescription
1 material
1.1 BHK-21 cells are derived from the BHK-21 suspension cell work of middle peasant Witter biotech inc foundation
Make cell bank, lot number BHK-21/W/S-201202 is used after the assay was approved through amplification culture.
1.2 serum newborn bovine serum are purchased from qualified supplier, according to middle peasant Witter biotech inc new life
Cow's serum internal control quality standard uses after the assay was approved.
1.3 reagents and equipment
Main agents: U.S. waters amino acid analysis chromatographic column (5 μm, 150 × 4.6mmol)；20 kinds of amino acid standard specimens；
Acetonitrile (chromatographically pure)；Sodium acetate (analysis is pure)；Acetic acid (analysis is pure)；Triethylamine (TEA)；PhNCS (PITC) etc..
Key instrument: cytoanalyze (German INNOVATIS company)；U.S.'s Waters high performance liquid chromatograph；NOVA
100 Biochemical Analyzer of PLUS；5L bioreactor (U.S. NBS)；Microscope (Japanese Olympus)；5L bioreactor (beauty
State NBS),
1.4 cell base culture solutions: it weighs a certain amount of MEM culture medium powder and is dissolved in a certain amount of distilled water (distillation coolant-temperature gage
Lower than 40 DEG C) in, dissolution is sufficiently stirred, with sodium hydroxide or salt acid for adjusting pH value to 6.95~7.15, last constant volume to obtain the final product
BHK-21 cell suspension cultures basic culture solution.
2 methods
2.1 main measuring methods
2.1.1 cell count and analysis
2.1.1.1 it samples: drawing cell with sterile suction pipe；
2.1.1.2 it is loaded: gently blowing and beating cell suspension with suction pipe, draw cell sample 20ul and CASY TON with pipettor
Cell analysis buffer 10ml is mixed repeatedly, is analyzed with cytoanalyze.
2.1.1.3 it detects: according to cytoanalyze measurement as a result, calculate Cell viability, density and cell growth ratio,
Determine the proliferative capacity of cell.
2.1.2 amino acid tests and analyzes: being detected using HPLC
2.2 cell culture and method
2.2.1 cell recovery and culture
1. taking one, cell from suspension BHK-21 cell work library, it is immediately placed in 40 DEG C of water-baths and thaws；
2. 1. 4min will be centrifuged under 1000rpm revolving speed by obtained cell, supernatant is abandoned, is added and contains 5% serum in right amount
Culture solution be resuspended, stationary culture passed for 2~3 generations in T100 cell square vase after recovery, and cell uses trypsase after covering with single layer
Digestion；
3. will 2. obtained cell be transferred in 250ml triangular flask, 37 DEG C of oscillations, which suspend, is cultivated, shaking speed 90r/
Min, pH value 7.0~7.2, per daily NaHCO3PH value is adjusted, 50h secondary culture is cultivated；
4. reaching 100ml, cell density 3.0 × 10 to cell culture volumes6Cells/ml, 93% or more Cell viability,
Amplify step by step, collect cell, be transferred to 5L bioreactor culture, setup parameter: temperature is 37.0~37.5 DEG C, pH value 7.0
~7.2, DO value are 60.0%~80.0%, and speed of agitator is 80~120r/min, cultivate 50h secondary culture.
2.2.2 BHK-21 cell amino acid, glucose and metabolic by-product demand behaviors
1) by 4. obtained cell in 5L bioreactor carries out batch experiments in above-mentioned steps 2, with cell density and
Cell viability is evaluation index, culture 0h, for 24 hours, 48h, 72h and 96h take cell sample, be centrifuged under the conditions of 4 DEG C, 1000rpm
5min leaves and takes cell supernatant；
2) above-mentioned 1) gained cell supernatant is subjected to Major Nutrient substance glucose and glutamine tests and analyzes, grape
Sugar and glutamine concentration are measured using 100 Biochemical Analyzer of NOVA PLUS；
3) above-mentioned 1) gained cell supernatant is carried out to the detection and analysis of Major Nutrient substance amino acid, amino acid concentration is adopted
With superelevation liquid phase pre-column derivatization, specific steps are as follows:
1. sample pretreatment: the cell culture medium detected to needs is taken with 1000r/min, 4 DEG C of pelleted by centrifugation 10min
It is clear spare；
2. the preparation of derivative reagent: take the TEA dilution in acetonitrile of the PITC and 144uL of 12uL to 1000uL respectively, it is spare；
3. analyte derivative reacts: taking supernatant 200uL, while taking each 100uL of PITC, TEA, vortex oscillation 30s, room respectively
Temperature extraction 1h, terminates 10min with isometric n-hexane immediately, takes supernatant 0.22 μm of membrane filtration, spare, the derivative side of sample
Method is same with standard items；
4. mobile phase: two kinds of solution gradient elutions of A and B.A liquid is sodium acetate solution；B liquid is acetonitrile solution, and C liquid is to wash
Liquid, i.e., 20% acetonitrile solution；Mobile phase before the use through 0.22 μm of membrane filtration, and with ultrasonic wave deaerate 10min；
5. standard curve is formulated: will be diluted containing the standard solution of 20 kinds of amino acid, concentration be respectively 0.1mmol/L,
0.25mmol/L,0.5mmol/L,1.0mmol/L；
6. balancing chromatographic column: first amino acid analysis column is cleaned with C liquid, then balances chromatographic column with A liquid, it is flat to baseline
Until weighing apparatus；
7. Run sample: loading volume 20uL, flow velocity 1ml/min, runing time 65min, detect wave by 30 DEG C of column temperature
Long 254nm；
8. rinsing instrument and chromatographic column, flush sequence is C liquid 60ml or more, B liquid 30ml or more；
9. data processing calculates amino acid content.
2.2.3 the optimization and application of culture medium prescription
According to the cell growth metabolism parameter that above-mentioned steps 2.2.2 is detected, combination cell metabolic fluxes monitoring result is determined
The content of the main each nutriment for influencing cell growth metabolism, at the end of making its cell culture, the content of nutriment is not
Lower than 5%, by-product ammonia density is less than 8mM in cellular process, lactic acid concn is less than the tolerable concentration limiting value of 19mM, into
The supplement or reduction of row Major Nutrient substance, to be adjusted to basic catalogue culture medium MEM.
3 results
After the influence of 3.1 amino acid, glucose and metabolic by-product cell proliferation measures cell inoculation by HPLC method
20 kinds of amino acid contents in 0h and 48h culture medium, and associational cells growth metabolism is horizontal, determines each needed for one cell of every growth
The amount of kind amino acid, as shown in Figure 1, being monitored in turn to the metabolism of amino acid in cell growth process.
Amount of amino acid needed for cell Proliferation, glucose amount and the variation of cell metabolism by-product concentration are respectively such as Fig. 2-figure
Shown in 4.
The variation of main metabolic substance concentration in 3.2 BHK-21 Cells in Batch incubations
The variation of main metabolic substance concentration during 1 batch experiments of table
3.3 culture medium prescriptions are determining and verify
In conjunction with above-mentioned cell metabolism stream monitoring result, containing for the main each nutriment for influencing cell growth metabolism is determined
ZN-MEM, the culture medium are named as to design culture medium prescription to the tolerable concentration of by-product in amount and cellular process
Including amino acid moiety, salt part is balanced, vitamin moieties, other additives, serum and distilled water, pH value 7.0~7.2,
Wherein, serum addition concentration is 1% (v/v), and the final concentration of other each raw materials in the medium is as shown in table 2 below:
Table 2
Application of the personalized culture medium of embodiment two under different culture scales
1 material
1.1BHK-21 cell is derived from the BHK-21 suspension cell work of middle peasant Witter biotech inc foundation
Cell bank, lot number BHK-21/W/S-201201 are used after the assay was approved through amplification culture.
1.2 serum newborn bovine serum are purchased from qualified supplier, according to middle peasant Witter biotech inc new life
Cow's serum internal control quality standard uses after the assay was approved.
1.3 culture medium
The low blood serum medium ZN-MEM of BHK-21 of the invention, the culture medium includes amino acid moiety, balances salt part,
Vitamin moieties, other additives, serum and distilled water, pH value 7.0~7.2, wherein it is 1% (v/ that serum, which adds concentration,
V), other final concentrations of each raw material in the medium are as shown in table 3 below:
The ZN-MEM culture medium prescription commission culture medium producer of development is processed, for preparing 50L culture solution,
Preparation method are as follows:
1. measuring the water for injection of 90%-95% total volume, 35~40 DEG C are down to temperature, weighs the training of equivalent 50L dry powder
Base is supported, i.e. 0.925kg dry powder (not containing glutamine, Pluronic F-68 and serum) is dissolved in wherein, and stirring is at least
50min is allowed to sufficiently dissolve；
2. glutamine 36.5g is added in 1. solution that above-mentioned steps obtain, 15min is stirred；
3. anti-shearing protective agent Pluronic F-68 50.0g is added in 2. solution that above-mentioned steps obtain, stir
15min；
4. 1% (v/v) serum is added in 3. solution that above-mentioned steps obtain, mix well；
5. a certain amount of 4.0mol/L sodium hydroxide is added in 4. solution that above-mentioned steps obtain or 1.0mol/L hydrochloric acid is molten
Liquid, adjusting pH value is 7.0~7.2；
6. cooling water for injection is added in 5. solution that above-mentioned steps obtain, it is settled to final volume 50L；
7. the filter core aseptic filtration in 0.22 μm of aperture of 6. solution that above-mentioned steps obtain, sets 4 DEG C of preservations.
1.4 reagents and equipment
Main agents: U.S. Waters amino acid analysis chromatographic column (5 μm, 150 × 4.6mmol)；20 kinds of amino acid standard specimens；
Acetonitrile (chromatographically pure)；Sodium acetate (analysis is pure)；Acetic acid (analysis is pure)；Triethylamine (TEA)；PhNCS (PITC)；
Key instrument: cytoanalyze (German INNOVATIS company)；Microscope (Japanese Olympus)；U.S. Waters
High performance liquid chromatograph；100 Biochemical Analyzer of NOVA PLUS, 5L bioreactor (U.S. NBS), 500L bioreactor
(autonomous Design entrusts the production of bioreactor producer).
2 methods
2.1 cell counts and analysis
2.1.1 it samples: drawing cell with sterile suction pipe；
2.1.2 it is loaded: gently blowing and beating cell suspension with suction pipe, with pipettor by cell sample 20ul and CASY TON cell
Analysis buffer 10ml is mixed repeatedly, is analyzed with cytoanalyze.
2.1.3 it detects: according to cytoanalyze measurement as a result, calculating Cell viability, density and cell growth ratio, really
Determine the proliferative capacity of cell.
2.2 cell recoveries and culture
1. taking one, cell from suspension BHK-21 cell work library, it is immediately placed in 40 DEG C of water-baths and thaws；
2. 1. 4min will be centrifuged under 1000rpm revolving speed by obtained cell, supernatant is abandoned, is added and contains 5% serum in right amount
Culture solution be resuspended, stationary culture passed for 2~3 generations in T100 cell square vase after recovery, and cell uses trypsase after covering with single layer
Digestion；
3. will 2. obtained cell be transferred in 250ml triangular flask, 37 DEG C of oscillations, which suspend, is cultivated, shaking speed 90r/
Min, pH value is 7.0~7.2, per daily 8%NaHCO3PH value is adjusted, after cell culture volumes reach specified quantity, is collected thin
Born of the same parents make cell seed in case subsequent.
2.3 BHK-21 cells are in 5L bioreactor secondary culture
The cell that above-mentioned 2.2 collect is transferred in 5L bioreactor and is cultivated, inoculum density is 0.50 × 106cells/
Ml, volume of culture 4000ml cultivate setup parameter: 37 DEG C of temperature, DO value be 68%, pH value 7.0, revolving speed 120r/min,
It automatically controls.Cultivate 48h, secondary culture 40 days (20 generation).
2.4BHK-21 cell is in 500L bioreactor secondary culture
The cell seed of above-mentioned collection is expanded to culture, inoculum density 0.55 × 10 on 500L bioreactor6A/
Ml, reactor control parameter: temperature is 37.0 DEG C, DO value is 70.0%, pH value 7.1, revolving speed 100r/min, automatic control
System.Cultivate 48h, secondary culture 40 days (20 generation).
3, result
Combination cell metabolic fluxes monitoring result, determine the main each nutriment for influencing cell growth metabolism content and
To the tolerable concentration of by-product in cellular process, 5L and 500L different scales cell suspension cultures are carried out to this culture medium
Application verification, culture cell stability passage situation under different scales as shown in figure 5, show to apply present invention determine that culture medium
Continuously BHK-21 cell 40 days (20 generation) of culture, density is 3.5 × 106A/ml or more, and its motility rate is above 94%, because
This, this culture medium ZN-MEM can be applied to BHK-21 cell large-scale culture.
Influence of the BHK21 cell of three different culture medium culture of embodiment to virus multiplication
1 material and method
1.1 material
1.1.1 cell
BHK-21-P cell is purchased from ATCC, source: Hamster Syrian Kidney, middle peasant Witter biotechnology share
Co., Ltd saves, and test cell has passed F18 generation.
1.1.2 culture medium
The commercialized low blood serum medium of BHK-21 cell, it may be assumed that BHK-21MEM, BHK-21GMEM, SLM BHK-21, point
It is not named as A, B and C culture medium；Culture medium ZN-MEM of the present invention is named as D culture medium, formula and the culture in embodiment two
Base phase is same.
1.1.3 medium additives
Glutamine, Pluronic F-68, ironic citrate, transferrins, putrescine, hormone, lipid material are purchased from SIGMA
Company, soybean protein hydrolyate, ultra-filtration plant peptone, yeast extract etc. are purchased from Beijing Reagent Company.
1.1.4 other reagents and equipment
The common agents such as sodium bicarbonate, sodium hydroxide, hydrochloric acid are that domestic analysis is pure,
Key instrument: cell counter (German INNOVATIS company), the 100 Biochemical Analyzer (U.S. NOVA PLUS
Nova Biomedical company), inverted microscope (Japanese Olympus) etc..
1.2 method
1.2.1 BHK-21 cell recovery and the culture that suspends
1. taking one, cell from suspension BHK-21 cell work library, it is immediately placed in 40 DEG C of water-baths and thaws；
2. 1. 4min will be centrifuged under 1000rpm revolving speed by obtained cell, supernatant is abandoned, is added and contains 5% serum in right amount
Culture solution be resuspended, stationary culture passed for 2~3 generations in T100 cell square vase after recovery, and cell uses trypsase after covering with single layer
Digestion；
3. will 2. obtained cell be transferred in 250ml triangular flask, 37 DEG C of oscillations, which suspend, is cultivated, shaking speed 90r/
Min, pH value 7.0~7.2, per daily 8%NaHCO3PH value twice is adjusted, reaches 100ml to cell culture volumes, takes above-mentioned be in
The cell of logarithmic growth phase is transferred to expand in 1000ml triangular flask by 1:6~1:10 and be cultivated.
1.2.2 the batch experiments of BHK-21 cell in different medium
Well-grown serum-free BHK-21 suspension cell is taken, with same initial density 0.60 × 106Cells/ml difference
It is inoculated in tetra- kinds of culture mediums of A, B, C and D, carries out batch experiments in the case where 37 DEG C, 100r/min, pH value are 7.0 condition of culture.?
Culture to 0h, for 24 hours, 48h, 72h, 96h and 120h take cell sample, morphological observation is carried out under inverted microscope, it is close with cell
Degree and Cell viability are evaluation index, analyze cell growth status.
1.2.3 the measurement of the cell metabolisms parameter such as Major Nutrient substance and metabolic by-product
Take 0h during above-mentioned batch experiments, for 24 hours, 48h and 96h cell sample, 800r/min is centrifuged, and supernatant is stayed
Liquid is analyzed using 100 Biochemical Analyzer of NOVA PLUS, detects BHK-21 cell under the conditions of different serum free mediums, carefully
Glutamine, glucose, ammonia, lactic acid concn in born of the same parents' culture solution.
1.2.4 the influence that the BHK21 cell of different culture medium culture is proliferated foot and mouth disease virus
The cell of A, B, C and D culture medium culture is taken, is inoculated with foot and mouth disease virus Re-A WH/09 type, O/MYA98/BY/ respectively
2010 and FMD/Asia1JSL/06 strain, sets control parameter: for temperature as 36.5~37.0 DEG C, pH value is 7.4~7.6, DO value
It is 60.0%~70.0%, speed of agitator is 40~60r/min.When cytopathy variability is 90% or more, harvest virus, record is received
The time of virus is obtained, routinely the TCID of method measurement harvest virus liquid50、LD50With Effective Antigens 146S content.
2 results
The comparison of 2.1 BHK-21 cell cell culture forms in different serum free mediums
BHK-21 cell cellular morphology in four kinds of culture mediums is as shown in Figure 6, it is seen that BHK-21 cell is in above-mentioned four breeding
It is cultivated in base, cellular morphology differs greatly, and the cellular morphology of the culture medium culture wherein optimized in D figure is trained better than other A, B and C
Base is supported, cell is bright, full, mellow and full, sharpness of border, and cell size is normal (generally at 10.0~11.0 μm).
Batch experiments cell growth curve compares 2.2 BHK-21 cells in different medium
The growth curve of BHK-21 cell batch experiments in four kinds of culture mediums is as shown in Figure 7.As can be seen from Figure 7, in batch
During the entire process of culture, D culture basal cell's growth of optimization is most fast, and Cell viability is high, in culture to 72h, maximum living cells
Density is up to 8.42 × 106Cells/ml, Cell viability 96.88%.As it can be seen that the D culture medium of optimization is more suitable for BHK-21 cell
Culture.
The measurement of the cell metabolisms parameter such as 2.3 Major Nutrient substances and metabolic by-product
Using 100 Biochemical Analyzer of NOVA PLUS, to culture 0h, for 24 hours, 48h and 96h cell supernatant carry out glutamy
The measurement of amine, glucose, ammonia and lactic acid concn.
The variation of glucose and lactic acid concn is as shown in figure 8, BHK-21 batch experiments mistake during BHK-21 batch experiments
The variation of journey glutamine and ammonia density is as shown in Figure 9.
From Fig. 8,9 as it can be seen that main nutrient of the BHK-21 cell under four kinds of culture medium condition of culture, in cell culture fluid
Matter glucose and glutamine concentration are gradually decreased with the extension of incubation time, it is very fast in culture decline early period, in after
Phase declines the trend slowed down, and the lactic acid and ammonia density in cell culture supernatant are constantly tired with the extension of incubation time
Product.
By comparing discovery, glucose and glutamine concentration are lower in D culture medium, and metabolic by-product ammonia and lactic acid are dense
Relatively low (when lactic acid and ammonia density reach 19mM, 8mM, serious inhibiting effect is generated to the growth of BHK-21 cell) is spent, is said
Glucose and glutamine concentration being optimal of ratio in bright D culture medium can promote thin to optimize cellular metabolic pathways
The proliferation of born of the same parents.
The influence that the BHK-21 cell of 2.4 different culture medium cultures is proliferated foot and mouth disease virus
The influence that the BHK-21 cell of 3 different culture medium culture of table is proliferated foot and mouth disease virus
Table 3 the result shows that: produce hoof-and-mouth disease venom with the BHK-21 suspension cells of above-mentioned four kinds of culture medium cultures
TCID50、LD50Reach the requirement of industrialized production, and the different shaped foot and mouth disease virus of homemade low blood serum medium D production
The cytopathy time shorten, Effective Antigens 146S content improve 43%, better than A, B, C be commercialized culture medium, this be FMD Gao Pin
Matter production of vaccine is laid a good foundation.
3 conclusions
BHK-21 cell is cultivated respectively with the personalized culture medium voluntarily developed and three kinds of commercialized low blood serum mediums,
Study the growth and virus multiplication situation of BHK-21 cell in different medium, the results showed that, D of the BHK-21 cell in optimization
It is cultivated in culture medium, is commercialized culture medium better than A, B, C, the cell of culture is full, bright, and vitro growth rates are very fast, cell
Metabolic by-product ammonia and lactic acid concn are low, and energy continuous-stable secondary culture, cell density improves 28% or so, the aftosa of production
Virus liquid Effective Antigens content improves 43%, can guarantee the production of high-quality vaccine.
Claims (8)
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| CN106676058B (en) * | 2016-12-09 | 2020-06-19 | 中农威特生物科技股份有限公司 | BHK21 suspension cell high-density fed-batch culture method and application thereof in foot-and-mouth disease virus proliferation |
| CN109628411B (en) * | 2018-12-26 | 2019-11-08 | 内蒙古金源康生物工程有限公司 | FMD antigen enhanced culture medium and preparation method and application thereof |
| CN110643568A (en) * | 2019-09-23 | 2020-01-03 | 山东甲骨文生物科技有限公司 | Low-serum culture medium for BHK-21 cell culture and corresponding virus production |
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