CN1121496C - Method for culturing anchorage dependent animal cell to produce virus by using magnetic stable fluidized bed - Google Patents
Method for culturing anchorage dependent animal cell to produce virus by using magnetic stable fluidized bed Download PDFInfo
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- CN1121496C CN1121496C CN00100230A CN00100230A CN1121496C CN 1121496 C CN1121496 C CN 1121496C CN 00100230 A CN00100230 A CN 00100230A CN 00100230 A CN00100230 A CN 00100230A CN 1121496 C CN1121496 C CN 1121496C
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/16—Particles; Beads; Granular material; Encapsulation
- C12M25/20—Fluidized bed
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/06—Magnetic means
Abstract
The present invention relates to a method for producing viruses by culturing anchorage dependent animal cells by a magnetic stable fluidized bed. After a culture system and a preprocessed micro-carrier are cleaned and sterilized, a cell culture medium is injected to soak the micro-carrier for a proper time. The inoculation is to make cells anchor on the micro-carrier and make the culture medium circulate and filled with oxygen. Parts of culture medium are antiquated, and simultaneously fresh culture media are replenished to carry out cell culture. A virus culture medium is replaced; seed viruses are inoculated. The viruses are completely adsorbed on the cells; the culture medium circulates. Liquid flowing from a culture medium outlet of the magnetic stable fluidized bed is switched to a harvest liquid outlet until to a harvest liquid storage tank in a proper time; the viruses are continuously or respectively harvested. When the present invention is used for culturing Vero cells to produce encephalitis B viruses, the cell density reaches more than 10<8> cells/ml; the virus titer reaches 8.5 TCID50.
Description
Technical field
The invention belongs to the animal cell culture technology, particularly relate to the method for the magnetic stabilization fluid bed cultivation anchorage dependent animal cell to produce virus of a kind of usefulness.
Background technology
Animal cell large-scale is cultivated has more and more important meaning in modern biotechnology and medicinal industry, be used for virus and then preparation vaccines such as production hepatitis B, encephalitis, mad dog.
Magnetic stabilization fluid bed (Magnetically stabilized fluidized bed) is to add magnetic field to common fluidized-bed, and the particle in the fluidized-bed also is magnetic.Particle forms chain in magnetic field, liquid during by fluidized-bed granular layer evenly expand, liquid is plug-flow.The pressure drop of magnetic stabilization fluid bed existing common fluidized-bed and fluidization characteristic have the liquid-flow and the solid-liquid contact performance of packed bed again.In addition,, can regulate the magnetic stabilization fluid bed flow rate of liquid upper limit, thereby magnetic stabilization fluid bed operating restraint is wide, it is wide to adapt to by regulating magneticstrength.Solid particulate is static relatively in magnetic stabilization fluid bed, intergranular collision and friction have been eliminated, pellet density in simultaneously magnetic stabilization fluid bed can reach more than 40% (volume fraction), only a little less than packed bed, than the high 1-2 of the airlift reactor that is used for an animal cell culture order of magnitude.Therefore,,, be expected to cell density is improved 1-2 the order of magnitude, thereby improve the ability that reactor is produced virus with magnetic stabilization fluid bed cultivation zooblast if microcarrier is made magnetic-particle.
Magnetic stabilization fluid bed owing to have unique advantage, in biochemical engineering, obtained many-sided application.Aspect immobilization bioreactor, as document " Biotechnology and Bioengineering " (Biotech.Bioeng.1981,23,2561-2567) literary composition describedly is embedded in urase and ferriferrous oxide particles in the polymethylmethacrylate altogether, adding under the action of a magnetic field, flow rate of liquid reaches 24 cm per minute and granular layer still keeps stable, and the same period, transformation efficiency can be compared with packed bed.Document " chemical industry journal " 1988 (1), 120-126 is described to be carrier with the polymethylmethacrylate that contains magnetic, and the embedding candida tropicalis is used for the processing of phenolic wastewater in the stationary magnetic field, improved speed of response, the degradation amount of unit volume reactor is obviously increased.Document " biotechnology progress " (Biotech.Prog., 1990,6,452-457) described with the magnetic stabilization fluid bed cultivation that is applied to the immobilization vegetable cell, cell and the common embedding of magnetic being made magnetic-particle, produce secondary metabolite, is fluidisation matrix with the substratum, the loss that causes because of particle collision has been eliminated in substratum circulation oxygenation.And to not appearing in the newspapers so far with magnetic stabilization fluid bed cultivation animal cell to produce virus.
Summary of the invention
When the objective of the invention is to overcome utilization microcarrier cultivation animal cell to produce virus, microcarrier particle running foul of each other in reactor, improve the packing density of microcarrier, reduce cells injury, improve the density of animal cell culture and then improve the efficient that reactor is produced virus, the method for the magnetic stabilization fluid bed cultivation anchorage dependent animal cell to produce virus of a kind of usefulness is provided.
The object of the present invention is achieved like this; Add longitudinal magnetic field common in liquid-solid fluid bed, Magnetosensitive microcarrier is housed in the fluidized-bed.Cell culture medium or virus culture base enter fluidized-bed from the lower end, leave from the upper end; The substratum that leaves can all return inlet through after the oxygenation, and batch formula that realizes is cultivated; Also can partly return or not return, to realize perfusion culture.Realize with helmholtz (Helmholtz) coil in magnetic field, logical direct current in the coil.Field direction comprises that up and down magnetic field can be uniform magnetic field, also can be the magnetic field of enhanced from top to bottom that gradient is arranged.There is filter screen bed body lower end, reveals to prevent particle; Under the filter screen back up pad is arranged; Under the back up pad liquid distributing board is arranged, make the liquid uniform distribution; Bed body lower end is a tapered bottom, and to eliminate the dead angle of fluidized-bed, this cone angle is 40 °-120 °; The material of filter screen, grid distributor and back up pad is with can steam sterilizing and do not pollute nutrient solution and be as the criterion, as pottery or stainless steel.There is the inoculation mouth bed body upper surface.There is the particle inlet bed body upper end, in order to add the Magnetosensitive microcarrier particle.Bed body upper surface or sidewall have perforate, are used for sensor installation (as measuring temperature, the pH value scope of solution acid alkalinity, dissolved oxygen scope).Bed body upper and lower ends has the gangway of sterilization steam.
The method of the used magnetic stabilization fluid bed cultivation anchorage dependent animal cell to produce virus of the present invention is as follows;
(1) under the closing state of magnetic field, cleans intrasystem all containers of magnetic stabilization fluid bed zooblast and virus culture, pipeline; Before the control device that starts magnetic stabilization fluid bed zooblast and virus culture system, set required pH value of solution value scope, dissolved oxygen scope and the temperature of reaction of cell cultures.
(2) with after the Magnetosensitive microcarrier pre-treatment by microcarrier add inlet join magnetic stabilization fluid bed in, in system, feed steam sterilizing by the sterilization steam-in then, or before feeding steam, add damping fluid to submergence Magnetosensitive microcarrier and feed steam sterilizing again; Described damping fluid is a phosphoric acid buffer.Or with Magnetosensitive microcarrier sterilize separately the back under gnotobasis, join in sterilized magnetic stabilization fluid bed; Wherein the add-on of Magnetosensitive microcarrier is counted 10%～95% of magnetic stabilization fluid bed working volume by apparent volume, and the coercive force of Magnetosensitive microcarrier is lower than 200 amperes per meter, and particle diameter is 50 μ m～2mm, and wet proportion is 1.1～2.5g/cm
3, magnetic stabilization fluid bed liquid apparent velocity is 5 cm per minute～25 cm per minute, magneticstrength is 3～100mT; Follow certain order during sterilization, guarantee all fully sterilizations of all containers, pipeline, element and microcarrier.
(3) after the cooling liquid in magnetic stabilization fluid bed is discharged by water port; To magnetic stabilization fluid bed in, inject cell culture medium to the submergence Magnetosensitive microcarrier but be no more than 95% of magnetic stabilization fluid bed working volume, immersion Magnetosensitive microcarrier appropriate time through millipore filtration by magnetic stabilization fluid bed culture medium inlet.
(4) add the cell seed liquor that has prepared by magnetic stabilization fluid bed inoculation mouth.
(5) feed sterile air or the oxygen-rich air that Magnetosensitive microcarrier is suspended by magnetic stabilization fluid bed sterilization steam-in through gas-filtering device, continue more than 5 seconds, make the Magnetosensitive microcarrier particle suspension and with the liquid thorough mixing, open magnetic field then, stopping to ventilate, close magnetic field after 20-30 minute,---ventilation---is opened magnetic field---and is stopped ventilation; Repeated to close magnetic field later on every 20-30 minute---process that stops to ventilate that ventilation---is opened magnetic field---, adherent complete on Magnetosensitive microcarrier until cell; Ventilation wherein is meant that aforesaid process gas-filtering device feeds sterile air or oxygen-rich air that Magnetosensitive microcarrier is suspended and also continues more than 5 seconds; Supply cell culture medium through millipore filtration and make it to be full of oxygenate apparatus and pipeline.
(6) control device and the oxygenate apparatus of magnetic stabilization fluid bed zooblast of startup and virus culture system allow cell culture medium circulate, and carry out cell cultivation process; According to the needs of actually operating, replenish fresh cell culture medium continuously or in batches through millipore filtration, the timing sampling analysis is until reaching required cell density or predetermined incubation time; Close the control device and the oxygenate apparatus of magnetic stabilization fluid bed zooblast and virus culture system, stop the circulation of cell culture medium; In cell cultivation process, every 2-12 hour magnetic field is closed, open again again.
(7) cell culture medium in the magnetic stabilization fluid bed culture systems is discharged, add washings to being full of culture systems through millipore filtration by the substratum inlet, soak culture systems or make the washings certain hour that in culture systems, circulates with washings, magnetic stabilization fluid bed zooblast and virus culture system are washed, discharge washings then; According to the prescription of the needs of culturing process decision washings and washing times 1-5 time or more times; Set required pH value of solution value scope, dissolved oxygen scope, the temperature of reaction of virus culture process; Process millipore filtration adding virus culture base posts the Magnetosensitive microcarrier of cell to submergence but is no more than 95% of magnetic stabilization fluid bed working volume; Start the temperature-control device of magnetic stabilization fluid bed zooblast and virus culture system, insert kind of a poison; Close magnetic field, feed the sterile air or the oxygen-rich air that can make the Magnetosensitive microcarrier suspension of posting cell by magnetic stabilization fluid bed sterilization steam-in through gas-filtering device, continue more than 5 seconds, make the Magnetosensitive microcarrier particle suspension that posts cell and with the liquid thorough mixing, open magnetic field then, stopping to ventilate, close magnetic field after 20-30 minute,---ventilation---is opened magnetic field---and is stopped ventilation; Repeated to close magnetic field later on---process that stops to ventilate that ventilation---is opened magnetic field---was adsorbed on cell fully until virus every 20-30 minute; Ventilation wherein is meant that aforesaid process gas-filtering device feeds the sterile air or the oxygen-rich air that can make the Magnetosensitive microcarrier suspension of posting cell and also continues more than 5 seconds; Supply the virus culture base through millipore filtration and make it to be full of oxygenate apparatus and pipeline; Start dissolved oxygen control device, pH control device and the oxygenate apparatus of magnetic stabilization fluid bed zooblast and virus culture system, allow the virus culture base circulate; The magnetic stabilization fluid bed culture medium is exported effusive liquid in good time and switch to results liquid and be exported to the results liquid storage tank, results virus continuously or in batches, and replenish the virus culture base through millipore filtration simultaneously is until viral harvest home: close corresponding pipeline;
(8) pumping into sterilized water through millipore filtration by magnetic stabilization fluid bed culture medium inlet ejects the raffinate in magnetic stabilization fluid bed to the results liquid storage tank by upper end results liquid outlet, or raffinate is directly put to the results liquid storage tank, or raffinate is discharged by water port by the substratum of lower end inlet; Close magnetic field, in system, feed steam sterilizing; The Magnetosensitive microcarrier that posts cell is added inlet water by outlet of results liquid or microcarrier wash to discharge and carry out aftertreatment, culturing process is finished.
Described virus culture base is meant the substratum that is fit to virus multiplication.
The present invention is for the density that improves animal cell culture and the efficient of reactor, the employed Magnetosensitive microcarrier that is used for animal cell culture should have response to magnetic field, be suitable for adherent, the growth of cell again, the leakage of magnetic can't be arranged, in order to avoid pollute nutrient solution and injury cell.
The Magnetosensitive microcarrier that is used for animal cell culture is the core-shell type structure, and kernel is a magnetosensitive nuclear, and magnetosensitive nuclear is made of the high molecular polymer of magnetic and parcel magnetic, and shell is the matrix that is suitable for cell attachment, growth.
The median size of used magnetic is the 0.1-5 micron, and said magnetic is γ-Fe (OH)
2, γ-Fe
2O
3Or Fe
3O
4Said high molecular polymer can be by monomer, initiator and the linking agent preparation that can form spherical particle through suspension polymerization commonly used, wherein monomer comprises the monomer that can form spherical particle through suspension polymerization, as phenylethylene, as vinylbenzene, to Vinyl toluene, a Vinyl toluene or alpha-methyl styrene, and methyl methacrylate.Initiator comprises the initiator that suspension polymerization is commonly used, as benzoyl peroxide (BPO) or Diisopropyl azodicarboxylate (AIBN).Linking agent comprises the linking agent that suspension polymerization is commonly used, as Vinylstyrene and triallyl isocyanurate.Shell matrix comprises the matrix that is fit to different types of cell attachment and growth, as gelatin, dextran etc.
The preparation of above-mentioned microcarrier is polymerization single polymerization monomer, linking agent, initiator and magnetic to be mixed by a certain percentage carry out polymerization, is dispersion agent with the gelatin solution, obtains containing the particle of magnetic through suspension polymerization, after washing, oven dry required magnetic nuclear; Magnetic nuclear is mixed with shell matrix, be suspended in the toluene liquid, the magnetic nuclear that is wrapped with gelatin is fully disperseed, cooling afterwards, crosslinked, reduction, wash, get Magnetosensitive microcarrier.Prepared microcarrier can comprise further that DEAE modifies or the finishing of derivatize, to change its charge distribution, wetting ability etc.
The median size of the Magnetosensitive microcarrier of preparing is 50 microns～2 millimeters, wet proportion 1.1-2.5g/cm
3, particle content is 30%-50% (wt).Microcarrier has the rigid dense structure.The microcarrier of shell nuclear formula structure, its shell is the matrix that helps cell attachment, growth, this structure had both made microcarrier that there is response in magnetic field, having completely cut off magnetic again contacts with the direct of cell, also prevented the leakage of magnetic in substratum, the injury of polluting nutrient solution and pair cell, cultivation results shows and is suitable for African green monkey kidney cell (Vero cell) adherent growth.
Other used auxiliary equipment of the present invention comprises: substratum storage tank, results liquid storage tank, sterilising plant, oxygenate apparatus, temperature-control device, pH value of solution value control device, dissolved oxygen control device and necessary connecting pipeline.Substratum storage tank, results liquid storage tank and sterilising plant are conventional design.Oxygenate apparatus is for to the substratum oxygen supply, it is connected in the circulation way of substratum, directly the liquid in oxygenate apparatus feeds oxygen-rich air or sterile air, or in oxygenate apparatus, adopt gas-exchange membrane to the substratum oxygen supply, by the air flow of dissolved oxygen sensor and dissolved oxygen control device control oxygen supply.Temperature-control device and pH value of solution value control device (comprising transmitter) but free choice of goods equipment.Temperature control mode has multiple, magnetic stabilization fluid bed cooling stick and heating rod can be installed in the lower end to small-sized, and cooling stick is a sleeve structure, interior logical cold water; Heating rod can be a sleeve structure, interior logical hot water or steam, or adopt the nichrome wire heating.Temperature control also can be adopted the mode of water jacket controlled temperature.To large-scale magnetic stabilization fluid bed, can vertical comb be installed at magnetic stabilization fluid bed inwall, also can use water jacket, or many cooling sticks and heating rod are installed, also can be in substratum circulation way mounting heat exchanger.The key of control pH value of solution value scope is acid or the alkali overrich of avoiding in the solution, to the large-scale magnetic stabilization fluid bed mode that can adopt spray.The control mode of most convenient is all to be installed in controlling elements on the oxygenate apparatus, so both can simplify the structure of fluidized-bed main body, can avoid the injury of the too high pair cell of too high and the sour or alkali concn of partial temperature again, its control mode can adopt above-mentioned the whole bag of tricks, depends on scale, manufacturing process, cost and the control strategy of oxygenate apparatus.
Magnetic stabilization fluid bed used material, with the substratum of sterilizing, not pollute, not damaging cells, easily be processed as suitable.To small-sized magnetic stabilization fluid bed, tank body can be used glass, available stainless steels such as top cover, interface, associated ping, filter screen, grid distributor.Magnetic stabilization fluid bedly can all adopt stainless steel to large-scale.
To small-sized magnetic stabilization fluid bed, the tank body upper end does not need expanding reach.To large-scale magnetic stabilization fluid bed, the upper end preferably is connected to expanding reach, carries secretly to reduce particulate.The internal diameter of expanding reach is 1.0-2.0 a times of inner major diameter, highly is 0.5-3.0 times of inner major diameter, and the cone angle of expanding reach bottom awl section is 40 °-120 °.
Being shaped as of magnetic stabilization fluid bed cross section is circular, square etc.With circular best, because can make full use of the uniform parts in magnetic field, and the wall effect minimum in the bed.
Magnetic stabilization fluid bed aspect ratio is 1: 1-20: 1, and internal diameter is 1 centimetre-100 centimetres.
The present invention uses Magnetosensitive microcarrier, particle is static relatively, intergranular collision and friction have been eliminated, improved the packing density of microcarrier, reduced cells injury, the volume fraction of Magnetosensitive microcarrier can reach more than 40%, thereby has improved the ability of the magnetic stabilization fluid bed volume production virus of density and unit of cell cultures.Produce encephalitis b virus with culture technique Cultivation of Vero of the present invention (African green monkey kidney cell), use homemade Magnetosensitive microcarrier, cell density reaches 10
8More than individual cell/ml, far above with the cell density that stirring-type or airlift reactor reached, virus titer reaches 8.5TCID in the virus results liquid
50
Description of drawings
Fig. 1. magnetic stabilization fluid bed zooblast of the present invention and virus culture system schematic;
Fig. 2. magnetic stabilization fluid bed structural representation of the present invention.1. fluid bed main bodys, 2. filter screens, 3. gripper shoes, 4. liquid distributing boards, 5. culture medium entrances 6. sterilization steam enter 7. drainings, 8. culture mediums outlet, 9. discarded culture mediums and export the culture medium that 10. pressure balance mouths, 11. exhaust outlets, 12. microcarriers add culture medium entrance 17. oxygenate apparatus of entrance 13. inoculations mouthful 14.Helmholtz coils 15. oxygenate apparatus 16. oxygenate apparatus and export 18. oxygen-enriched air or 19. pH sensor 20. dissolved oxygen sensors, 21. temperature sensor 22. control device, 23. acid of pure oxygen entrance or alkali storage tank 24. temperature control executing agencies, 25. oxygen-enriched air or pure oxygen source 26. constant flow pumps 27. air cleaners 28. gas circuit valves 29. liquid levels 30. fresh cultures 31. steam 32. air
Embodiment
Fig. 1 draws according to controlling elements and transmitter all are installed on the oxygenate apparatus, and its controlling elements and transmitter also can be installed on the magnetic stabilization fluid bed main body; Do not draw among the figure form, hand-hole and nonproductive essential but valve, pipeline and import and export etc. that need in maintenance of the equipment, maintenance or when cleaning; Do not draw among Fig. 1 for miscellaneous equipment, transmitter and the sampling unit analyzing, detect, control, as tails assay, medium component analysis etc.; Can select mechanical defoaming device in the oxygenate apparatus for use or adopt defoamer, not draw; The mode of connection of parts, equipment is not drawn, and as ring flange, bolt, nut, larynx hoop, weld etc. and specification thereof, size, these depend on equipment scale and manufacturing process; Material, model, the specification of connecting pipeline, valve, transmitter, strainer etc. all do not mark; Control device among Fig. 1 only is a synoptic diagram, can choose the product of different class, function and specification as required; Painting magnetic stabilization fluid bed main body among the figure is magnetic stabilization fluid bed that expanding reach is arranged, and magnetic stabilization fluid bedly can not want expanding reach to small-sized; The cooling or the type of heating of oxygenate apparatus do not draw, and these depend on equipment scale, manufacturing process and cost; Gas dispersion mode in the oxygenate apparatus is not drawn, and can use grid distributor, baffle plate, mechanical stirring or membrane module; Substratum storage tank and results liquid storage tank do not draw; Direct supply does not draw; Tensimeter among the figure on each tank body top cover does not draw; Sterilization steam-in, outlet and the water port of substratum storage tank, results liquid storage tank, oxygenate apparatus are not drawn; For fully sterilize essential auxiliary line, valve of all containers, pipeline, valve in the assurance system do not draw; Under meter and bactericidal device necessary in the pipeline do not draw.
Fig. 2 is the magnetic stabilization fluid bed structural representation of the present invention.Substratum enters fluidized-bed from the lower end, leaves from the upper end.In magnetic stabilization fluid bed 1 Magnetosensitive microcarrier is housed, adds longitudinal magnetic field with Helmholtz coil 14.The top of magnetic stabilization fluid bed main body 1 is the taper expanding reach, and purpose is that speed reduces after making liquid leave granular layer, reduces particle entrainment; The bottom of magnetic stabilization fluid bed main body 1 is taper, to eliminate the dead angle; There is filter screen 2 lower end of magnetic stabilization fluid bed main body 1, reveals to prevent particle; Filter screen has back up pad 32 times, to support filter screen 2; Back up pad has liquid distributing board 43 times, makes the liquid uniform distribution; The material of filter screen 2, back up pad 3 and liquid distributing board 4 is with can steam sterilizing and do not pollute nutrient solution and be as the criterion, as pottery or stainless steel.The lower end of magnetic stabilization fluid bed main body 1 is provided with 5,6,7 three interfaces, and the 5th, substratum inlet, the 6th, sterilization steam-in, the 7th, water port.The side of magnetic stabilization fluid bed expanding reach is provided with 8,9 two interfaces, and the 8th, the substratum outlet, the 9th, contain the results liquid outlet (removing to gather in the crops liquid storage tank) of virus.Magnetic stabilization fluid bed upper surface is provided with the venting port 11 of steam or air, is provided with microcarrier and adds inlet 12, in order to add the microcarrier particle, is provided with inoculation mouthfuls 13, can make the inoculation mouth with 12 during manual inoculation.
Magnetic stabilization fluid bed structure and operating procedure parameter are as follows; 10 centimetres of magnetic stabilization fluid bed inner major diameter are high 50 centimetres; 15 centimetres of expanding reach internal diameters are high 15 centimetres; The connection portion of expanding reach and main body is high 5 centimetres; The tapering of main body lower end is high 4 centimetres; Cultured cells is 137 generation Vero cells, and the product of intending results is an encephalitis b virus; Cell culture medium is for adding the M199 substratum of 8% foetal calf serum, and the virus culture base is for adding the MEM substratum of 1% human serum albumin; The particle diameter of used microcarrier is 100 μ m-150 μ m, and coercive force is 20 amperes per meter, and wet proportion is 1.3g/cm
3Use the oxygen bottle oxygen supply; The magnetic induction density in magnetic field is 12mT; Superficial liquid velocity in the bed is 15 cm per minute; Cell inoculation density is 5 * 10
6Individual cell/ml; Temperature is set at 37 ℃ during culturing cell, and the pH value of solution value is set at 7.0, and dissolved oxygen is set at 30% air saturation; Planting malicious titre is 8.0TCID
50, connecing the poison amount is 1: 100; Temperature is set at 37 ℃ when cultivating virus, and the pH value of solution value is set at 7.3, and dissolved oxygen is set at 30% air saturation.
(1) under the closing state of magnetic field, cleans intrasystem all containers of magnetic stabilization fluid bed zooblast and virus culture, pipeline; Set required pH value of solution value scope, dissolved oxygen scope and the temperature of reaction of cell cultures;
(2) with 1.6 kilograms of Magnetosensitive microcarriers with the phosphoric acid buffer of pH value of solution value 7.0 in 37 ℃ of swellings 3 hours, give a baby a bath on the third day after its birth time with phosphoric acid buffer, add magnetic stabilization fluid bedly then by No. 12 mouths, add the phosphoric acid buffer of 1 liter of pH value 7.0 again.In system, feed sterilization steam, sterilized 30 minutes;
(3) after the system cools liquid in magnetic stabilization fluid bed is discharged by No. 7 mouths; In magnetic stabilization fluid bed, inject 2 liters of cell culture mediums through millipore filtration by No. 5 mouths, soaked Magnetosensitive microcarrier 12 hours;
(4) add the Vero cell suspension (seed liquor) that has prepared by No. 13 mouths;
(5) through millipore filtration by No. 6 mouths by 0.2 liter/minute flow aerating oxygen 3 minutes, make particle suspension and with the liquid thorough mixing, open magnetic field then, stop ventilation; Repeat the process of " close magnetic field---ventilation---is opened magnetic field---and stopped ventilation " every half an hour, to inoculating back 3 hours; That " ventilation " wherein is meant is aforesaid " through millipore filtration by No. 6 mouths by 0.2 liter/minute flow aerating oxygen 3 minutes "; Supplying cell culture medium through millipore filtration makes and is full of oxygenate apparatus and pipeline;
(6) start-up control device and oxygenate apparatus allow cell culture medium circulate; The fresh cell culture medium of beginning continuous supplementation after 12 hours, what discard equivalent simultaneously exports effusive cell culture medium by substratum; Culture medium supplemented speed increased progressively once every 12 hours, was followed successively by 10ml/ minute, 15ml/ minute, 20ml/ minute, 30ml/ minute, 40ml/ minute, 60ml/ minute, 80ml/ minute, 120ml/ minute, 160ml/ minute.Insert cell and stop after 120 hours replenishing cell culture medium, the control device and the oxygenate apparatus of closing magnetic stabilization fluid bed zooblast and virus culture system stop the circulation of cell culture medium; In cell cultivation process, every 6 hours magnetic field is closed, open again again; This moment, cell density was 1.2 * 10
8Individual cell/ml;
(7) with the cell culture medium emptying in the magnetic stabilization fluid bed culture systems, use Earle ' s liquid washing system 2 times, use the MEM washing system again 1 time; Washing process is: the injection washing liq is full of system and washing liq speed with 10cm/ minute in magnetic stabilization fluid bed was circulated 2 minutes, stops circulation and emptying, magnetic field is closed opened in 5 seconds again then.Set required pH value of solution value scope, dissolved oxygen scope, the temperature of reaction of virus culture process; In magnetic stabilization fluid bed, inject 2 liters in virus culture base through millipore filtration by No. 5 mouths, start the temperature-control device of magnetic stabilization fluid bed zooblast and virus culture system, by the kind poison of inoculation mouthful access encephalitis b virus; Close magnetic field, through millipore filtration by No. 6 mouths by 0.2 liter/minute flow aerating oxygen 3 minutes, make particle suspension and with the liquid thorough mixing, open magnetic field then, stop ventilation; The process that repeated aforesaid " close magnetic field---ventilation---is opened magnetic field---and stopped ventilation " every 25 minutes is to connecing back 24 hours of poison, supplies the virus culture base then and makes and be full of oxygenate apparatus and pipeline; Start dissolved oxygen control device, pH control device and the oxygenate apparatus of magnetic stabilization fluid bed zooblast and virus culture system, allow the virus culture base circulate; Access kind of poison begins after 72 hours through the fresh virus culture base of millipore filtration continuous supplementation, gathers in the crops the flowing liquid (containing virus) of equivalent to gathering in the crops liquid storage tank by No. 9 mouths simultaneously.The additional speed of virus culture base is 20ml/ minute, continuously results virus.Insert kind of poison and stop results virus after 72 hours.Close corresponding pipeline.The virus titer of results liquid is 8.5TCID
50
(8) raffinate in magnetic stabilization fluid bed is drained into the results liquid storage tank by No. 5 mouths; Close magnetic field, in system, feed the sterilization steam sterilizing; + microcarrier is washed discharge by No. 12 saliva, carry out aftertreatment; Culturing process finishes.
Claims (3)
1. method with magnetic stabilization fluid bed cultivation anchorage dependent animal cell to produce virus is characterized in that steps of the method are:
(1) under the closing state of magnetic field, cleans intrasystem all containers of magnetic stabilization fluid bed zooblast and virus culture, pipeline; Before the control device that starts magnetic stabilization fluid bed zooblast and virus culture system, set required pH value of solution value scope, dissolved oxygen scope and the temperature of reaction of cell cultures;
(2) with after the Magnetosensitive microcarrier pre-treatment by microcarrier add the inlet join magnetic stabilization fluid bed in, then by the sterilization steam-in in system, feed steam sterilizing; Or adding damping fluid to submergence Magnetosensitive microcarrier feeds steam sterilizing again before feeding steam, and described damping fluid is a phosphoric acid buffer; Or with Magnetosensitive microcarrier sterilize separately the back under gnotobasis, join in sterilized magnetic stabilization fluid bed; Wherein the add-on of Magnetosensitive microcarrier is counted 10%～95% of magnetic stabilization fluid bed working volume by apparent volume, and the coercive force of Magnetosensitive microcarrier is lower than 200 amperes per meter, and particle diameter is 50 μ m～2mm, and wet proportion is 1.1～2.5g/cm
3, magnetic stabilization fluid bed liquid apparent velocity is 5 cm per minute～25 cm per minute, magneticstrength is 3～100mT;
(3) after the cooling liquid in magnetic stabilization fluid bed is discharged by water port; To magnetic stabilization fluid bed in, inject cell culture medium to the submergence Magnetosensitive microcarrier but be no more than 95% of magnetic stabilization fluid bed working volume, immersion Magnetosensitive microcarrier appropriate time through millipore filtration by magnetic stabilization fluid bed culture medium inlet;
(4) add the cell seed liquor that has prepared by magnetic stabilization fluid bed inoculation mouth;
(5) feed sterile air or the oxygen-rich air that Magnetosensitive microcarrier is suspended by magnetic stabilization fluid bed sterilization steam-in through gas-filtering device, continue more than 5 seconds, make the Magnetosensitive microcarrier particle suspension and with the liquid thorough mixing, open magnetic field then, stopping to ventilate, close magnetic field after 20-30 minute,---ventilation---is opened magnetic field---and is stopped ventilation; Repeated to close magnetic field later on every 20-30 minute---process that stops to ventilate that ventilation---is opened magnetic field---, adherent complete on Magnetosensitive microcarrier until cell; Ventilation wherein is meant that aforesaid process gas-filtering device feeds sterile air or oxygen-rich air that Magnetosensitive microcarrier is suspended and also continues more than 5 seconds; Supply cell culture medium through millipore filtration and make it to be full of oxygenate apparatus and pipeline;
(6) control device and the oxygenate apparatus of magnetic stabilization fluid bed zooblast of startup and virus culture system allow cell culture medium circulate, and carry out cell cultivation process; According to the needs of actually operating, replenish fresh cell culture medium continuously or in batches through millipore filtration, the timing sampling analysis is until reaching required cell density or predetermined incubation time; Close the control device and the oxygenate apparatus of magnetic stabilization fluid bed zooblast and virus culture system, stop the circulation of cell culture medium; In cell cultivation process, every 2-12 hour magnetic field is closed, open again again;
(7) cell culture medium in the magnetic stabilization fluid bed culture systems is discharged, add washings to being full of culture systems through millipore filtration by the substratum inlet, soak culture systems or make the washings certain hour that in culture systems, circulates with washings, magnetic stabilization fluid bed zooblast and virus culture system are washed, discharge washings then; Set required pH value of solution value scope, dissolved oxygen scope, the temperature of reaction of virus culture process; Process millipore filtration adding virus culture base posts the Magnetosensitive microcarrier of cell to submergence but is no more than 95% of magnetic stabilization fluid bed working volume; Start the temperature-control device of magnetic stabilization fluid bed zooblast and virus culture system, insert kind of a poison; Close magnetic field, feed the sterile air or the oxygen-rich air that can make the Magnetosensitive microcarrier suspension of posting cell by magnetic stabilization fluid bed sterilization steam-in through gas-filtering device, continue more than 5 seconds, make the Magnetosensitive microcarrier particle suspension that posts cell and with the liquid thorough mixing, open magnetic field then, stopping to ventilate, close magnetic field after 20-30 minute,---ventilation---is opened magnetic field---and is stopped ventilation; Repeated to close magnetic field later on---process that stops to ventilate that ventilation---is opened magnetic field---was adsorbed on cell fully until virus every 20-30 minute; Ventilation wherein is meant that aforesaid process gas-filtering device feeds the sterile air or the oxygen-rich air that can make the Magnetosensitive microcarrier suspension of posting cell and also continues more than 5 seconds; Supply the virus culture base through millipore filtration and make it to be full of oxygenate apparatus and pipeline; Start dissolved oxygen control device, pH control device and the oxygenate apparatus of magnetic stabilization fluid bed zooblast and virus culture system, allow the virus culture base circulate; The magnetic stabilization fluid bed culture medium is exported effusive liquid in good time and switch to results liquid and be exported to the results liquid storage tank, results virus continuously or in batches, and replenish the virus culture base through millipore filtration simultaneously, until viral harvest home; Close corresponding pipeline;
(8) pumping into sterilized water through millipore filtration by magnetic stabilization fluid bed culture medium inlet ejects the raffinate in magnetic stabilization fluid bed to the results liquid storage tank by upper end results liquid outlet, or raffinate is directly put to the results liquid storage tank, or raffinate is discharged by water port by the substratum of lower end inlet; Close magnetic field, in system, feed steam sterilizing; The Magnetosensitive microcarrier that posts cell is added inlet water by outlet of results liquid or microcarrier wash to discharge and carry out aftertreatment, culturing process is finished.
2. the method for the magnetic stabilization fluid bed cultivation anchorage dependent animal cell to produce virus of usefulness as claimed in claim 1 is characterized in that described virus culture base is meant the substratum that is fit to virus multiplication.
3. the method for the magnetic stabilization fluid bed cultivation anchorage dependent animal cell to produce virus of usefulness as claimed in claim 1 is characterized in that the washing times in the described step (7) is 1-5 time.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00100230A CN1121496C (en) | 2000-01-10 | 2000-01-10 | Method for culturing anchorage dependent animal cell to produce virus by using magnetic stable fluidized bed |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN00100230A CN1121496C (en) | 2000-01-10 | 2000-01-10 | Method for culturing anchorage dependent animal cell to produce virus by using magnetic stable fluidized bed |
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| CN1303924A CN1303924A (en) | 2001-07-18 |
| CN1121496C true CN1121496C (en) | 2003-09-17 |
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| FR3027678B1 (en) * | 2014-10-24 | 2016-12-30 | Viovy Jean Louis | METHOD FOR DETECTING ORGANISMS IN A DILUTED SAMPLE |
| PL227303B1 (en) * | 2015-04-30 | 2017-11-30 | Zachodniopomorski Univ Technologiczny W Szczecinie | Two-chamber reactor for magnetic supporting of chemical processes and the system with this reactor |
| CN110684655B (en) * | 2019-10-18 | 2023-03-17 | 江苏大学 | Microalgae separation gradient magnetic stabilization fluidized bed device and microalgae harvesting method thereof |
| CN112094819B (en) * | 2020-08-13 | 2022-10-14 | 浙江美保龙生物技术有限公司 | Full suspension culture method of chicken infectious bursal disease virus |
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