CN104046588A - Micro-carrier culture method of bioreactor of MDCK (Madin Darby Canine Kidney) cells and application thereof - Google Patents
Micro-carrier culture method of bioreactor of MDCK (Madin Darby Canine Kidney) cells and application thereof Download PDFInfo
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Abstract
The invention discloses a micro-carrier culture method of a bioreactor of MDCK (Madin Darby Canine Kidney) cells and discloses an application of MDCK cells cultured by the culture method in preparation of influenza vaccines for people and avian influenza vaccines. The method comprises the following specific steps: culturing seed cells by a square bottle or a spinner bottle; after digesting the seed cells, inoculating the cells to the bioreactor equipped with the micro-carrier for amplification culture; inoculating a corresponding avian influenza or influenza virus for viral multiplication culture; and after over 70% of cells have lesion, harvesting a viral culture solution for a conventional vaccine preparation process. The method disclosed by the invention has the beneficial effects that the technical defects in production of chick embryos that production period is long, operation is tedious, the workload large and contamination rate is high and avian influenza bursts can be fundamentally overcome. The existing vaccine process is technically upgraded, the production cost of the vaccine is lowered, the output and quality of the vaccine are improved, and the method has an application prospect in actual production.
Description
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Technical field
The present invention relates to field of biological pharmacy, be specifically related to bio-reactor microcarrier cultural method and the application thereof of mdck cell.
Background technology
Influenza (Influenza), is the Acute respiratory infectious disease being caused respectively by first (A), second (B), third (C) three type influenza viruses, often causes in various degree popular, comprises being very popular in the world, local outbreak and distributing.Bird flu (Bird Flu or Avian Influenza) is the abbreviation of avian influenza, it is a kind of acute infectious disease being caused by a kind of hypotype of influenza A virus (also claiming avian influenza virus), also can infect the mankind, common people infects bird flu mortality ratio and is about 33%.No matter be that desirable medicine is not found in influenza or bird flu so far, vaccine inoculation is still and prevents that now disease from occurring and the most effective popular means.
China influenza vaccines and avian influenza vaccine are produced the main chicken embryo production technique that adopts, there is many defects such as production cycle length, complex operation, large, the easy pollution of workload in chicken embryo production technique, as bird flu large-scale outbreak, also will there is very large problem in the supply of chicken embryo.If the technique that adopts passage cell to produce vaccine will not be subject to the restriction of the natural factors such as chicken embryo, and with short production cycle, technique is simple, as Influenza Outbreak meeting rapid reaction, in the short period of time, can provide a large amount of vaccine products.In addition; the passage cell of adherent type can be cultivated with the mass-producing of bio-reactor microcarrier; in production, can Growth of Cells and viral proliferation be controlled within the scope of optimum condition automatically; improve cell density and virus titer; realize large batch quantity, little difference between batch; product has better stability and security; a large amount of saving labor force, production site and energy consumptions; reduce production costs, compare and there is clear superiority with the technique of spinner culture passage cell virus of proliferation with the technique of the direct virus of proliferation of chicken embryo.
Mdck cell (Madin-Daby Canine Kidney Cells, MDCK) by Madin, set up in the separated cultivation of the female bent frame dog renal tissue of 1958 Nian Cong U.S. Cocker Spaniel with Darby, because its virus infection efficiency is high, propagation fast, and be difficult for variation, mdck cell system is acknowledged as and is suitable for one of 3 kinds of clones that influenza virus copies most.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, and a kind of cultural method that utilizes bio-reactor microcarrier to cultivate mdck cell is provided.
To achieve these goals, technical scheme provided by the invention is: the bio-reactor microcarrier cultural method of mdck cell, comprises the following steps:
1) microcarrier is processed:
The PBS damping fluid that is 7.4 with pH by Cytodex series microcarrier soaks 3-24h, and cleans twice, and autoclaving is cooling standing, after cleaning, adds the cell culture fluid containing bovine serum of same volume standby with the cell culture fluid that contains bovine serum;
GF series microcarrier is soaked to 3-24h by the purified water that contains tween-80, after the PBS buffer solution for cleaning that is 7.4 with pH again, autoclaving, cooling standing, after cleaning with the cell culture fluid that contains bovine serum, add the cell culture fluid containing bovine serum of same volume standby;
2) MDCK seed cell recovery:
From liquid nitrogen, take out frozen mdck cell, quick-thawing recovery cell in the water-bath of 37 ℃-39 ℃, after recovery, with the cell culture fluid that contains bovine serum, standing in square vase to be cultured to Growth of Cells more than 90% fine and close to square vase aufwuchsplate;
3) enlarged culturing of seed cell:
Until step 2) in Growth of Cells when more than 90% fine and close to square vase aufwuchsplate, use cell dissociation buffer peptic cell, according to the sub-bottle ratio enlarged culturing of (1:2)-(1:10) to the required cell count of spinner culture; Inoculate spinner culture, until Growth of Cells, arrive rolling bottle aufwuchsplate when more than 90% fine and close, use cell dissociation buffer peptic cell, according to the sub-bottle ratio enlarged culturing of (1:2)-(1:10) to the required cell count of bioreactor culture;
4) bio-reactor microcarrier is cultivated:
According to throwing in the ratio of inoculating 5-50 cell on 2-25g microcarrier, each microcarrier in every liter of volume of culture, by being inoculated into after the mdck cell digestion of spinner culture, in bio-reactor, being cultured to 90% microcarrier surface and forming fine and close individual layer.
Further, the bio-reactor microcarrier cultural method of above-mentioned mdck cell, in the Cytodex series microcarrier treating processes of described step 1), every gram of microcarrier soaks with the consumption of PBS damping fluid for being greater than 20mL; The PBS damping fluid consumption that every gram of microcarrier cleans use is at every turn for being greater than 20mL, and the enchylema consumption that contains bovine serum that every gram of microcarrier cleans use is at every turn for being greater than 20mL; The described cell culture fluid that contains bovine serum is the high sugared nutrient solution of the DMEM containing volumn concentration 2%-10% bovine serum; Described bovine serum is newborn calf serum or foetal calf serum.
Further, the bio-reactor microcarrier cultural method of above-mentioned mdck cell, in the GF series microcarrier treating processes of described step 1), described in contain tween-80 purified water be to contain the purified water that volumn concentration is 0.01% tween-80; Every gram of microcarrier soaks with the consumption of the purified water that contains tween-80 for being greater than 20mL; The PBS damping fluid consumption that every gram of microcarrier cleans use is at every turn for being greater than 20mL, and the enchylema consumption that contains bovine serum that every gram of microcarrier cleans use is at every turn for being greater than 20mL; The described cell culture fluid that contains bovine serum is the high sugared nutrient solution of the DMEM containing volumn concentration 2%-10% bovine serum; Described bovine serum is newborn calf serum or foetal calf serum.
Further, the bio-reactor microcarrier cultural method of above-mentioned mdck cell, described step 2) in, described in contain bovine serum cell culture fluid be the high sugared nutrient solution of DMEM that contains volumn concentration 8%-10% bovine serum; Described culture temperature is 37 ℃.
Further, the bio-reactor microcarrier cultural method of above-mentioned mdck cell, in described step 3), cell dissociation buffer is that mass percent concentration is 0.25% trypsin solution; Cell culture fluid is the high sugared nutrient solution of DMEM that contains volumn concentration 8%-10% bovine serum; The rotating speed of spinner culture is 2-15rpm/min; Culture temperature is 37 ℃.
Further, the bio-reactor microcarrier cultural method of above-mentioned mdck cell, in described step 4), microcarrier is Cytodex series microcarrier or the GF series microcarrier of processing in step 1); Cell dissociation buffer is that mass percent concentration is 0.25% trypsin solution; Cell culture fluid is the high sugared nutrient solution of DMEM that contains volumn concentration 8%-10% bovine serum; The culture condition of reactor is: rotating speed 20-50rpm, 37 ℃ of temperature, pH value 7.2-7.4, dissolved oxygen 20-80%.
Second object of the present invention has been to provide the mdck cell of the bio-reactor microcarrier cultural method cultivation of above-mentioned mdck cell and used the application in influenza vaccines preparation people.
The 3rd object of the present invention has been to provide the application in preparing avian influenza vaccine of mdck cell that the bio-reactor microcarrier cultural method of above-mentioned mdck cell cultivates.
Further, above-mentioned application, comprises the following steps:
A) virus inoculation:
Mdck cell 90% microcarrier surface until bioreactor culture forms after fine and close individual layer, stop stirring 3-5min, make microcarrier naturally sink to reactor bottom, extract out and add again half maintenance medium of original fluid after all cell culture fluids, according to TPCK pancreatin 1-20 μ g/ml, virus infection plural number 0.01-1 virus inoculation, cultivate after 2h, maintenance medium is mended to consistent with original fluid;
B) venom results:
Sampling observation of cell pathology effect CPE per hour from connecing malicious the 10th hour of cultivating, when there is pathology effect in 80% above cell, aseptic collection all cells maintenance medium, sampling and measuring blood clotting valency or mensuration hemagglutinin content after freeze thawing once, when blood clotting valency is 2
9or hemagglutinin content while reaching 2 μ g/ml by bird flu or people manufactures with influenza vaccines and inspection procedure is carried out.
Further, above-mentioned application, in described step a), maintenance medium is the high sugared nutrient solution of DMEM that contains volumn concentration 1%-3% bovine serum; Described TPCK pancreatin is serum-free ox pancreas source VIII type pancreatin; The culture condition of described reactor is: rotating speed 20-50rpm, 33 ℃-35 ℃ of temperature, pH value 7.4-7.6, dissolved oxygen 20-80%.
Beneficial effect of the present invention is:
The present invention adopts the technique of bio-reactor microcarrier culturing cell virus of proliferation, with square vase or spinner culture seed cell, after seed cell digestion, be inoculated into amplification culture in the bio-reactor that has microcarrier, inoculate corresponding bird flu or influenza virus and carry out virus multiplication cultivation, after 80% above cell produces pathology, gather in the crops virus-culturing fluid and carry out conventional seedling technique.This technique fundamentally solves chicken embryo and exists in producing production cycle length, complex operation, workload greatly, easily to pollute and meet the defective workmanships such as Avian Influenza, when existing vaccine technique is carried out to technology upgrading, reduced the production cost of vaccine, improve vaccine output and quality, in actual production, there is application prospect.Tradition chicken embryo technique is pressed 8 yuan, results 10ml unit price venom by each SPF chicken embryo, refuse chicken embryo is processed the first calculating of every Kg50, the priming cost of producing 1000L unit price venom is about 1,150,000 yuan, from hatching to venom, results need 13-15 days time, and production process will carry out nearly 100,000 hatchings, inoculation and results, workload is large, and each link manual operation uncertain factor is many, affect vaccine quality, and can not produce continuously.Employing passage cell is produced, press 6 yuan/liter of substratum, 1000 yuan/liter of serum, 40,000 yuan/Kg of Cytodex series microcarrier, 10,000 yuan/Kg of GF series microcarrier, microcarrier are thrown in density and are pressed 5g/ liter, add pancreatin etc., the priming cost of producing 1000L unit price venom with Cytodex series microcarrier is about 310,000 yuan, and GF series microcarrier is about 160,000 yuan, the whole automated operations of production process, artificial interference factor is few, and can produce continuously, within while normally producing every 5-7 days, can produce a collection of.
Embodiment
embodiment 1:
1, material source and specification:
Microcarrier: Cytodex1 type, GE company, lot number 10047670, every gram containing 3.1 * 10
6individual carrier.
Mdck cell: Gansu Province's System in Animal Cell Biotechnology Technical Research Center provides.
DMEM substratum (dry powder): Invitrigen Corporation, article No.: 02-5062EJ, by specification preparation.
Trypsin dry powder): Invitrigen Corporation, article No.: 27250, being made into according to a conventional method mass percent concentration is 0.25%.
TPCK pancreatin (ox pancreas source VIII type pancreatin): Sigma company, article No.: T8802, lot number: 059K8710.
New-born calf serum: Lanzhou people's marine life Engineering Co., Ltd, lot number: 20120716.
Avian influenza strain: H5N1 hypotype Re-5 strain, is provided by Harbin Veterinary Medicine Inst., China Academy of Agriculture.(experiment completes in Jilin dawn Bioisystech Co., Ltd)
Chemical reagent is analytical pure.
2, plant and instrument:
1) incubator, Thermo Fisher Scientific Corporation, model: 3111;
2) Rotary Machine, Lanzhou Lanfei Biochemistry Equipment Co., Ltd., model: VIG;
3) bio-reactor, New Brunswick Scientific, model BIOFLO CELLIGEN 310, culture tank effective volume volume 5L;
3, processing method:
1) microcarrier is prepared:
(microcarrier is thrown in density: 5g/L), be 7.4 without Ca with 1000mlpH to take 25g Cytodex1 type microcarrier
2+, Mg
2+pBS damping fluid soak 4h, and with without Ca
2+, Mg
2+pBS buffer solution for cleaning twice, each cleaning used damping fluid 1000ml; 121 ℃, 15min steam sterilizing, naturally cooling is standing, adds 1000ml to contain the high sugared nutrient solution of DMEM of volumn concentration 10% bovine serum standby with 1000ml containing the high sugared nutrient solution of the DMEM of volumn concentration 3% bovine serum after cleaning;
2) recovery of MDCK seed cell is cultivated:
(cell generation is P65, cell total amount 5 * 10 in mdck cell strain frozen in ordinary method recovery liquid nitrogen
6individual), be inoculated in square vase (T75) static cultivations 72h containing the high sugared nutrient solution of the DMEM of volumn concentration 10% bovine serum, arrive 100% densification of square vase aufwuchsplate, culture temperature: 37 ℃ to Growth of Cells;
3) enlarged culturing of seed cell:
3.1 square vase enlarged culturing:
Get the mdck cell of recovery incubation growth densification in above-mentioned square vase, with 0.25% trypsin solution according to a conventional method after peptic cell, being inoculated in 4 square vases (T75) static cultivation 72h goes down to posterity after to Growth of Cells to 100% densification of square vase aufwuchsplate again and is cultured in 16 square vases (T75), after cultivating 72h, Growth of Cells is to 100% densification of square vase aufwuchsplate, culture temperature: 37 ℃, nutrient solution is the high sugared nutrient solution of the DMEM containing volumn concentration 10% bovine serum;
3.2 rolling bottle enlarged culturing:
The cell of above-mentioned flask culture is inoculated in 1 15L rolling bottle after peptic cell and is cultivated according to a conventional method with 0.25% trypsin solution, cultivate 72h Growth of Cells to 100% densification of rolling bottle aufwuchsplate, again the passage of spinner culture is cultured in 4 15L rolling bottles and continues to cultivate 48h Growth of Cells to more than 95% densification of rolling bottle aufwuchsplate, culture temperature: 37 ℃, Rotary Machine rotating speed is 8rpm/min;
4) bio-reactor microcarrier is cultivated:
Digesting the mdck cell that is cultured to more than 95% densification of aufwuchsplate in above-mentioned 3 15L rolling bottles, all after digestion, be inoculated in the bio-reactor of getting microcarrier ready and cultivate, is 1.5 * 10 through counting cells number
9individual, through counting the cell count of receiving on each microcarrier, be 14,6h before cultivating, nutrient solution is 3L, rotating speed 30rpm, temperature: 37 ℃, pH7.25, dissolved oxygen: 50%, to cultivate after 6h, nutrient solution is mended to 5L, rotating speed 25rpm, temperature: 37 ℃, pH7.3, dissolved oxygen: 50%; At 24h, 48 h, 60 h and the 72h that cultivate, change respectively 2.5 liters of liquid, stop stirring 3-5min and make microcarrier naturally sink to reactor bottom, extract out and add again half maintenance medium of original fluid after all cell culture fluids;
5) virus inoculation:
Mdck cell is cultivated 84h in bio-reactor, 90% microcarrier surface forms after fine and close individual layer, stopping stirring 3min makes microcarrier naturally sink to reactor bottom, after extracting all cell culture fluids out, add again 2.5L to contain the high sugared maintenance medium of DMEM of volume percentage amounts 1% new-born calf serum, according to the amount of 3 μ g/ml, adding TPCK pancreatin and M.O.I is that after 0.1 inoculation avian influenza strain (H5N1 hypotype Re-5 strain) is cultivated 2h, maintenance medium is mended to 5L, 2h before cultivating, bioreactor culture condition is rotating speed: 30rpm, temperature: 33 ℃, pH7.5, dissolved oxygen: 50%, cultivate after 2h, bioreactor culture condition is rotating speed: 25rpm, temperature: 33 ℃, pH7.5, dissolved oxygen: 50%,
6) venom results:
Sampling observation of cell CPE per hour from meeting the malicious 10h cultivating, when 15h sampling is observed, 90% cell has come off, sampling and measuring blood clotting valency after aseptic collection all cells maintenance medium freeze thawing once, blood clotting valency is 2
9.
Finally it should be noted that: the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
- The bio-reactor microcarrier cultural method of 1.MDCK cell, is characterized in that, comprises the following steps:1) microcarrier is processed:The PBS damping fluid that is 7.4 with pH by Cytodex series microcarrier soaks 3-24h, and cleans twice, and autoclaving is cooling standing, after cleaning, adds the cell culture fluid containing bovine serum of same volume standby with the cell culture fluid that contains bovine serum;GF series microcarrier is soaked to 3-24h by the purified water that contains tween-80, after the PBS buffer solution for cleaning that is 7.4 with pH again, autoclaving, cooling standing, after cleaning with the cell culture fluid that contains bovine serum, add the cell culture fluid containing bovine serum of same volume standby;2) MDCK seed cell recovery:From liquid nitrogen, take out frozen mdck cell, quick-thawing recovery cell in the water-bath of 37 ℃-39 ℃, after recovery, with the cell culture fluid that contains bovine serum, standing in square vase to be cultured to Growth of Cells more than 90% fine and close to square vase aufwuchsplate;3) enlarged culturing of seed cell:Until step 2) in Growth of Cells when more than 90% fine and close to square vase aufwuchsplate, use cell dissociation buffer peptic cell, according to the sub-bottle ratio enlarged culturing of (1:2)-(1:10) to the required cell count of spinner culture; Inoculate spinner culture, until Growth of Cells, arrive rolling bottle aufwuchsplate when more than 90% fine and close, use cell dissociation buffer peptic cell, according to the sub-bottle ratio enlarged culturing of (1:2)-(1:10) to the required cell count of bioreactor culture;4) bio-reactor microcarrier is cultivated:According to throwing in the ratio of inoculating 5-50 cell on 2-25g microcarrier, each microcarrier in every liter of volume of culture, by being inoculated into after the mdck cell digestion of spinner culture, in bio-reactor, being cultured to 90% microcarrier surface and forming fine and close individual layer.
- 2. the bio-reactor microcarrier cultural method of mdck cell according to claim 1, is characterized in that, in the Cytodex series microcarrier treating processes of described step 1), every gram of microcarrier soaks with the consumption of PBS damping fluid for being greater than 20mL; The PBS damping fluid consumption that every gram of microcarrier cleans use is at every turn for being greater than 20mL, and the enchylema consumption that contains bovine serum that every gram of microcarrier cleans use is at every turn for being greater than 20mL; The described cell culture fluid that contains bovine serum is the high sugared nutrient solution of the DMEM containing volumn concentration 2%-10% bovine serum; Described bovine serum is newborn calf serum or foetal calf serum.
- 3. the bio-reactor microcarrier cultural method of mdck cell according to claim 1, it is characterized in that, in the GF series microcarrier treating processes of described step 1), described in contain tween-80 purified water be to contain the purified water that volumn concentration is 0.01% tween-80; Every gram of microcarrier soaks with the consumption of the purified water that contains tween-80 for being greater than 20mL; The PBS damping fluid consumption that every gram of microcarrier cleans use is at every turn for being greater than 20mL, and the enchylema consumption that contains bovine serum that every gram of microcarrier cleans use is at every turn for being greater than 20mL; The described cell culture fluid that contains bovine serum is the high sugared nutrient solution of the DMEM containing volumn concentration 2%-10% bovine serum; Described bovine serum is newborn calf serum or foetal calf serum.
- 4. the bio-reactor microcarrier cultural method of mdck cell according to claim 1, is characterized in that described step 2) in, described in contain bovine serum cell culture fluid be the high sugared nutrient solution of DMEM that contains volumn concentration 8%-10% bovine serum; Described culture temperature is 37 ℃.
- 5. the bio-reactor microcarrier cultural method of mdck cell according to claim 1, is characterized in that, in described step 3), cell dissociation buffer is that mass percent concentration is 0.25% trypsin solution; Cell culture fluid is the high sugared nutrient solution of DMEM that contains volumn concentration 8%-10% bovine serum; The rotating speed of spinner culture is 2-15rpm/min; Culture temperature is 37 ℃.
- 6. the bio-reactor microcarrier cultural method of mdck cell according to claim 1, is characterized in that, in described step 4), microcarrier is Cytodex series microcarrier or the GF series microcarrier of processing in step 1); Cell dissociation buffer is that mass percent concentration is 0.25% trypsin solution; Cell culture fluid is the high sugared nutrient solution of DMEM that contains volumn concentration 8%-10% bovine serum; The culture condition of reactor is: rotating speed 20-50rpm, 37 ℃ of temperature, pH value 7.2-7.4, dissolved oxygen 20-80%.
- 7. the mdck cell of cultivating according to the bio-reactor microcarrier cultural method of the arbitrary described mdck cell of claim 1-6 is used the application in influenza vaccines preparation people.
- 8. the application of the mdck cell of cultivating according to the bio-reactor microcarrier cultural method of the arbitrary described mdck cell of claim 1-6 in preparing avian influenza vaccine.
- 9. according to the application described in claim 7 or 8, it is characterized in that, comprise the following steps:A) virus inoculation:Mdck cell 90% microcarrier surface until bioreactor culture forms after fine and close individual layer, stop stirring 3-5min, make microcarrier naturally sink to reactor bottom, extract out and add again half maintenance medium of original fluid after all cell culture fluids, according to TPCK pancreatin 1-20 μ g/ml, virus infection plural number 0.01-1 virus inoculation, cultivate after 2h, maintenance medium is mended to consistent with original fluid;B) venom results:Sampling observation of cell pathology effect CPE per hour from connecing malicious the 10th hour of cultivating, when there is pathology effect in 80% above cell, aseptic collection all cells maintenance medium, sampling and measuring blood clotting valency or mensuration hemagglutinin content after freeze thawing once, when blood clotting valency is 2 9or hemagglutinin content while reaching 2 μ g/ml by bird flu or people manufactures with influenza vaccines and inspection procedure is carried out.
- 10. application according to claim 9, is characterized in that, in described step a), maintenance medium is the high sugared nutrient solution of DMEM that contains volumn concentration 1%-3% bovine serum; Described TPCK pancreatin is serum-free ox pancreas source VIII type pancreatin; The culture condition of described reactor is: rotating speed 20-50rpm, 33 ℃-35 ℃ of temperature, pH value 7.4-7.6, dissolved oxygen 20-80%.
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