CN1238495C - Aimal cell multipore micro carrier immobilized high efficiency culturing method and its culturing medium - Google Patents
Aimal cell multipore micro carrier immobilized high efficiency culturing method and its culturing medium Download PDFInfo
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Abstract
The present invention discloses a high-efficiency culture method for immobilization of porous microcarriers of animal cells, which comprises cellulose porous microcarrier pretreatment, cell inoculation, cell acclimation and cell culture, wherein the treated porous microcarriers and a serum-containing culture medium are added into a blending jar, and cell suspension is inoculated into the blending jar according to the required inoculum density and cultured; the cell suspension growing on the porous microcarriers is progressively amplified and cultured in the blending jar, and the content of the serum in the culture medium is gradually reduced to 0%; the cell suspension is transferred into a reactor and progressively amplified and cultured, and part of the microcarriers are periodically updated to culture the cells without the serum. The present invention has the advantages of high cell culture density, convenient amplification of culture scale, long cell culture time, low price of the serum-free medium and high reactor productive capacity. The cell culture time can generally reach 100 days more or less. The present invention is suitable for the culture of adherent cells and suspension cells.
Description
Technical field
The present invention relates to zooblast porous microcarrier immobilization high-efficient culture method and substratum thereof, more specifically say so and utilize porous microcarrier the zooblast immobilization, serum-free high-density culture zooblast in bio-reactor, efficiently express the method and the employed substratum of gene engineering product or other biological goods, belong to cell engineering field and field of biological pharmacy.
Background technology
With genetically engineered and cell-fusion techniques is that the modern biotechnology of sign has formed a new high-tech industry--modern biotechnology pharmaceutical industry in medicine industry.According to statistics, the sales volume of calendar year 2001 whole world modern biotechnology medicine surpasses 30,000,000,000 dollars, and annual speed increment with 15%-20%, and the rate of increase of conventional medicine is 5%-8%.The animal cell large-scale culture technique is a technology platform of the most critical in the bio-pharmaceuticals industry, the biological products that are used for clinical treatment that get the Green Light over half are all produced by cell culture technology at present, and, exploitation along with macromole functional protein and Ren Yuan (change) antibody, the progress of gene therapy and cell therapy, and the research and development of organizational project and artificial creature's organ product, cell culture technology will be brought into play bigger effect in bio-pharmaceuticals industrialization field.
The biological products of producing with the animal cell large-scale culture technique comprise all therapeutic monoclonal antibodies, virus vaccines, Interferon, rabbit, t-PA, EPO, thrombin, G-CSF, fusion rotein (as Enbrel), tissue engineering product and other biological goods.Very most of biological products of drugs approved by FDA are all produced by animal cell expression.Mainly contain three kinds of expression system express recombinant proteins of bacterium (E.coli), yeast, mammalian cell (as Chinese hamster ovary cell, i.e. Chinese hamster ovary celI) at present.Prokaryotic expression systems such as bacterium are bred soon, are easy to cultivate, but expressed protein lacks the modification after transcribing, as the environment etc. that lacks protein restriction restriction enzyme site, disulfide linkage, special glycosylation, phosphorylation, amidation and form the accurate three-dimensional structure of natural protein.And many proteic biological activitys with transcribe after modification relevant, and the prokaryotic system expressed proteins is generally intracellular product, needs smudge cells could extract product to bring difficulty for the separation and purification of product, also is subjected to the pollution of external source toxin simultaneously easily.And the albumen of eukaryotic expression system such as expressing cho cell has the modification after transcribing, all very approximate on 26S Proteasome Structure and Function with human body self excretory native protein, thereby the U.S. FDA tendency adopts eukaryotic expression system to produce pharmaceutical grade protein in 21 century.Nearly all albumen with procaryotic cell expression all can adopt eukaryotic expression system production, otherwise it is then exactly so, albumen with the animal cell expression system expression all is exocytosis, the separation and purification process of product is very simple, but, because cell large scale culture technique more complicated still is in development at present and improves the stage, thereby many recombinant proteins is still selected prokaryotic expression system production for use.Calendar year 2001 global marketing volume ranks preceding 20 biological products, and with account for 11 kinds of CHO animal cell expression, sales volume accounts for 65% of sum.This shows the importance of extensive high-efficient culture technology in field of biological pharmacy of animal cell expression product.
The demand for development of technology is increasing with the proportion of the biological products that the zooblast eukaryotic expression system is produced.Prokaryotic expression system generally is used for small molecules, protein production simple in structure, need not after albumen is transcribed to modify, as Regular Insulin.And eukaryotic expression system is mainly used in and produces macromole, baroque albumen, and the modification after transcribing has material impact to proteic biological activity, as (EPO) such as tissue-type plasminogen activator (tPA), erythropoietin.A development trend of biological products is the bigger functional protein of exploitation molecular weight and human antibody, gene therapy product, treatment usefulness vaccine and tissue engineering product, and these products are generally all produced by the animal cell large-scale culture technique.Especially treatment is the key areas of biological products research and development in nearest 5 years with the exploitation of antibody drug.At present nearly all chimeric antibody and humanized antibody all are to use expressing cho cell, and the production that detects with the mouse resource monoclonal antibody also can obtain through cultivating hybridoma.Therefore, along with the development of bio-pharmaceuticals industry, the biological products proportion of producing by the animal cell large-scale culture technique is with increasing.
The animal cell expression production throughput can not be met the need of market.The animal cell culture technical sophistication does not also have a kind of high-efficient culture technology of generally acknowledged standard of comparison so far.Because the needs of each drugmaker's technical know-how, in the paper of publishing, be difficult to see really be applied to the technology and the technology of producing.In general, nearly all drugmaker all adopts batch formula culture process similar to fermentation using bacteria, and this culture process technical difficulty is relatively low, but the throughput of reactor is also very low, has limited the production and supply of animal cell expression product greatly.Dosage is at the medicine such as the Enbrel in hundreds of milligram～a few gram/people/year, and the manufacturer has used 6 12, and the bio-reactor production of 500L, annual production are lower than 10 kilograms, and as seen the production efficiency of its reactor is not high.According to Bains﹠amp; Company estimates, 10 years from now on, far can not satisfy the demand in market with the biological products of mammalian cell production.
In order to improve the production efficiency of reactor, reduce running cost, ideal is commercial should to have following characteristics with industrialized animal cell culture technology: " high-density, long-time, high expression level on a large scale, ".With the reactor high efficiency production biological products than small-scale is the emphasis of animal cell large-scale culture technique research always, since the 1980's, the animal cell large-scale culture technique becomes one of of paramount importance research contents in biochemical engineering field, but owing to there is following difficult point, so far still do not have a kind of sophisticated cell high-efficient culture technique, these difficult points comprise:
(1) a little less than zooblast is highly brittle, how to solve the contradiction of oxygen supply and cell injury.This is the key constraints of cell high-density culture.Improve oxygen transfer rate and generally can increase mixture strength, thereby may cause damage by pair cell.The factor that causes the cell physical abuse comprises that mainly mechanical collision, fluid shearing, bubble cohesion break etc., and the main method of solution is: add protective material such as Pluronic F68, serum etc.; The film embedding is cultivated as hollow fiber membrane reactor; Adopt the porous cell upholder to cultivate, as porous microcarrier, sintered ceramic etc.; Improve structure of reactor, make and stir gentleness, bubble and cell are isolated, as cage aeration reactor, still aerobic reactor, and the two reactors etc. that rise of gas-liquid.
(2) how to solve mass transfer and mix the contradiction of amplifying with process.This is the key that realizes that animal cell large-scale is cultivated.Should guarantee to have good mass transfer mixed performance and gentle cell growing environment after the reactor amplification culture.Many type of reactor such as fixed-bed reactor and hollow fiber membrane reactor have good cell cultures effect in small scale experiments, but all are difficult to use in commercialization, scale operation, and major cause is can not amplification culture.What can accomplish amplification culture at present generally is to adopt the cell suspension culture technology.Once thinking in the past that the porous microcarrier cultivation was difficult to amplify step by step.
(3) how to solve the apoptosis problem of cell long-period cultured continuously.This is the key that keeps the cell long-period high-density culture.When cells in vitro was cultivated, the envrionment conditions of bringing out apoptotic major cause and be the cell growth was more abominable, as nutritive deficiency, and the accumulation of meta-bolites, pH and dissolved oxygen control are improper, and the mechanical stirring shearing force is bigger etc., especially easier generation in serum free medium.Apoptosis has influenced the cell cultures time greatly, thereby seriously reduces the throughput and the utilising efficiency of reactor, has increased running cost and labour intensity.Just because of this, apoptotic research was just becoming a heat subject in the cell culture technology during pair cell was cultivated in recent years.For addressing this problem, many scholars attempt utilizing the strain of genetic engineering technique engineered cells, to suppress apoptotic gene bcl-2 transfection in host cell, or in cell culture medium, add the expensive apoptotic factor of inhibition, expect to suppress or delay apoptosis, the life-span that prolongs individual cells is to prolong incubation time, but effect is also not obvious at present, there is the effect instability, cost is expensive, time expand, do not grown deficiencies such as (prolonging 2-3 days), do not see that application is arranged in mass cell is cultivated.
(4) how to solve cell under conditions of high density, owing to have density effect, the expression level decline problem of individual cells.How to keep or improve the expression level of cell under the high-density growth condition, be related to the concentration of product and the utilization ratio of output and substratum.Cell activities rely interdependent internal structure be one of complicated nonlinear systems of nature, it is not nearly enough simply coming the design and running bio-reactor with traditional chemical reaction engineering principle, especially cultivate the more complicated zooblast of structure (cell surface has the acceptor relevant with cell communication, information between cell and the environment, between cell and the cell and the transmission of the cell interior growth, propagation, metabolism and the product that directly influence cell express).Many documents attempt utilizing variation and other outside stimuluss of variation of temperature, nutrient solution osmotic pressure, improve the expression level of cell.
(5) research and development of serum free medium.Produce biological products and have many unfavorablely with containing blood serum medium, cultivate cost and opportunities for contamination as increasing, increase purifying difficulty and cost, increase the product quality control index, therefore, the biological products of the clinical use of scale operation should use serum free medium as far as possible.
(6) cell retention Study on Technology.Because the level of individual cells expression product is certain, improves cell density and just can improve production concentration in the supernatant, thereby improve the throughput of reactor.And to obtain higher cell culture density, prerequisite is will be with cell retention in reactor.The method of entrapped cell mainly contains three classes: (A) partition method, as centrifugal, rotary filter on the throne, recirculation reactor, ultrasonic wave isolation etc.; (B) entrapping method (closed) is as hollow fiber membrane reactor, microencapsulation, membrane reactor; (C) wrapped folder method (open) is as porous ceramics, non-woven fabrics cultivation, sponges/foams matrix, porous microcarrier etc.
(7) zooblast is criticized formula cultivation and the selection of cultured continuously and the foundation of growth kinetics of cells model.Select training method to determine according to the concrete characteristics of cell and product.From culture effect, cultured continuously cell density height, the cultured continuously cell density of general entrapped cell is all 10
7/ ml, cultivate high about 10 times than batch formula, thereby reactor throughput is also cultivated high more than 10 times than batch formula, and the labour intensity of cultured continuously is little, production cost is low, but prerequisite is whole continuous culture system, comprises that reactor Controlling System, cell retention system etc. must guarantee the long-play normal reliable.But most biopharmaceutical companys all also adopt batch formula training mode, because the batch type suspended cultivation operating ratio of zooblast is more simple and reliable, easily implementation procedure is amplified, do not relate to the problem that apoptosis and high-density culture expression level descend, the suspension cell individuality is less, be subjected to the influence of hydrodynamic shear less relatively, than the technology that is easier to use for reference fermentation using bacteria.This also illustrates does not also have a kind of ideal cultured continuously technology to select for drugmaker at present.But the batch type suspended cultivation throughput of zooblast is low, and equipment and factory building investment are high, and production cost is also than higher.To produce t-PA is example, and the scale of zooblast reactor reaches 6 * 6000L, and corresponding purifier apparatus is also very huge, promptly requires the purifier apparatus can disposable processing cells and supernatant several ten thousand liters.With the cost of this explained hereafter lg engineered protein about 10,000 dollars, and annual production has only 10 feather weight levels, and batch formula training method is not suitable for unsettled proteinic production, as the production of uPA.The product of animal cell expression such as various humanized antibody, Enbrel, t-PA etc., dosage is generally all in milligram level even gram level (except the EPO), therefore, meet the need of market, adopt batch scale of formula cultivation zooblast reactor all very big, as the reactor scale of producing Enbrel reached 12,500L, but output still can not be met the need of market.
Be the emphasis of animal cell large-scale culture technique research than reactor high efficiency production biological products on a small scale always, this direction more tallies with the national condition, and does not have the patent of the mature technology of the long-term cultured continuously of animal cell large-scale high-density.
Summary of the invention
The porous microcarrier immobilization high-efficient culture method that the purpose of this invention is to provide a kind of cultured continuously zooblast.
Another object of the present invention provides a kind of serum free medium with low cost, quality controllable, safe in utilization.
For achieving the above object, the present invention is by the following technical solutions:
Zooblast porous microcarrier immobilization high-efficient culture method comprises the steps:
(1) Mierocrystalline cellulose porous microcarrier pre-treatment: behind phosphoric acid buffer (PBS) the immersion 4h of Mierocrystalline cellulose porous microcarrier with 0.1mol/L, pH7.0, the PBS that inclines is again with PBS washing 3 times.Behind 121 ℃ of autoclaving 30min, sucking-off PBS, standby with 24 ℃ of postposition of DMEM/F12 substratum washing;
(2) cell inoculation: add the porous microcarrier of handling well in the blender jar and contain blood serum medium, cell suspension is inoculated in the blender jar by required inoculum density cultivates, culture condition is 37.0 ℃ ± 0.1 ℃ of a temperature, pH value 7.0 ± 0.05, rotating speed 20-100r/min;
(3) cell domestication: will be grown in cell suspension on porous microcarrier amplification culture step by step in blender jar, and the serum content in the substratum dropped to 0% gradually, culture condition is 37.0 ℃ ± 0.1 ℃ of a temperature, pH value 7.0 ± 0.05, rotating speed 20-100r/min, to cell density greater than 5 * 10
6Cell/mL changes liquid measure and is generally 1/2-1 volume of culture;
(4) cell cultures: cell suspension is transferred in the reactor amplification culture step by step, uses still ventilation oxygen-supplying system, adopt batch formula to change the liquid continuous culture process, regular update part microcarrier, pair cell carries out serum-free culture.
Described Mierocrystalline cellulose porous microcarrier is Cytopore-2.
The concentration of porous microcarrier is 1-4g/L in described (2) cell inoculation step.
The concentration of blood serum medium is 2% in described (2) cell inoculation step.
The inoculum density of cell suspension is 2 * 10 in described (2) cell inoculation step
5-5 * 10
5Cell/mL.
Transfer in described (4) cell culture step is meant that the porous microcarrier that uses pipeline will cover with cell directly imports in the next stage reactor.
Add an amount of substratum and treated porous microcarrier in advance in the reactor in described (4) cell culture step.
Described (4) cell culture step adopts batch formula to change the liquid cultured continuously and is meant and changes a liquid 1-1.2 working volume by the cell retention system every day, and microcarrier and cell retention in reactor, are gathered in the crops and contained the supernatant of product and add the equivalent fresh culture.
The serum-free culture of described (4) cell culture step is meant that using serum-free growth medium and serum-free to express substratum successively cultivates the cell in the reactor.
Described zooblast is attached cell such as Chinese hamster ovary celI or suspension cell such as hybridoma.
The serum free medium that is used for zooblast porous microcarrier immobilization high-efficient culture, this serum free medium comprise growth form serum free medium and expression type serum free medium, and the composition and the content thereof of every 10L substratum see the following form respectively:
Growth form serum-free culture based component and content thereof (every 10L substratum)
| Composition | The amount of taking by weighing | Composition | The amount of taking by weighing |
| DMEM/F12(1∶1) | 156g | Glucose | 20g-60g |
| Sodium bicarbonate | 10g-20g | Peptone | 10g |
| Glutamine | 5g | 6-aminocaprolc acid | 6.5g |
| Proline(Pro) | 110mg | Leucine | 500mg |
| Aspartic acid | 70mg | Halfcystine | 320mg |
| Methionine(Met) | 600mg | Serine | 300mg |
| Glycine | 75mg | Folic acid | 100mg |
| Vitamins B 1 | 500mg | Vitamins B 2 | 100mg |
| Vitamins B 6 | 500mg | Vitamins B 12 | 50mg |
| Vitamins C | 1000mg | Inositol | 2000mg |
| Niacinamide | 500mg | Regular Insulin | 800U |
| Transferrins,iron complexes | 25mg | Thanomin | 20mg |
| FeSO 4·7H 2O | 10mg | ZnSO 4 | 20mg |
Expression type serum-free culture based component and content thereof (every 10L substratum)
| Composition | The amount of taking by weighing | Composition | The amount of taking by weighing |
| DMEM/F12(1∶1) | 156g | Glucose | 20g-60g |
| Sodium bicarbonate | 10g-20g | Peptone | 10g |
| Glutamine | 5g | 6-aminocaprolc acid | 6.5g |
| Proline(Pro) | 110mg | Leucine | 500mg |
| Aspartic acid | 70mg | Halfcystine | 320mg |
| Methionine(Met) | 600mg | Serine | 300mg |
| Glycine | 75mg | Folic acid | 100mg |
| Vitamins B 1 | 1000mg | Vitamins B 2 | 200mg |
| Vitamins B 6 | 1000mg | Vitamins B 12 | 100mg |
| Vitamins C | 2000mg | Inositol | 2000mg |
| Niacinamide | 500mg | Regular Insulin | 400U |
| FeSO 4·7H 2O | 10mg | ZnSO 4 | 20mg |
The present invention adopts porous microcarrier immobilized cell culture technique, and the protection cell is avoided physical abuse, and attached cell or suspension cell are trapped in the reactor, improves cell culture density; The rule of utilizing cell to shift automatically between porous microcarrier is set up the technological method that simple and reliable cell cultivation process amplifies; Adopt still ventilation oxygen-supplying system, solve in cell cultivation process that foam that direct ventilation oxygen-supplying brings spreads unchecked and the problem of damaging cells; The serum free medium of development reduces cell cultures cost, opportunities for contamination and subsequent product purifying cost and difficulty; Adopt batch formula to change the liquid continuous culture process, in time remove the refuse accumulation in the culturing process, reduce the ratio of product degradation in batch formula culturing process, and improve the throughput of reactor; Adopt the method for regular update part microcarrier, zooblast is shifted between microcarrier mutually, improve the microenvironment of cell growth, solve cell long-period cultured cells apoptosis problem.
Advantage of the present invention is: adopt the biochemical engineering technology, the technology of having set up a kind of simple, reliable long-term cultured continuously of large scale and high density zooblast and having efficiently expressed, adopt this technology to cultivate zooblast (1) cell culture density height, general culture density＞2 * 10
7/ ml; (2) can make things convenient for the amplification culture scale; (3) solved the apoptosis problem in the cultivation, incubation time is long, generally can reach about 100 days, and cell viability remains on more than 95%; (4) serum free medium is cheap, and can grow and expression by fine sustenticular cell; (5) the throughput height of reactor, expression level are 5 μ g/10
6The engineering cell of cells/d adopts the technology among the present invention to cultivate, and yearly capacity can reach 10g/L reactor/year, and more present reactor throughput improves about 100 times; (6) both can be used for the cultivation of attached cell such as Chinese hamster ovary celI, can be used for the cultivation of suspension cell such as hybridoma again.
The invention will be further described below in conjunction with embodiment, is not limitation of the present invention.
Embodiment
Embodiment 1, Cytopore-2 Mierocrystalline cellulose porous microcarrier are cultivated zooblast
One, the pre-treatment of Cytopore-2 Mierocrystalline cellulose porous microcarrier
(Pharmacia Biotech AB, Sweden are that a kind of physicochemical characteristic is fit to animal cell large-scale cultured cells upholder very much CatNo.17-1271-02) to Cytopore-2 Mierocrystalline cellulose porous microcarrier, and its main physicochemical characteristic sees Table 1.
Behind preceding phosphoric acid buffer (PBS) immersion 4h with 0.1mol/L, pH7.0, the PBS that inclines is again with PBS washing 3 times.Behind 121 ℃ of autoclaving 30min, sucking-off PBS, standby with 24 ℃ of postposition of DMEM/F12 substratum washing.
Table 1Cytopore-2 porous microcarrier physicochemical characteristic
| Material particle diameter d 50Effective surface area density aperture swelling coefficient electric density | 100% Mierocrystalline cellulose 200-280 μ m 1.1m 2The about 1.8meq/g of the about 30 μ m 40mL/g dry of/g dry 1.03g/mL |
Cytopore porous microcarrier physical strength height, recyclable repeated use behind the culturing cell.Recovery method is as follows: (1) has the porous microcarrier of cell to place the 2000mL bottle minister in about 300mL, adds 1000mL water, with forced oscillation after 2 minutes, and with 150 order stainless steel filtering net filtering liquid, the cell and the cell debris that come off with preliminary removal.(2) microcarrier is suspended in the citric acid solution of 0.2mol/L again, placed room temperature 6 hours, and, make the most cells cracking in the porous microcarrier inside and come off out, with 150 mesh filter screen filtering liquid every vibration in 1 hour 2 minutes.(3) repeat the first step, water repeatedly cleans porous microcarrier, until treat after the microcarrier sedimentation liquid limpid transparent till.If necessary, can use the citric acid solution washing by soaking once more.(4) with 5% sodium hydrogen carbonate solution washing by soaking once, wash with water thereafter three times.(5) microscopically is observed, if porous microcarrier inside also has more dead cell, available thick pancreatin solution soaking was O.2% hatched 3 hours and swayed 2 minutes every half an hour in 37 ℃, and water cleans to liquid limpid transparent afterwards.(4) the usefulness deionized water wash is three times, and is after the 0.1M PBS damping fluid of usefulness pH 7.0～7.5 washs three times again, standby at 121 ℃ of following autoclavings.(5) if necessary, add in the solution that contains 0.02mol/L-0.05mol/L DEAE hydrochloride, 0.2mol/L NaOH, put 60 ℃ and constantly stir 10h down, make its crosslinked-DEAE weakly-basic anion cation exchange groups, to promote cell attachment.Wash once with 1% citric acid solution crosslinked back, uses distilled water wash again three times, uses 0.1mol/L phosphoric acid buffer (PBS) washing 3 times thereafter, behind 121 ℃ of steam sterilizing 30min.(6) sucking-off PBS, with the DMEM/F12 substratum (U.S. Hyclone company produces, and CatNo.SH30004.04) washing is 2 times, again with the immersion of DMEM/F12 substratum put 4 ℃ standby.
Two, cell inoculation
The porous microcarrier handled well the concentration by 1-1.5g/L is added in the blender jar, and adds the substratum that contains 1% serum.Attached cell of in square vase and rolling bottle, cultivating such as Chinese hamster ovary celI, trysinization through 0.25%, make cell suspension with substratum piping and druming, direct inoculation is in blender jar, and the suspension cells of cultivating in square vase such as hybridoma can directly be made cell suspension inoculation with substratum piping and druming and cultivate in blender jar.General inoculum density needs greater than 2 * 10
5Cell/mL, preferably inoculum density is 5 * 10
5Cell/mL.In the 5h of inoculation back, mixing speed is generally (20-40) r/min, to allow cell more fully contact porous microcarrier, rotating speed can be increased to (50-60) r/min subsequently, and culture temperature is 37.0 ℃ ± 0.1 ℃.
Three, cell domestication
To be grown in cell suspension on porous microcarrier amplification culture step by step in blender jar, cultivate 3d after, can change liquid in right amount according to glucose concn in the substratum, culture condition is 37.0 ℃ ± 0.1 ℃ of a temperature, pH value 7.0 ± 0.05, rotating speed 50-60r/min, to cell density greater than 5 * 10
6Cell/mL changes liquid measure and is generally 1/2-1 volume of culture; And the serum content in the substratum dropped to 0% gradually, to tame cell adapted serum free medium.
Four, cell cultures
Cell suspension is transferred to 7.5L Biostat CT reactor by blender jar, and (working volume is 5L, B.Braun company, Germany), thereafter transfer to 30L Biostat UC reactor (working volume is 20L, B.Braun company, Germany) amplification culture step by step in the amplification culture reactor step by step, use still ventilation oxygen-supplying system, adopt batch formula to change the liquid continuous culture process, regular update part microcarrier, pair cell carries out serum-free culture.
Because cell can shift between carrier that covers with cell and empty carrier automatically, during every grade of amplification culture, in more massive reactor, add earlier an amount of substratum and treated porous microcarrier in advance, the porous microcarrier that covers with cell directly enters in the next stage reactor by pipeline, and the inoculation and the amplification of cell cultures are helped in the digestion that need not pancreatin.This seed cell preparation method and reactor cell inoculation method are very easy, and can strict guarantee aseptic, for the scale of porous microcarrier cell cultures is provided by the condition that provides.
Five, the dyeing of grown cell and counting in the porous microcarrier
1.MTT staining: take out behind the sample with 5mg/ml MTT solution-dyed, put 37 ℃ of following 2h-4h after, examine under a microscope, it is long that the porous microcarrier of cell is arranged is black, the porous microcarrier of growing cells still is not this white.
2. crystal violet staining assay: 5ml is in the 10ml test tube in sampling, treat after the porous microcarrier sedimentation with suction pipe sucking-off supernatant as much as possible, thereafter the 0.1mol/L citric acid solution that contains 0.1% Viola crystallina that adds equivalent, blow and beat, sway with suction pipe every 1h, place 37 ℃ after following 3 hours, shake up and treat that microcarrier slightly after the sedimentation, draws appropriate amount of fluid with suction pipe on blood counting chamber near liquid level, number goes out to be dyed the cell check figures of intense violet color.
The preparation of embodiment 2, serum free medium
Cell culture density is greater than 2 * 10
6Behind cell/mL, can use serum free medium gradually instead.Serum free medium has two kinds: a kind of for the growth form serum free medium, when cell density greater than 2 * 10
6Behind cell/mL, with this culture medium culturing Chinese hamster ovary celI, its specific growth rate is about 0.22d
-1Another kind is the expression type serum free medium, when cell density greater than 10
7Behind cell/mL, with this culture medium culturing Chinese hamster ovary celI, its specific growth rate is about 0.11d
-1, lower growth velocity helps keeping the relatively stable of cell density under the high-density culturing condition, to keeping dissolved oxygen level and cellular expression levels favourable.
1, the preparation of growth form serum free medium:
(1) composition and content see Table 2.
Table 2 preparation 10L growth form serum-free culture based component table look-up
| Composition | The amount of taking by weighing | Composition | The amount of taking by weighing |
| DMEM/F12(1∶1) | 156g | Glucose | 20g-60g |
| Sodium bicarbonate | 10g-20g | Peptone | 10g |
| Glutamine | 5g | 6-aminocaprolc acid | 6.5g |
| Proline(Pro) | 110mg | Leucine | 500mg |
| Aspartic acid | 70mg | Halfcystine | 320mg |
| Methionine(Met) | 600mg | Serine | 300mg |
| Glycine | 75mg | Folic acid | 100mg |
| Vitamins B 1 | 500mg | Vitamins B 2 | 100mg |
| Vitamins B 6 | 500mg | Vitamins B 12 | 50mg |
| Vitamins C | 1000mg | Inositol | 2000mg |
| Niacinamide | 500mg | Regular Insulin | 800U |
| Transferrins,iron complexes | 25mg | Thanomin | 20mg |
| FeSO 4·7H 2O | 10mg | ZnSO 4 | 20mg |
(2) preparation method
Take by weighing each composition in the aseptic apyrogenic container of 10L by table 2, add 10L pure water stirring and dissolving, with 0.22 μ m membrane filtration degerming.
2, the preparation of expression type serum free medium:
(1) composition and content see Table 3.
Table 3 preparation 10L expression type serum-free culture based component table look-up
| Composition | The amount of taking by weighing | Composition | The amount of taking by weighing |
| DMEM/F12(1∶1) | 156g | Glucose | 20g-60g |
| Sodium bicarbonate | 10g-20g | Peptone | 10g |
| Glutamine | 5g | 6-aminocaprolc acid | 6.5g |
| Proline(Pro) | 110mg | Leucine | 500mg |
| Aspartic acid | 70mg | Halfcystine | 320mg |
| Methionine(Met) | 600mg | Serine | 300mg |
| Glycine | 75mg | Folic acid | 100mg |
| Vitamins B 1 | 1000mg | Vitamins B 2 | 200mg |
| Vitamins B 6 | 1000mg | Vitamins B 12 | 100mg |
| Vitamins C | 2000mg | Inositol | 2000mg |
| Niacinamide | 500mg | Regular Insulin | 400U |
| FeSO 4·7H 2O | 10mg | ZnSO 4 | 20mg |
(2) preparation method
Take by weighing each composition in the aseptic apyrogenic container of 10L by table 3, add 10L pure water stirring and dissolving, with 0.22 μ m membrane filtration degerming.
When cultivating the hybridization cancer cells with serum free medium, at cell density greater than 5 * 10
6Use the expression type substratum behind the cells/mL instead, can on the basis of table 3, add 5 * 10
-5The mol/L mercaptoethanol.
Embodiment 3, bio-reactor high-density long-term cultivation gene recombination Chinese hamster ovary celI also efficiently express gene recombination uPA (rhPro-UK)
One, embodiment 1 is seen in Mierocrystalline cellulose porous microcarrier pre-treatment.
Two, cell inoculation: the porous microcarrier handled well the concentration by 1-1.5g/L is added in the blender jar, and adds the substratum that contains 1% serum.The gene recombination Chinese hamster ovary celI of the attached cell Pro-UK that cultivates in square vase and rolling bottle is that CL-11G (sees Chinese patent, the patent No. is ZL96119836.2, international monopoly Main classification C12N15/58), trysinization through 0.2%, make cell suspension with substratum piping and druming, direct inoculation is in blender jar, and inoculum density needs greater than 2 * 10
5Cell/mL, preferably inoculum density is 5 * 10
5Cell/mL.In the 5h of inoculation back, mixing speed is generally (20-40) r/min, to allow cell more fully contact porous microcarrier, rotating speed can be increased to (50-60) r/min subsequently, and culture temperature is 37.0 ℃ ± 0.1 ℃.
Three, cell domestication: will be grown in cell suspension on porous microcarrier amplification culture step by step in blender jar, after cultivating 3d, can change liquid in right amount according to glucose concn in the substratum, culture condition is 37.0 ℃ ± 0.1 ℃ of a temperature, pH value 7.0 ± 0.05, rotating speed 50-60r/min, to cell density greater than 5 * 10
6Cell/mL changes liquid measure and is generally 1/2-1 volume of culture; And the serum content in the substratum dropped to 0% gradually, to tame cell adapted serum free medium.
Four, cell cultures: will tame good seed cell and be inoculated in the still ventilating/stirring reactor of 30L Biostat UC (working volume is 20L, B.Braun, Germany), inoculum density is generally 10
6Cell/mL, earlier with serum-free growth form substratum, control pH=7.0 ± 0.05, dissolved oxygen (DO)=20%-40%, temperature=37.0 ℃ ± 0.1 ℃, mixing speed is 70rpm-90rpm, the concentration of porous microcarrier is the 2g/L-4g/L substratum.Adopt batch formula to change liquid cultured continuously mode, at cell density greater than 10
7Behind cell/mL, use serum-free expression type substratum instead, change liquid 1-1.2 working volume by the cell retention system every day, and microcarrier is trapped in the reactor, and results contain the supernatant of product and add the equivalent fresh culture.Be the vigor of maintenance long-term cultured, and keep the expression level of cell under the high-density culturing condition, after covering with cell in the porous microcarrier, before cellular expression levels sharply descends, partly change the porous microcarrier that covers with cell with new porous microcarrier.The replacing amount of microcarrier and change frequency visual cell's expression level and decide.Usually,, can keep cell and well grow more than 1 month if once change the porous microcarrier of 1/2-2/3, and if upgrade microcarrier about 1/4 at every turn, generally every about 15d, just need the removable parts microcarrier.Adopt this training mode, cell density can maintain (1-2) * 10 always
7Cell/mL, the viable cell ratio can maintain 90%-95%, and Pro-UK concentration can maintain 50mg/L-100mg/L in the culture supernatant, and can gather in the crops supernatant 20L-25L every day, can obtain product (1-2.5) g/d, and whole incubation time can maintain more than 3 months.
Embodiment 4, bio-reactor high-density culture mouse-mouse hybridoma 3G7-anti-Pro-UK produce the former monoclonal antibody of antiurokinase
One, embodiment 1 is seen in Mierocrystalline cellulose porous microcarrier pre-treatment.
Two, cell inoculation: the porous microcarrier handled well the concentration by 1-1.5g/L is added in the blender jar, and adds the substratum that contains 1% serum.With the mouse-mouse hybridoma of secretion antiurokinase original antibody is that the 3G7-anti-scuPA cell suspension (preserve by China Committee for Culture Collection of Microorganisms common micro-organisms center, the No. 13, North No.1 Row, Zhongguancun, Haidian District, Beijing City, 100080, phone: 010-62542758, preservation day is on September 8th, 2003, preserving number is CGMCC No.0999, the classification called after 3G7-anti-scuPA mouse of suggestion-mouse hybridoma system).Direct inoculation is in blender jar, and inoculum density needs greater than 5 * 10
5Cell/mL.In the 5h of inoculation back, mixing speed is generally (20-40) r/min, to allow cell more fully contact porous microcarrier, rotating speed can be increased to (50-60) r/min subsequently, and culture temperature is 37.0 ℃ ± 0.1 ℃.
Three, cell domestication
To be grown in cell suspension on porous microcarrier amplification culture step by step in blender jar, cultivate 3d after, can change liquid in right amount according to glucose concn in the substratum, culture condition is 37.0 ℃ ± 0.1 ℃ of a temperature, pH value 7.0 ± 0.05, rotating speed 50-60r/min, to cell density greater than 5 * 10
6Cell/mL changes liquid measure and is generally 1/2-1 volume of culture; And the serum content in the substratum dropped to 0% gradually, to tame cell adapted serum free medium.
Four, cell cultures
Cell suspension direct inoculation after the domestication is cultivated in 5L stirring reactor (Biostat CT5, B.Braun company, Germany), and inoculum density is generally 10
6Cell/mL, earlier with serum-free growth form substratum, control pH=7.0 ± 0.05, dissolved oxygen (DO)=20%-40%, temperature=37.0 ℃ ± 0.1 ℃, mixing speed is 70rpm-90rpm, the concentration of porous microcarrier is the 2g/L-4g/L substratum.Adopt batch formula to change liquid cultured continuously mode, at cell density greater than 10
7Behind cell/mL, use serum-free expression type substratum instead, change liquid 1-1.2 working volume by the cell retention system every day, and microcarrier is trapped in the reactor, and results contain the supernatant of product and add the equivalent fresh culture.Adopt the training mode among the present invention, cell density can reach 1 * 10
7Cell/mL, the viable cell ratio can maintain 90%-95%, and antibody titers generally can reach 1 in the culture supernatant: 150000-1: 300000, whole incubation time can maintain more than 2 months.
Claims (9)
1, zooblast porous microcarrier immobilization high-efficient culture method comprises the steps:
(1) Mierocrystalline cellulose porous microcarrier pre-treatment: behind the phosphoric acid buffer immersion 4h of Mierocrystalline cellulose porous microcarrier with 0.1mol/L, pH7.0, the dephosphorization acid buffer that inclines is again with phosphoric acid buffer washing 3 times; Behind 121 ℃ of autoclaving 30min, the sucking-off phosphoric acid buffer, standby with 24 ℃ of postposition of DMEM/F12 substratum washing;
(2) cell inoculation: add the porous microcarrier of handling well in the blender jar and contain blood serum medium, cell suspension is inoculated in the blender jar by required inoculum density cultivates, culture condition is 37.0 ℃ ± 0.1 ℃ of a temperature, dissolved oxygen 20%-40%, pH value 7.0 ± 0.05, rotating speed 20-100r/min;
(3) cell domestication: will be grown in cell suspension on porous microcarrier amplification culture step by step in blender jar, and the serum content in the substratum dropped to 0% gradually, culture condition is 37.0 ℃ ± 0.1 ℃ of a temperature, dissolved oxygen 20%-40%, pH value 7.0 ± 0.05, rotating speed 20-100r/min, to cell density greater than 5 * 10
6Cell/mL changes liquid measure and is generally 1/2-1 volume of culture;
(4) cell cultures: cell suspension is transferred in the reactor amplification culture step by step, uses still ventilation oxygen-supplying system, adopt batch formula to change the liquid continuous culture process, regular update part microcarrier, pair cell carries out serum-free culture;
Described zooblast is attached cell or suspension cell;
Described serum free medium comprises growth form serum free medium and expression type serum free medium, and the composition of every 10L substratum and content thereof are as follows respectively:
Growth form serum-free culture based component and content thereof
DMEM/F12,1: 1 156g glucose 20g-60g
Sodium bicarbonate 10g-20g peptone 10g
Glutamine 5g 6-aminocaprolc acid 6.5g
Proline(Pro) 110mg leucine 500mg
Aspartic acid 70mg halfcystine 320mg
Methionine(Met) 600mg Serine 300mg
Glycine 75mg folic acid 100mg
Vitamins B
1The 500mg vitamins B
2100mg
Vitamins B
6The 500mg vitamins B
1250mg
Vitamins C 1000mg inositol 2000mg
Niacinamide 500mg pancreas islet 0 plain 800U
Transferrins,iron complexes 25mg thanomin 20mg
FeSO
4·7H
2O 10mg ZnSO
4 20mg；
Expression type serum-free culture based component and content thereof
DMEM/F12,1: 1 156g glucose 20g-60g
Sodium bicarbonate 10g-20g peptone 10g
Glutamine 5g 6-aminocaprolc acid 6.5g
Proline(Pro) 110mg leucine 500mg
Aspartic acid 70mg halfcystine 320mg
Methionine(Met) 600mg Serine 300mg
Glycine 75mg folic acid 100mg
Vitamins B
1The 1000mg vitamins B
2200mg
Vitamins B
6The 1000mg vitamins B
12100mg
Vitamins C 2000mg inositol 2000mg
Niacinamide 500mg Regular Insulin 400U
FeSO
4·7H
2O 10mg ZnSO
4 20mg。
2, zooblast porous microcarrier immobilization high-efficient culture method according to claim 1, it is characterized in that: described Mierocrystalline cellulose porous microcarrier is Cytopore-2.
3, zooblast porous microcarrier immobilization high-efficient culture method according to claim 1, it is characterized in that: the concentration of porous microcarrier is 1-4g/L in described (2) cell inoculation step.
4, zooblast porous microcarrier immobilization high-efficient culture method according to claim 1, it is characterized in that: the concentration of blood serum medium is 2% in described (2) cell inoculation step.
5, zooblast porous microcarrier immobilization high-efficient culture method according to claim 1, it is characterized in that: the inoculum density of cell suspension is 2 * 105-in described (2) cell inoculation step
5* 10
5Cell/mL.
6, zooblast porous microcarrier immobilization high-efficient culture method according to claim 1 is characterized in that: the transfer in described (4) cell culture step is meant that the porous microcarrier that uses pipeline will cover with cell directly imports in the next stage reactor.
7, zooblast porous microcarrier immobilization high-efficient culture method according to claim 1 is characterized in that: add an amount of substratum and treated porous microcarrier in advance in the reactor in described (4) cell culture step.
8, zooblast porous microcarrier immobilization high-efficient culture method according to claim 1, it is characterized in that: described (4) cell culture step adopts batch formula to change the liquid cultured continuously and is meant and changes a liquid 1-1.2 working volume by the cell retention system every day, in reactor, results contain the supernatant of product and add the equivalent fresh culture with microcarrier and cell retention.
9, be used for the serum free medium of zooblast porous microcarrier immobilization high-efficient culture, it is characterized in that: this serum free medium comprises growth form serum free medium and expression type serum free medium, and the composition of every 10L substratum and content thereof are as follows respectively:
Growth form serum-free culture based component and content thereof
DMEM/F12,1: 1 156g glucose 20g-60g
Sodium bicarbonate 10g-20g peptone 10g
Glutamine 5g 6-aminocaprolc acid 6.5g
Proline(Pro) 110mg leucine 500mg
Aspartic acid 70mg halfcystine 320mg
Methionine(Met) 600mg Serine 300mg
Glycine 75mg folic acid 100mg
Vitamins B
1The 500mg vitamins B
2100mg
Vitamins B
6The 500mg vitamins B
1250mg
Vitamins C 1000mg inositol 2000mg
Niacinamide 500mg Regular Insulin 800U
Transferrins,iron complexes 25mg thanomin 20mg
FeSO
4·7H
2O 10mg ZnSO
4 20mg；
Expression type serum-free culture based component and content thereof
DMEM/F12,1: 1 156g glucose 20g-60g
Sodium bicarbonate 10g-20g peptone 10g
Glutamine 5g 6-aminocaprolc acid 6.5g
Proline(Pro) 110mg leucine 500mg
Aspartic acid 70mg halfcystine 320mg
Methionine(Met) 600mg Serine 300mg
Glycine 75mg folic acid 100mg
Vitamins B
1The 1000mg vitamins B
2200mg
Vitamins B
6The 1000mg vitamins B
12100mg
Vitamins C 2000mg inositol 2000mg
Niacinamide 500mg Regular Insulin 400U
FeSO
4·7H
2O 10mg ZnSO
4 20mg。
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| CN103045530B (en) * | 2012-12-14 | 2015-06-17 | 山东滨州沃华生物工程有限公司 | Method for suspension culturing cell |
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| CN112442484A (en) * | 2020-11-25 | 2021-03-05 | 大连普瑞康生物技术有限公司 | Method for large-scale cell culture based on porous nanoscale temperature-sensitive soft colloid |
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