CN105861422A - Method for preparing MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and MDCK cell line - Google Patents
Method for preparing MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and MDCK cell line Download PDFInfo
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Abstract
The invention discloses a method for preparing an MDCK (Madin-Darby canine kidney) cell line adaptive to serum-free full-suspension culture and the MDCK cell line. The method includes steps of 1), discarding culture media for cultivating MDCK cells, cleaning cell layers by the aid of pancreatin solution, then discarding the pancreatin solution, adding pancreatin solution into the MDCK cells again, digesting the MDCK cells, stopping digesting the MDCK cells after the cells are rounded, centrifuging cell suspension and then discarding supernatant to obtain cell clusters; 2), re-suspending the cell clusters obtained at the step 1) by the aid of serum-free culture media to obtain cell re-suspension; 3), arranging the cell re-suspension obtained at the step 2) in a shaking table, cultivating the cell re-suspension in culture tanks, diluting the cell re-suspension by the aid of the serum-free culture media and carrying out passage on the cell re-suspension to obtain the MDCK cell line. The method and the MDCK cell line have the advantages that the MDCK cell line is high in cell density and activity and uniform in size, individually grows in a dispersion manner, is plump in form and is suitable for serum-free suspension culture by the aid of bioreactors.
Description
Technical field
The present invention relates to cell line and cultivate domestication field, be specifically related to a kind of MDCK adapting to the full suspension culture of serum-free thin
The preparation method of born of the same parents system and the mdck cell system that obtained by this preparation method.
Background technology
Dog kidney epithelia cell (Madin-Darby canine kidney cell, MDCK) be considered to be best suited for first,
One of cell line that influenza B virus produces, can be used for replacing chick embryo culture influenza virus.Have now been developed
Mdck cell system is utilized to replace the microcarrier adhere-wall culture manufacturing technique method of chick embryo culture influenza virus, although this process
With accessible certain production scale, but still there is certain defect: 1, microcarrier is difficult to repeatedly use, and causes
Production cost improves；2, the culture density of attached cell is limited by adherent area, causes the decline of viral yield；3, adherent
Cultivate and generally need to add serum to help cell attachment and growth, need to carry out changing liquid, complex process, and can introduce former
The pollution of body, chlamydia or animal proteinum, the safety to vaccine product causes potential threat.And use serum-free entirely to suspend training
Support the technology of mdck cell, the shortcoming that adhere-wall culture can be overcome, however it is necessary that and mdck cell is tamed.Indirect method is tamed
Serum free suspension is cultivated mdck cell and is divided into two stages to carry out, and first, tames adherent MDCK cell with serum-free medium, makes
It can be in adherent growth under serum-free condition；Then domestication is continued with serum-free medium so that it is need not the suspension of carrier
Grow under state.Prior art has the culture medium using SIMF8 commercialization, has with indirect replacement the circulation of blood serum medium to tame and docile
Change method carries out the domestication of mdck cell suspension culture, the method for domesticating more than 20 weeks, it is thus achieved that MDCK.SUS2 suspension growth
Cell；Additionally, use the serum-free medium of SFM4Mega Vir commercialization respectively with directly adapting to depletion of blood therapy for clearing away heat (time-consuming 3 weeks)
With indirectly adapting to depletion of blood therapy for clearing away heat (time-consuming 2 months) domestication, mdck cell can be grown under suspension culture pattern.But pass through
The MDCK suspension cell that above method domestication obtains all tames the process of suspension culture after adapting to serum-free culture again, mostly
There is domestication time length, complex steps, cell conglomeration or cell size inequality first-class defect, do not reach high-density growth requirement.
Summary of the invention
For the deficiencies in the prior art, an object of the present invention is to provide a kind of full suspension culture of serum-free that adapts to
The preparation method of mdck cell system, this preparation method is tamed with shorter cycle and less generation and is obtained the suitable of full suspension culture
Answering the mdck cell system of the full suspension culture of serum-free, this mdck cell density is high, and in single dispersion growth, form is full, size
Homogeneous, activity is big, it is adaptable to the large-scale production of biological product.
For achieving the above object, the present invention adopts the following technical scheme that a kind of MDCK adapting to the full suspension culture of serum-free
The preparation method of cell line, comprises the following steps:
1), when mdck cell culture medium culturing to the cell confluency degree needing serum adhere-wall culture being reached 80～90%, abandon
Go to cultivate the culture medium of mdck cell, after cleaning cellular layer with trypsin solution, discard solution；Again add trypsin solution to covering
Mdck cell carries out digesting 5～15min；After cell rounding, add the culture medium containing hyclone terminate digestion；Then collect
Cell suspension, and abandon supernatant after centrifugal for cell suspension, it is thus achieved that cell mass；
2) by step 1) in the cell mass serum-free medium that obtains carry out resuspended to cell density 1.3～1.6 ×
106Cells/mL, it is thus achieved that cell re-suspension liquid；
3) by step 2) the middle cell re-suspension liquid addition square vase obtained, at rotating speed 30rpm, temperature 37 DEG C, 5%CO2Bar
It is placed in shaking table and puts in incubator cultivation under part, after 2 cultures, the cell re-suspension liquid of cultivation is proceeded to shaking flask, rotating speed
It is promoted to 120rpm；With fresh serum-free medium, cell re-suspension liquid being diluted to cell density every 48h is 1.3～1.6
×106Cells/mL, passes on, it is thus achieved that adapt to the mdck cell system of the full suspension culture of serum-free.
A kind of preferably scheme as the present invention: the described culture medium for cultivating mdck cell is for containing 10% new born bovine
The DMEM culture medium of serum.
A kind of preferably scheme as the present invention: step 1) in, the described culture medium containing hyclone is containing 10% tire
The culture medium of Ox blood serum.
A kind of preferably scheme as the present invention: described step 3) in, mdck cell was at least passaged to the 6th generation.
A kind of preferably scheme as the present invention: in described serum-free medium, the concentration of each component is:
Basal metabolism nutrient:
Nucleotide:
Vitamin:
Inorganic salt:
Acid-base value buffer agent:
Sodium bicarbonate 1000～3000mg/L；
Acid-base value indicator:
Phenol red 5～15mg/L；
Proliferation of influenza virus accelerator:
Other additive:
A kind of preferably scheme as the present invention: described shearing force protective agent is blocked polyethers F68.
A kind of preferably scheme as the present invention: described anti-cell conglomeration agent is dextran sulfate.
A kind of preferably scheme as the present invention: described serum-free medium is prepared by following steps:
1) mixed liquor is prepared: wear into fine powder after being mixed by raw material, then dissolved in 10～30 DEG C of solvents by gained fine powder,
Obtain mixed liquor；
2) regulation pH: the pH to 6.3～6.7 of regulation mixed liquor, obtain serum-free medium after constant volume.
Further object is that a kind of mdck cell system of offer, the cell density of this mdck cell system is high, in
Single dispersion growth, form is full, and size is homogeneous, and activity is big, it is adaptable to bioreactor serum free suspension is cultivated.
For achieving the above object, the present invention is adopted the following technical scheme that and is obtained by above-mentioned preparation method.
The beneficial effects of the present invention is:
1, the preparation method of the present invention uses one-step method, serum directly will be needed to paste with shorter cycle and less generation
The cell of the cultivation of wall tames into the full suspension cell of serum-free, and domestication process only needs 2 weeks, is far superior to existing skill in efficiency
Art；
2, the serum-free medium that the preparation method of the present invention is used does not contains animal serum, low cost；Support that MDCK is mono-
The full suspension culture of high cell densities, its definite ingredients, easily preparation and easy to use；
3, the serum-free medium of the present invention coordinates the preparation method of the present invention, is effectively shortened mdck cell by adherent
Cell tames into the full suspension cell domestication time of serum-free, improves the efficiency of production, it is thus achieved that high-quality full suspension is thin
Born of the same parents；
4, the cell density of the mdck cell system of the present invention is high, and in single dispersion growth, form is full, and size is homogeneous, lives
Property big, it is adaptable to bioreactor serum free suspension cultivate.
Accompanying drawing explanation
Fig. 1 is mdck cell aspect graph under adhered state in embodiment 1；
Fig. 2 is the mdck cell aspect graph in embodiment 1 under serum-free medium is tamed；
Fig. 3 is to use Hyclone company serum-free medium SFM4 Mega Vir by direct method for domesticating in embodiment 1
The MDCKS cellular morphology figure of the suspension culture obtained；
Fig. 4 is that the business serum-free medium SMI F8 indirect method domestication using the exploitation of Gibco company in embodiment 1 obtains
MDCK.SUS2 cellular morphology figure；
Fig. 5 is viable cell density and the cytoactive curve chart of embodiment 2MDCK cell.
Detailed description of the invention
Below, in conjunction with detailed description of the invention, the present invention is described further:
The preparation method of a kind of mdck cell system adapting to the full suspension culture of serum-free, comprises the following steps:
1) the DMEM culture medium culturing needing mdck cell 10% new-born calf serum of serum adhere-wall culture is converged to cell
Right when reaching 80～90%, discard the culture medium cultivating mdck cell, after cleaning cellular layer with trypsin solution, discard solution；Again
Secondary addition trypsin solution carries out digesting 5～15min to covering mdck cell；Add containing 10% hyclone after cell rounding
Culture medium terminates digestion；Then collect cell suspension, and abandon supernatant after centrifugal for cell suspension, it is thus achieved that cell mass；
2) by step 1) in the cell mass serum-free medium that obtains carry out resuspended to cell density 1.3～1.6 ×
106Cells/mL, it is thus achieved that cell re-suspension liquid；
3) by step 2) the middle cell re-suspension liquid addition square vase obtained, at rotating speed 30rpm, temperature 37 DEG C, 5%CO2Bar
It is placed in shaking table and puts in incubator cultivation under part, after 2 cultures, the cell re-suspension liquid of cultivation is proceeded to shaking flask, rotating speed
It is promoted to 120rpm；With fresh serum-free medium, cell re-suspension liquid being diluted to cell density every 48h is 1.3～1.6
×106Cells/mL, passes on, and is at least passaged to for the 6th generation, it is thus achieved that adapt to the mdck cell system of the full suspension culture of serum-free.
Wherein, in serum-free medium, the concentration of each component is:
Basal metabolism nutrient:
Nucleotide:
Vitamin:
Inorganic salt:
Shearing force protective agent:
Blocked polyethers F68 500～2500mg/L；
Anti-cell conglomeration agent:
Dextran sulfate 20～150mg/L；
Acid-base value buffer agent:
Sodium bicarbonate 1000～3000mg/L；
Acid-base value indicator:
Phenol red 5～15mg/L；
Proliferation of influenza virus accelerator:
Other additive:
Wherein, nucleotide is selected hypoxanthine and thymidine, can promote that the nucleotide of mdck cell synthesizes, it is ensured that cell
Growth；Hypoxanthine and thymidine composition are the highest, can cell growth inhibiting；
Wherein, other additives are selected ferric ammonium citrate, plays its original effect in order to substitute transferrins, do not affect
Cell growth and iron metabolism, and the animal proteinum composition in serum-free medium can be reduced, reduce culture medium cost and to production
Uncertainty and insecurity；Ferric ammonium citrate relies on bivalent metal ion passage DMT1 to absorb ferrum, and transferrins is by turning
Human Placental Ferritin Receptor absorbs ferrum, and the former improves the mdck cell absorption rate to ferrum than the latter；Ferric ammonium citrate constituent concentration mistake
Height can suppress mdck cell to grow；Concentration is the lowest, the mdck cell incomplete absorption to ferrum；
Wherein, in other additive, the concentration of insulin is 2～15mg/L, can promote glucose metabolism, it is ensured that MDCK is thin
The growth of born of the same parents and the activity of maintenance mdck cell；
Wherein, in other additive, the concentration of soy hydrolyzate is 1000～5000mg/mL, can guarantee that vitamin, metal
The supply of other cofactors such as ion, aminoacid, improves mdck cell to amino acid whose picked-up；
Described serum-free medium is prepared by following steps:
1) mixed liquor is prepared: wear into fine powder after being mixed by raw material, then dissolved in 10～30 DEG C of solvents by gained fine powder,
Obtain mixed liquor；
2) regulation pH: the pH to 6.3～6.7 of regulation mixed liquor, obtain serum-free medium after constant volume.
Specific embodiment:
Embodiment 1
The preparation method of a kind of mdck cell system adapting to the full suspension culture of serum-free, comprises the following steps:
1) the DMEM culture medium culturing needing mdck cell 10% new-born calf serum of serum adhere-wall culture is converged to cell
Right when reaching 80～90%, discard the culture medium cultivating mdck cell, after cleaning cellular layer with trypsin solution, discard solution；Again
Secondary addition trypsin solution carries out digesting 5～15min to covering mdck cell；Add containing 10% hyclone after cell rounding
Culture medium terminates digestion；Then collect cell suspension, and abandon supernatant after centrifugal for cell suspension, it is thus achieved that cell mass；
2) by step 1) in the cell mass serum-free medium that obtains carry out resuspended to cell density 1.3～1.6 ×
106Cells/mL, it is thus achieved that cell re-suspension liquid；
3) by step 2) the middle cell re-suspension liquid addition square vase obtained, at rotating speed 30rpm, temperature 37 DEG C, 5%CO2Bar
It is placed in shaking table and puts in incubator cultivation under part, after 2 cultures, the cell re-suspension liquid of cultivation is proceeded to shaking flask, rotating speed
It is promoted to 120rpm；With fresh serum-free medium, cell re-suspension liquid being diluted to cell density every 48h is 1.3～1.6
×106Cells/mL, passes on, and passes on for the 6th generation, it is thus achieved that adapt to the mdck cell system of the full suspension culture of serum-free.
Wherein, in serum-free medium, the concentration of each component is:
Basal metabolism nutrient:
Nucleotide:
Vitamin:
Inorganic salt:
Shearing force protective agent:
Blocked polyethers F68 500～2500mg/L；
Anti-cell conglomeration agent:
Dextran sulfate 20～150mg/L；
Acid-base value buffer agent:
Sodium bicarbonate 1000～3000mg/L；
Acid-base value indicator:
Phenol red 5～15mg/L；
Proliferation of influenza virus accelerator:
Other additive:
Described serum-free medium is prepared by following steps:
1) mixed liquor is prepared: wear into fine powder after being mixed by raw material, then dissolved in 10～30 DEG C of solvents by gained fine powder,
Obtain mixed liquor；
2) regulation pH: the pH to 6.5 of regulation mixed liquor, obtain serum-free medium after constant volume.
Compare as Figure 1-4 by adhere-wall culture and the mdck cell form obtained by the present embodiment:
Fig. 1 is that under adhered state, mdck cell is attached at culture medium surface, in paving stone shape；
Fig. 2 is the form of the mdck cell of full suspension culture that the domestication of the present embodiment serum-free medium obtains, cell in
Single dispersed, without clustering phenomena, cellular morphology is complete, and border is smooth clearly, and size is homogeneous；
Fig. 3 is the suspension using Hyclone company serum-free medium SFM4 Mega Vir to be obtained by direct method for domesticating
The MDCKS cell cultivated, image credit: Zhang Liangyan, Yao Zhidong, waits suspension domestication and the Preliminary Applications of .MDCK cell. biological skill
Art communication .2013,24 (3): 382-384；In figure visible, multiple cell aggregationes are agglomerating, rare individual cells, and cell size is uneven
One；
Fig. 4 is that the business serum-free medium SMIF8 indirect method domestication using the exploitation of Gibco company obtains
MDCK.SUS2 cell；Image credit: V.Lohr, Y.Genzel, et al.A new MDCK suspension line
cultivated in a fully defined med ium in stirred-tank and wave
bioreactor.Vaccine.2010,28(3):6256-6264；Seen in figure, in this serum-free medium during suspension culture
Cellular morphology is also in assembling shape, but agglomerate is less, cell size heterogeneity, and state is the most deficient.
At the present embodiment, the mdck cell of adhere-wall culture being domesticated for suspension culture state, it is raw that this cell is single dispersion
Long, cellular morphology is full, and size is homogeneous；Cell quality is high.
Embodiment 2
The preparation method of a kind of mdck cell system adapting to the full suspension culture of serum-free, comprises the following steps:
1) the DMEM culture medium culturing needing mdck cell 10% new-born calf serum of serum adhere-wall culture is converged to cell
Right when reaching 80～90%, discard the culture medium cultivating mdck cell, after cleaning cellular layer with trypsin solution, discard solution；Again
Secondary addition trypsin solution carries out digesting 5～15min to covering mdck cell；Add containing 10% hyclone after cell rounding
Culture medium terminates digestion；Then collect cell suspension, and abandon supernatant after centrifugal for cell suspension, it is thus achieved that cell mass；
2) by step 1) in the cell mass serum-free medium that obtains carry out resuspended to cell density 1.3～1.6 ×
106Cells/mL, it is thus achieved that cell re-suspension liquid；
3) by step 2) the middle cell re-suspension liquid addition square vase obtained, at rotating speed 30rpm, temperature 37 DEG C, 5%CO2Bar
It is placed in shaking table and puts in incubator cultivation under part, after 2 cultures, the cell re-suspension liquid of cultivation is proceeded to shaking flask, rotating speed
It is promoted to 120rpm；With fresh serum-free medium, cell re-suspension liquid being diluted to cell density every 48h is 1.3～1.6
×106Cells/mL, passes on.
Wherein, in serum-free medium, the concentration of each component is:
Basal metabolism nutrient:
Nucleotide:
Vitamin:
Inorganic salt:
Shearing force protective agent:
Blocked polyethers F68 1600mg/L；
Anti-cell conglomeration agent:
Dextran sulfate 50mg/L；
Acid-base value buffer agent:
Sodium bicarbonate 2200mg/L；
Acid-base value indicator:
Phenol red 8mg/L；
Proliferation of influenza virus accelerator:
Other additive:
Described serum-free medium is prepared by following steps:
1) mixed liquor is prepared: wear into fine powder after being mixed by raw material, then dissolved in 10～30 DEG C of solvents by gained fine powder,
Obtain mixed liquor；
2) regulation pH: the pH to 6.5 of regulation mixed liquor, obtain serum-free medium after constant volume.
The viable cell density of the mdck cell that the present embodiment obtains and cytoactive be as shown in Figure 5: needs serum adhere-wall culture
Mdck cell in serum-free medium, tamed for 6 generations after (13d after domestication), cell growth is gradually stable, cytoactive
It is maintained at more than 95%.Thus may certify that, make to need the mdck cell of serum adhere-wall culture by the preparation method of the present embodiment
Adapt to suspension culture and stable growth only needs 2 weeks, substantially reduce mdck cell and tamed into the complete outstanding of serum-free by attached cell
The floating cell domestication time.
Embodiment 3
The preparation method of a kind of mdck cell system adapting to the full suspension culture of serum-free, comprises the following steps:
1) the DMEM culture medium culturing needing mdck cell 10% new-born calf serum of serum adhere-wall culture is converged to cell
Right when reaching 80～90%, discard the culture medium cultivating mdck cell, after cleaning cellular layer with trypsin solution, discard solution；Again
Secondary addition trypsin solution carries out digesting 5～15min to covering mdck cell；Add containing 10% hyclone after cell rounding
Culture medium terminates digestion；Then collect cell suspension, and abandon supernatant after centrifugal for cell suspension, it is thus achieved that cell mass；
2) by step 1) in the cell mass serum-free medium that obtains carry out resuspended to cell density 1.3～1.6 ×
106Cells/mL, it is thus achieved that cell re-suspension liquid；
3) by step 2) the middle cell re-suspension liquid addition square vase obtained, at rotating speed 30rpm, temperature 37 DEG C, 5%CO2Bar
It is placed in shaking table and puts in incubator cultivation under part, after 2 cultures, the cell re-suspension liquid of cultivation is proceeded to shaking flask, rotating speed
It is promoted to 120rpm；With fresh serum-free medium, cell re-suspension liquid being diluted to cell density every 48h is 1.3～1.6
×106Cells/mL, passes on.
Wherein, in serum-free medium, the concentration of each component is:
Basal metabolism nutrient:
Nucleotide:
Vitamin:
Inorganic salt:
Shearing force protective agent:
Blocked polyethers F68 1000mg/L；
Anti-cell conglomeration agent:
Dextran sulfate 25mg/L；
Acid-base value buffer agent:
Sodium bicarbonate 2200mg/L；
Acid-base value indicator:
Phenol red 8mg/L；
Proliferation of influenza virus accelerator:
Other additive:
Described serum-free medium is prepared by following steps:
1) mixed liquor is prepared: wear into fine powder after being mixed by raw material, then dissolved in 10～30 DEG C of solvents by gained fine powder,
Obtain mixed liquor；
2) regulation pH: the pH to 6.4 of regulation mixed liquor, obtain serum-free medium after constant volume.
Embodiment 4
The preparation method of a kind of mdck cell system adapting to the full suspension culture of serum-free, comprises the following steps:
1) the DMEM culture medium culturing needing mdck cell 10% new-born calf serum of serum adhere-wall culture is converged to cell
Right when reaching 80～90%, discard the culture medium cultivating mdck cell, after cleaning cellular layer with trypsin solution, discard solution；Again
Secondary addition trypsin solution carries out digesting 5～15min to covering mdck cell；Add containing 10% hyclone after cell rounding
Culture medium terminates digestion；Then collect cell suspension, and abandon supernatant after centrifugal for cell suspension, it is thus achieved that cell mass；
2) by step 1) in the cell mass serum-free medium that obtains carry out resuspended to cell density 1.3～1.6 ×
106Cells/mL, it is thus achieved that cell re-suspension liquid；
3) by step 2) the middle cell re-suspension liquid addition square vase obtained, at rotating speed 30rpm, temperature 37 DEG C, 5%CO2Bar
It is placed in shaking table and puts in incubator cultivation under part, with fresh serum-free medium, cell re-suspension liquid is diluted every 48h
It is 1.3～1.6 × 10 to cell density6Cells/mL, passes on.
Wherein, in serum-free medium, the concentration of each component is:
Basal metabolism nutrient:
Nucleotide:
Vitamin:
Inorganic salt:
Shearing force protective agent:
Blocked polyethers F68 2200mg/L；
Anti-cell conglomeration agent:
Dextran sulfate 100mg/L；
Acid-base value buffer agent:
Sodium bicarbonate 2200mg/L；
Acid-base value indicator:
Phenol red 8mg/L；
Proliferation of influenza virus accelerator:
Cholesterol 6.26mg/L；
Other additive:
Described serum-free medium is prepared by following steps:
1) mixed liquor is prepared: wear into fine powder after being mixed by raw material, then dissolved in 10～30 DEG C of solvents by gained fine powder,
Obtain mixed liquor；
2) regulation pH: the pH to 6.7 of regulation mixed liquor, obtain serum-free medium after constant volume.
The culture medium that embodiment 2-4 obtains is carried out attribute testing:
1, instrument: Bio-Bundle bioreactor (purchased from Applikon Biotechno logy company of Holland), tank body
Volume is 3L；
2, the mdck cell system for comparison passes through commercialization serum-free medium SFM4 Mega Vir (purchased from Hyclone
Company) cultivate obtain；
3, cultural method: with 0.5 × 106The cell density inoculating cell of cells/mL in bioreactor, 37 DEG C,
5%CO2Under conditions of carry out batch cultivating, every 24h sampling carries out viable count, and calculates cell growth rate；Result such as table
1, shown in table 2:
The highest viable cell density of table 1 (106cells/mL)
Table 2 cell growth rate and doubling time
Compared with the mdck cell of matched group commercialization serum-free medium SFM4 Mega Vir suspension culture, use this
The serum-free medium that thered is provided of invention, the viable cell density supported in incubation has and significantly increases；Additionally, non-
Exponential phase of growth cell specific growth rate from comparison 0.57d-1Maximum rises to the 0.91d in embodiment 2-1, cell doubles
Time then foreshortens to the 0.32d embodiment 2 from the 0.79d maximum of comparison.The preparation method of the visible employing present invention obtains
Mdck cell, is all greatly improved at cell growth rate and cytoactive.
For a person skilled in the art, can technical scheme as described above and design, make other each
Plant corresponding change and deformation, and all these changes and deforms the protection model that all should belong to the claims in the present invention
Within enclosing.
Claims (9)
1. the preparation method of the mdck cell system adapting to the full suspension culture of serum-free, it is characterised in that comprise the following steps:
1), when mdck cell culture medium culturing to the cell confluency degree needing serum adhere-wall culture being reached 80～90%, training is discarded
Support the culture medium of mdck cell, after cleaning cellular layer with trypsin solution, discard solution；Again add trypsin solution to covering MDCK
Cell carries out digesting 5～15min；After cell rounding, add the culture medium containing hyclone terminate digestion；Then cell is collected
Suspension, and abandon supernatant after centrifugal for cell suspension, it is thus achieved that cell mass；
2) by step 1) in the cell mass serum-free medium that obtains carry out resuspended to cell density 1.3～1.6 ×
106Cells/mL, it is thus achieved that cell re-suspension liquid；
3) by step 2) the middle cell re-suspension liquid addition square vase obtained, at rotating speed 30rpm, temperature 37 DEG C, 5%CO2Under conditions of put
In shaking table and put in incubator cultivate, after 2 cultures, the cell re-suspension liquid of cultivation is proceeded to shaking flask, rotating speed is promoted to
120rpm；Every 48h with fresh serum-free medium cell re-suspension liquid is diluted to cell density be 1.3～1.6 ×
106Cells/mL, passes on, it is thus achieved that adapt to the mdck cell system of the full suspension culture of serum-free.
2. the preparation method of the mdck cell system adapting to the full suspension culture of serum-free as claimed in claim 1, it is characterised in that:
Step 1) in, the described culture medium for cultivating mdck cell is the DMEM culture medium containing 10% new-born calf serum.
3. the preparation method of the mdck cell system adapting to the full suspension culture of serum-free as claimed in claim 1, it is characterised in that:
Step 1) in, the described culture medium containing hyclone is the culture medium containing 10% hyclone.
4. the preparation method of the mdck cell system adapting to the full suspension culture of serum-free as claimed in claim 1, it is characterised in that:
Described step 3) in, mdck cell was at least passaged to the 6th generation.
5. the preparation method of the mdck cell system adapting to the full suspension culture of serum-free as claimed in claim 1, it is characterised in that:
In described serum-free medium, the concentration of each component is:
Basal metabolism nutrient:
Nucleotide:
Vitamin:
Inorganic salt:
Shearing force protective agent: 500～2500mg/L；
Anti-cell conglomeration agent: 20～150mg/L；
Acid-base value buffer agent:
Sodium bicarbonate 1000～3000mg/L；Acid-base value indicator:
Phenol red 5～15mg/L；
Proliferation of influenza virus accelerator:
Other additive:
6. the preparation method of the mdck cell system adapting to the full suspension culture of serum-free as claimed in claim 5, it is characterised in that:
Described shearing force protective agent is blocked polyethers F68.
7. the preparation method of the mdck cell system adapting to the full suspension culture of serum-free as claimed in claim 5, it is characterised in that:
Described anti-cell conglomeration agent is dextran sulfate.
8. the preparation method of the mdck cell system adapting to the full suspension culture of serum-free as claimed in claim 1, it is characterised in that:
Described serum-free medium is prepared by following steps:
1) mixed liquor is prepared: wear into fine powder after being mixed by raw material, then dissolved in 10～30 DEG C of solvents by gained fine powder, obtain
Mixed liquor；
2) regulation pH: the pH to 6.3～6.7 of regulation mixed liquor, obtain serum-free medium after constant volume.
9. the mdck cell system adapting to the full suspension culture of serum-free, it is characterised in that: arbitrary described by claim 1-8
Preparation method obtain.
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| CN109402040A (en) * | 2017-08-15 | 2019-03-01 | 上海倍谙基生物科技有限公司 | Suitable serum-free suspends the bhk cell and its acclimation method of culture entirely |
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| CN108865967A (en) * | 2017-05-11 | 2018-11-23 | 华威特（江苏）生物制药有限公司 | A kind of quick domestication attached cell is the method for full suspension cell line |
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| CN110669722A (en) * | 2019-10-23 | 2020-01-10 | 长春生物制品研究所有限责任公司 | Serum-free full-suspension domestication method for MDCK cells |
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| CN114438019B (en) * | 2022-03-11 | 2023-01-03 | 上海荣盛生物药业股份有限公司 | Domestication method of MDCK cell line |
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