CN109609436A - A kind of no CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely - Google Patents
A kind of no CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
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- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
Abstract
The invention discloses a kind of no CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely, comprising the following steps: recovery mdck cell carries out amplification cultivation；The cell of acquisition is subjected to the domestication of serum-free adhere-wall culture；The cell suspension of acquisition is placed in constant-temperature shaking incubator and is allowed to from 5%CO2Environment gradually adapts to no CO2The full suspension of environment is cultivated；Utilize orthogonal test L9 (34) determine the critical process control parameter for taming successfully full suspended culture cell system；The procedure parameter of acquisition is carried out straight line to cell to amplify step by step, establishes (2L-10L-100L-500L) large-scale production model of the cell line.Mdck cell system density and vigor height, competence for added value are strong, and cellular morphology is good, establishes large-scale production model not by CO2Environmental condition limitation, fills up mdck cell without CO2Environment scale suspend culture blank, can scale straight line amplification, for mdck cell scale suspend culture convenient and fast training method is provided, for the biological products such as influenza virus vaccine prepare technical support is provided.
Description
Technical field
It suspends the present invention relates to cell line and cultivates domestication and production technology field, and in particular to a kind of no CO2Environment
The foundation of the mdck cell preparation method of suspension serum-free cell system and large-scale production model entirely.
Background technique
Influenza virus seriously endangers always the health of the mankind and animal, suffers huge economic loss per class is made one every year,
Then seem increasingly important with vaccine immunity come flu-prevention virus disease.However, traditional influenza vaccines are mainly with chicken embryo
For the inactivated vaccine of medium preparation, there are many deficiencies for this traditional vaccine manufacture source chicken embryo, therefore it is desirable to utilize
A kind of cell medium replaces chick embryo culture production of Influenza virus influenza vaccines, realizes that the upgrading of influenza vaccines production technology is changed
Generation.
Canine kidney cells MDCK is usually the epithelioid cell grown with adherent manner, has influenza virus easy infection, culture
Easy, proliferation is fastly and influenza virus is not easy the features such as making a variation, and is acknowledged as being most suitably adapted for production A type and Type B influenza disease at present
One of important cells system of malicious vaccine, therefore mdck cell is the optimum cell instead of chick embryo culture influenza virus；So far,
In spite of many reports about using microcarrier large-scale culture mdck cell, but the training method must be with expensive micro- load
Body is support, and when cell Proliferation also suffers from limitation, while operating procedure is also cumbersome, high production cost and needs are certain density
Serum, it is difficult to realize large-scale culture.Opposite microcarrier adhere-wall culture method, mdck cell suspend culture with many excellent entirely
Point: the growth of 1. cells is not limited by surface, it is easy to accomplish the large-scale culture of cell；2. without expensive microcarrier and digestion
Liquid can reduce the chance of operating procedure and pollution and save manpower；3. it is conducive to nutriment, gas etc. to come into full contact with cell,
Easily controllable key culture parameters；4. easily batch secondary culture is carried out in continuous closed system, convenient for expanding production step by step
Scale improves production efficiency；5. can be reduced the dosage of serum, it might even be possible to serum free medium culture cell, wherein without blood
Clear culture can eliminate many unfavorable factors of serum bring, the mycoplasma that may bring into such as the use of serum, virus or other
Pathogen causes potential hazard to cell；Difference between serum batch leads to the quality difference etc. between product batch.
In addition, the mdck cell culture being rarely reported at present, it all need to be in 5%CO2In the environment of carry out, the culture to cell
Equipment and environmental requirement are high, cause cell it is at high cost, inconvenient for operation when large-scale culture in closed bioreactor,
CO2Concentration it is not easy to control and be easy the presence for the defects of changing the pH value of culture solution.
Summary of the invention
Mdck cell is tamed regarding to the issue above, and an object of the present invention is to tame a kind of mdck cell adaptation serum-free
Without CO2Full suspension culture environment keeps the mdck cell system density and vigor high, and it is in single dispersed that competence for added value is strong, and form is good
Good, size is uniform, and the smooth of the edge is clear, not conglomeration, and the large-scale production for biological products such as influenza virus vaccines provides technology
It supports.
To achieve the above object, the present invention adopts the following technical scheme: a kind of no CO2The mdck cell of environment suspends nothing entirely
The preparation method of serum cell line, comprising the following steps:
1) recovery is purchased from the good MDCK attached cell of ATCC growth conditions, with the DMEM culture solution culture containing 10%FBS,
After it grows to 85%~90%, T25 square vase (F10) is passaged to by 1:3；
2) the square vase attached cell that will be obtained in step 1) carries out the free serum culture of mdck cell using gradually adjustment procedure
It is suitable to tame the speed of growth to vitro growth rates, cell state and cell in step 1), cell state for the adherent domestication of base
When, obtain the mdck cell suspension for being adapted to serum free medium；
3) by the cell suspension obtained in step 2) with 0.67~0.95 × 106The inoculum density of cells/mL accesses
In 250mL conical flask, first in 37 DEG C, 5%CO after inoculation2Incubator in static gas wave refrigerator, then with lower revolving speed constant temperature vibration
Culture 2h is swung, finally with the revolving speed constant-temperature shaking culture of 70r/min to 72h；Just start domestication when every 72h replace culture medium but
Regardless of biography, a point biography culture is carried out after specific cell growth rate tends towards stability；Divide when passing culture every the fresh serum-free of 72h
Cell density is diluted to 1.2~1.4 × 10 by culture medium6Cells/mL, and revolving speed is promoted to 120rpm, secondary culture until
Mdck cell is in 37 DEG C, 5%CO2The ability for sticking bottle wall, cell multiplication factor, cell density and work are lost in constant temperature incubation incubator
When the indexs such as rate tend towards stability substantially, show that the cell line is adapted to substantially in 37 DEG C, 5%CO2Incubator in suspend culture.
4) cell suspension obtained in step 3) is utilized into sterile porous plug, and cultural method is passed with dividing in step 3),
First in 37 DEG C, 5%CO2Incubator in suspension shaken cultivation 48h, be then transferred to no CO2Constant-temperature shaking culture is for 24 hours in incubator；It
It reduces when a point biography is cultivated every time afterwards in CO2Incubation time in incubator increases in no CO2Incubation time in incubator；Passage until
Cell is entirely without CO2The indexs such as multiplication factor, cell density and motility rate in incubator tend towards stability, cellular morphology it is good (it is round,
It is full bright, uniform in size, not conglomeration) when, show to have obtained no CO2The mdck cell of environment suspension serum-free cell system entirely.
Preferably, free serum culture basis representation described in step 2) does not add serum completely；
Preferably, 250mL conical flask described in step 3) is using preceding by 150 DEG C of vial silication agent dry roasting 50min
Specially treated；
Preferably, first in 37 DEG C, 5%CO in the step 3)2Incubator in suspend domestication operating procedure be it is static
Cultivate 30min, 50r/min constant-temperature shaking culture 2h, 70r/min constant-temperature shaking culture to 72h；
Preferably, mdck cell was at most cultivated into for 6 generations regardless of biography in the step 3), a point biography cultivates at least 4 generations；
Preferably, in mdck cell at least 6 generation of secondary culture, will be obtained without CO in the step 4)2The MDCK of environment is complete
Suspension serum-free cell system.
It is another object of the present invention to establish a kind of no CO2The mdck cell of environment suspension serum-free cell system entirely
Large-scale production model, which can increase substantially the density and motility rate of cell, so as to largely improve
Production efficiency and cellular morphology can be made good, suitable for carrying out the serum free suspension training of scale in bioreactors at different levels
It supports, has filled up the blank of domestic and international mdck cell scale suspension culture, while can the amplification of scale straight line.
To achieve the above object, the present invention adopts the following technical scheme: the present invention selects L9 (34) orthogonal arrage tested,
The crucial culture parameters such as temperature (T), pH value, DO, mixing speed (Agit) are chosen, using cell density and motility rate as evaluation index,
The critical process control parameter of mdck cell scale suspension free serum culture entirely is determined, to establish the scale of the cell line
Metaplasia produces model.
Preferably, the large-scale production model of mdck cell established by the present invention suspension serum-free cell system entirely hangs
Floating key to training process control parameter: cultivation temperature is 36.5 DEG C~37.0 DEG C, pH value is 6.8~7.0, DO value be 55.0%~
60.0%, speed of agitator is 105rpm~120rpm.
Preferably, the 2L-10L-100L-500L straight line that the present invention establishes the cell line amplifies no CO step by step2Environment rule
Modelling produces model.
Compared with prior art, the beneficial effects of the present invention are: serum-free used in 1, preparation method of the invention
Culture medium does not add animal blood serum completely, and the production cost and purifying difficulty of vaccine can be effectively reduced；2, it is prepared using the present invention
Mdck cell system density and vigor it is high, competence for added value is strong, is in single dispersed, form is good, full, and size is uniform, edge
Smooth clear, the large-scale production of the biological products such as infected by influenza vaccine has important application value；3, present invention preparation
Mdck cell tie up to no CO2In the environment of good can adapt to full suspension free serum culture, so as to reduce production cost,
Convenient for operating and controlling；4, mdck cell provided by the invention suspends no CO entirely2The large-scale production model of serum-free, Ke Yi great
Amplitude improves the density and motility rate of cell, and cell is made to double number up to 10 times or more, motility rate up to 93% or more；5, the present invention establishes
The large-scale production model of the cell line is not by CO2The limitation of environmental condition is suitable for the (2L-10L- in bioreactors at different levels
The culture of scale serum free suspension 100L-500L) is carried out, domestic and international mdck cell can be filled up and trained without CO2 environment scale suspension
Feeding blank, at the same can scale straight line amplification, for mdck cell scale suspend cultivate provide more convenient and fast culture
Mode, also the preparation for biological products such as influenza virus vaccines provides technical support, has a extensive future.
Detailed description of the invention
Fig. 1 is to have mdck cell (F10) aspect graph under serum adhered state；
Fig. 2 is adherent successful mdck cell (F20) aspect graph of domestication of serum free medium；
Fig. 3 is 5%CO2Aspect graph is tamed in the suspension of mdck cell (F26) in incubator；
Fig. 4 is 5%CO2Aspect graph is tamed in the suspension of mdck cell (F30) in incubator；
Fig. 5 is no CO2The serum-free that suspends entirely in environment tames successful mdck cell (F36) aspect graph；
Fig. 6 is suspension culture growth curve comparison diagram of the mdck cell suspension culture to F40 and F36；
Fig. 7 suspends in 2L, 10L, 100L and 500L bioreactor for mdck cell and cultivates growth curve comparison diagram.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's every other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
A kind of no CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely, comprising the following steps:
One, basic skills
1. material and method
1.1 material
Cell used is MDCK (CCL-34) cell purchased from ATCC；
Culture medium is that the DMEM purchased from GIBCO company is good for Gansu is purchased from along biological commercialization serum free medium；
Fetal calf serum (FBS) is purchased from GIBCO company；
Vial silication agent is purchased from Hubei xinsihai Chemical Co., Ltd..
1.2 method
1.2.1 mdck cell square vase adhere-wall culture
After recovery is purchased from the good MDCK attached cell of ATCC growth conditions, with the DMEM for containing 10% fetal calf serum (FBS)
Culture discards culture solution after culture is paved with T25 square vase bottom 85%~90% to cell；With containing 0.25% (W/V) trypsase
Solution is discarded after cleaning cellular layer with the trypsin solution of 0.03% (W/V) EDTA；Trypsin solution is added again and covers mdck cell
It is discarded after rocking uniformly, is placed in 37 DEG C of incubators and digests 5~10min；When quicksand like sliding occurs in cell, square vase is patted to cell
It completely falls off；Digestion is terminated with the DMEM containing 10%FBS, and is passaged to T25 square vase by 1:3 and carries out serum-free domestication (F10).
1.2.2 serum-free acclimation method
The attached cell that will be obtained in step 1.2.1, the serum-free for carrying out mdck cell using gradually adjustment procedure is adherent to tame and docile
Change；Mdck cell is gradually tamed by the DMEM containing 10%FBS first to the DMEM culture containing 3%FBS；Then contained by changing
The DMEM culture medium of 3%FBS and the mixed proportion of serum free medium are tamed, culture medium mixed proportion during domestication
Change and be divided into two steps: the first step is tamed after being mixed by the serum free medium of 70% DMEM culture medium containing 3%FBS and 30%,
Second step by 30% DMEM culture medium containing 3%FBS and 70% serum free medium mixing after tame；Finally by mdck cell
After the method digestion in step 1.2.1, completely with serum free medium culture, domestication to vitro growth rates, cell state
When the speed of growth, cell state are suitable in the DMEM culture medium containing 10%FBS with cell, show that mdck cell is trained in serum-free
It supports and is tamed successfully on base.
1.2.3 5%CO2Acclimation method in incubator
In order to prevent cell adherent, the silication agent of 250mL pyramidal cells culture bottle is handled first；Secondly by step
1.2.2 after the successful mdck cell digestion dilution of the adherent domestication of the serum-free obtained in, with 0.67~0.95 × 106cells/mL
Inoculum density access silication agent treated 250mL conical flask in, volume of culture 50mL；First in 37 DEG C, 5% after inoculation
CO2Incubator in static gas wave refrigerator 30min, then with the revolving speed constant-temperature shaking culture 2h of 50r/min, finally with 70r/min turn
Fast constant-temperature shaking culture is to 72h；Every 72h replacement culture medium but regardless of biography when just starting to tame, to guarantee enough cells
Number, (about regardless of pass 6 times) carries out point passing a culture after specific cell growth rate tends towards stability；Divide and is used when passing culture every 72h
Cell density is diluted to 1.2~1.4 × 10 by fresh serum free medium6Cells/mL, and revolving speed is promoted to 120rpm,
For secondary culture until cell loses the ability for sticking bottle wall, the indexs such as cell multiplication factor, cell density and motility rate tend to be steady substantially
Periodically, show that the cell is adapted to substantially in 37 DEG C, 5%CO2Incubator in suspend culture.
1.2.4 without CO2Acclimation method in incubator
The cell suspension that step 1.2.3 is obtained utilizes sterile porous plug, passes cultural method with dividing in step 1.2.3,
First in 37 DEG C, 5%CO2Incubator in suspension shaken cultivation 48h, be then transferred to no CO2Constant-temperature shaking culture is for 24 hours in incubator；It
Make cell in CO when a point biography is cultivated every time afterwards2Incubation time in incubator reduces 12h, and the incubation time in no CO2 incubator increases
Add 12h；Finally until cell is entirely without CO2The indexs such as multiplication factor, cell density and motility rate in incubator tend towards stability, cell
When form good (round, full bright, uniform in size, not conglomeration), show to have obtained no CO2The mdck cell of environment suspends entirely
Serum-free cell system.
2. result
The 2.1 adherent domestications of mdck cell serum-free
There is the adherent domestication of serum to serum-free adherent training from the DMEM containing 10%FBS mdck cell using gradually adjustment procedure
When supporting, the average specific growth rate of cell is in 0.510 ± 0.009d when 10 generations (after domestication the 30th day) is cultivated in domestication-1, cell work
Power is 95.37 ± 0.35%, with specific cell growth rate (0.517d of the cell in the DMEM culture medium containing 10%FBS-1), it is thin
Born of the same parents' vigor (95.89%) quite, shows that mdck cell is tamed successfully on serum free medium.Discovery is in this hair when microscopic observation
The serum-free MDCK attached cell (F20) that bright middle domestication obtains: adherent uniform but adherent more loose, cell edges are less clear
It is clear.Mdck cell serum-free domestication front and back cellular morphology, such as attached drawing 1 and attached drawing 2.
2.2 5%CO2The suspension domestication of mdck cell in incubator
Cell is in 5%CO2The culture that suspends is switched to by adhere-wall culture from F20 in incubator, just starts to suspend when taming regardless of biography
After cultivating for 6 generations, i.e., cell density is up to 2.815 × 10 when domestication is to F266Cells/mL, specific cell growth rate is up to 0.78d-1(being shown in Table 1)；Individual cells are rounded, bright when F26 known to attached drawing 3, but cell conglomeration is more serious, cell number is less.
Carry out point passing culture from F26, after point passing 4 generations of culture, i.e., when domestication is to F30 cell density up to 3.497 ×
106Cells/mL, vitro growth rates tend towards stability, and specific cell growth rate is up to 0.83d-1(being shown in Table 1)；
Cell loses the ability for sticking bottle wall when suspending domestication to F30, shows to obtain adaptation substantially in 37 DEG C, 5%
CO2Suspend the mdck cell of culture in incubator.Under the microscope: by attached drawing 4 it is found that F30 cell is in single, full bright, side more
Edge is neat, and cell clustering phenomena makes moderate progress, and is in the small agglomerate of several cytoadherences more.
2.3 without CO2The full suspension domestication of mdck cell in incubator
Cell was continuously suspended from F30 after 6 generations of domestication, can be obtained no CO2The full suspension serum-free mdck cell system of environment.
By attached drawing 5 it is found that the not conglomeration substantially of F36 cell, is in single dispersed, full bright, round, neat in edge, size is uniform, carefully
Born of the same parents' suspension is bright.
The indexs such as cell multiplication factor, cell density and motility rate tend towards stability when suspending culture domestication to F36, and cell is complete
Lose the ability for sticking bottle wall；Cell density is 4.1 × 10 when domestication is to F366Cells/mL, motility rate 90.4%, cell
Specific growth rate is 0.89d-1(being shown in Table 1), to show that the present invention has obtained no CO2The mdck cell of environment suspends serum-free entirely
Cell line.
Cell multiplication factor, density and survival rate test result during the suspension domestication of table 1
Embodiment 2
In order to determine critical process control parameter cell proliferation of the MDC K cell entirely during suspension free serum culture
It influences, the present invention utilizes orthogonal test, chooses the key parameters such as temperature (T), pH value, DO, mixing speed (Agit), close with cell
Degree and motility rate are evaluation index, to establish the large-scale production model of the cell line.
A kind of no CO2The foundation of the mdck cell of environment suspension serum-free cell system large-scale production model entirely, including with
Lower step:
1) experimental design: L9 (3 is selected4) orthogonal arrage tested (the not reciprocation between Consideration), experimental factor
And level is shown in Table 2；
2 process parameter optimizing of table level and factor
2) successful suspension cell will be tamed in embodiment 1 every time to be diluted cell density with fresh serum free medium
To 0.65 × 106Cells/mL, and (Sai Duolisi, model: B-DCU II) is transferred in 2L bioreactor, cultivation cycle is
72h；In no CO2In the environment of by the experimental design in step 1), suspension culture is carried out in 2L bioreactor；Every for 24 hours
Appropriate suspended culture cell is taken, by cytoanalyze CASY TT, the maximum viable cell density and motility rate of cell are detected, with this
For evaluation index, the mdck cell critical process control such as the optimum temperature of suspension free serum culture, pH, DO and mixing speed entirely is determined
Parameter processed, to establish the large-scale production model of the cell line.
3) result and analysis
3 test result of table (one)
4 test result of table (two)
Show that the optimum level group of mdck cell suspension culture is combined by 3 test result of table (one) in step 3)
A2B2C3D3, which can effectively improve the density of cell, i.e., when cultivation temperature, pH value, DO value and mixing speed are respectively 37.0
DEG C, 7.0,60%, 120rpm when cell Proliferation it is most fast；But A is shown by 4 test result of table (two) in step 3)1B1C2D2Group
The motility rate highest that mdck cell can be made to suspend culture is closed, i.e. the combination can effectively improve the motility rate of cell.It is thin by comprehensively considering
2 kinds of evaluation indexes of born of the same parents' density and motility rate, so that it is determined that in scale suspension culture production technology, which hangs cell entirely out
The optimum condition of 4 critical process control parameters of the floating appropriate to the occasion selection of free serum culture are as follows: 36.5 DEG C of temperature~37.0 DEG C, pH value
For 6.8~7.0, DO value be 55.0%~60.0%, speed of agitator is 105rpm~120rpm.
Embodiment 3
A kind of no CO2The suspension batch secondary culture of the mdck cell of environment suspension serum-free cell system entirely, including it is following
Step:
Successful F36 cell will be tamed in embodiment 1 is diluted to 0.65 × 10 with fresh serum free medium6cells/
ML, and according to the optimised process control parameter determined in embodiment 2: 36.5 DEG C~37.0 DEG C of temperature, pH value 6.8~7.0, DO value
55.0%~60.0%, speed of agitator 105rpm~120rpm carries out suspension batch secondary culture in 2L bioreactor, training
Supporting the period is 72h；The form of microscopic observation cell is sampled when passage and detects cell maximum viable cell density and motility rate.Work as cell
After density and motility rate improve and the speed of growth tends towards stability, cellular morphology normal (round, full bright, neat in edge, size
When uniformly), Cells in Batch can be carried out under the technique of foundation by showing to tame successfully full suspension serum-free cell system in embodiment 1
Secondary culture.
Cell density and motility rate tend towards stability when the present embodiment is tamed to F40, compared with F36 cell, are increased by the cell of Fig. 6
It grows curve graph and shows that cell density and motility rate increase substantially；Cell density is from 4.1 × 106Cells/mL rises to 6.7 ×
106Cells/mL, Cell viability increase to 93.8% from 90.4%, and the scale production process for showing that the present invention establishes can make to tame and docile
Its density of the cell line and motility rate changed significantly improve, and can carry out suspension batch secondary culture.
Embodiment 4
A kind of no CO2The mdck cell of environment full suspension serum-free cell system large-scale production model amplification verifying, packet
Include following steps:
Collect the cell in embodiment 3 cell is equally diluted to 0.65 with fresh serum free medium ×
106Cells/mL, and also the cell straight line of collection exists step by step according to the optimised process control parameter determined in embodiment 2
Compliance test result, cultivation cycle 72h are amplified in 10L, 100L, 500L bioreactor；Equally in amplification passage step by step
It samples the form of microscopic observation cell and detects cell maximum viable cell density and motility rate.When cellular morphology is normal (round, full
Bright, neat in edge is uniform in size), and the full suspension culture in this level Four bioreactor of 2L, 10L, 100L and 500L
When effect no significant difference, show large-scale production model established by the present invention can straight line amplify step by step.
The result shows that: when the cell straight line collected in 2L bioreactor is amplified to 10L, 100L, 500L step by step, with
F40 cell is compared, and is shown under the condition of suspension culture of scales at different levels by the cell Proliferation curve graph of Fig. 7, the mdck cell system
Full suspension free serum culture effect in 2L and 10L, 100L, 500L bioreactor no significant difference, the density of cell and
Motility rate is substantially close to show a kind of no CO established by the present invention2The mdck cell of environment suspension serum-free cell system entirely
Large-scale production model can straight line amplify step by step.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (9)
1. a kind of no CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely, which is characterized in that including following
Step:
1) recovery is purchased from the good MDCK attached cell of ATCC growth conditions, with the DMEM culture solution culture containing 10%FBS, to it
After growing to 85%~90%, T25 square vase (F10) is passaged to by 1:3；
2) the square vase attached cell that will be obtained in step 1) is pasted using the serum free medium that gradually adjustment procedure carries out mdck cell
It is suitable to tame the speed of growth to vitro growth rates, cell state and cell in step 1), cell state for wall culture domestication
When, obtain the mdck cell suspension for being adapted to serum free medium；
3) by the cell suspension obtained in step 2) with 0.67~0.95 × 106The inoculum density of cells/mL accesses 250mL taper
In bottle, preceding 72h is in 37 DEG C, 5%CO after inoculation2Incubator in static gas wave refrigerator for a period of time after, with lower revolving speed again constant temperature shake
Culture 2h is swung, later with the revolving speed constant-temperature shaking culture of 70r/min；Every 72h replacement culture medium but regardless of biography, to cell than giving birth to
Long rate carries out a point biography culture after tending towards stability；When point passing culture every 72h with fresh serum free medium by cell density
It is diluted to 1.2~1.4 × 106Cells/mL, and revolving speed is promoted to 120rpm, secondary culture until mdck cell 37 DEG C,
5%CO2The ability for sticking bottle wall is lost in constant temperature incubation incubator, the indexs such as cell multiplication factor, cell density and motility rate become substantially
When stablizing, show that the cell line is adapted to substantially in 37 DEG C, 5%CO2Incubator in suspend culture.
4) cell suspension obtained in step 3) is utilized into sterile porous plug, and with point biography cultural method in step 3), first existed
37 DEG C, 5%CO2Incubator in suspension shaken cultivation 48h, be then transferred to no CO2Constant-temperature shaking culture is for 24 hours in incubator；Later every time
Divide and reduces when passing culture in CO2Incubation time in incubator increases in no CO2Incubation time in incubator；Passage is until cell exists
Entirely without CO2The indexs such as multiplication factor, cell density and motility rate in incubator tend towards stability, and cellular morphology is good (round, full
It is bright, uniform in size, not conglomeration) when, show to have obtained no CO2The mdck cell of environment suspension serum-free cell system entirely.
2. one kind is as described in claim 1 without CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely,
Be characterized in that: free serum culture basis representation described in step 2) does not add serum completely.
3. one kind is as described in claim 1 without CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely,
Be characterized in that: 250mL conical flask described in step 3) does the special of roasting 50min by 150 DEG C of vial silication agent using preceding
Processing.
4. one kind is as described in claim 1 without CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely,
It is characterized in that: first in 37 DEG C, 5%CO in the step 3)2Incubator in suspend domestication operating procedure be static gas wave refrigerator
30min, 50r/min constant-temperature shaking culture 2h, 70r/min constant-temperature shaking culture are to 72h.
5. one kind is as described in claim 1 without CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely,
It is characterized in that: mdck cell being divided regardless of at most 6 generations of culture are passed and pass culture at least 4 generations in the step 3).
6. one kind is as described in claim 1 without CO2The preparation method of the mdck cell of environment suspension serum-free cell system entirely,
It is characterized in that: by mdck cell at least 6 generation of secondary culture in the step 4), will obtain without CO2The MDCK of environment suspends nothing entirely
Serum cell line.
7. a kind of no CO2The foundation of the mdck cell of environment suspension serum-free cell system large-scale production model entirely, feature exist
In, comprising the following steps: the present invention selects L9 (34) orthogonal arrage tested, choose temperature (T), pH value, DO, mixing speed
(Agit) etc. crucial culture parameters determine that mdck cell scale suspends no blood entirely using cell density and motility rate as evaluation index
The critical process control parameter cultivated clearly, to establish the large-scale production model of the cell line.
8. one kind is as claimed in claim 7 without CO2The mdck cell of environment suspension serum-free cell system large-scale production model entirely
Foundation, it is characterised in that: suspension key to training parameters of technique process: cultivation temperature is 36.5 DEG C~37.0 DEG C, pH value 6.8
~7.0, DO value is 55.0%~60.0%, speed of agitator is 105rpm~120rpm.
9. one kind is as claimed in claim 7 without CO2The mdck cell of environment suspension serum-free cell system large-scale production model entirely
Foundation, it is characterised in that: the 2L-10L-100L-500L straight line that the present invention establishes the cell line amplifies no CO step by step2Environment
Large-scale production model.
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Cited By (1)
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