CN106754635A - A kind of Chinese hamster ovary celI cultural method - Google Patents
A kind of Chinese hamster ovary celI cultural method Download PDFInfo
- Publication number
- CN106754635A CN106754635A CN201510809964.5A CN201510809964A CN106754635A CN 106754635 A CN106754635 A CN 106754635A CN 201510809964 A CN201510809964 A CN 201510809964A CN 106754635 A CN106754635 A CN 106754635A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture
- chinese hamster
- hamster ovary
- ovary celi
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The present invention provides a kind of Chinese hamster ovary celI cultural method, and the method first realizes the maximization of cell culture density, reduces the volume of cell culture, reduces follow-up labour intensity, and quality stable homogeneous, production process linking is tight, easily manipulation.The present invention carries out fluid infusion in cell culture and virus propagation process, it is ensured that the nutriment needed for cell propagation, and the utilization rate of nutrient solution is improve again, cost-effective.According to adenovirus carrier vaccine production technology of the invention, virus titer can reach 8 × 108TCID50/mL。
Description
Technical field
The present invention relates to cell engineering field, and in particular to a kind of Chinese hamster ovary celI cultural method.
Background technology
Though Chinese hamster ovary celI can be carried out big as microbial cell in the bioreactor of manual control condition
Scale evaluation, but its eucaryotic cell structure and cultural character have marked difference compared with microbial cell.Zooblast
More much bigger than microbial cell, acellular wall, mechanical strength is low, sensitive to shearing force, and accommodative ability of environment is poor；
Doubling time is long, slow-growing, easily by microorganism pollution, antibiotic must be used during culture；Incubation oxygen demand
Few, cell is inter-adhesive in incubation exists in cluster form；Primary cultured cell typically breeding 50 generations be
Degeneration and death；Metabolite has bioactivity, and production cost is high, but added value is also high.
In the prior art, experiment confirms cell under the excessively multipair Chinese hamster ovary celI monolayer cultivation mode of passage number of times
Growth and the expression quantity of adenovirus vector generate obvious influence, but under suspension training method
The growth of Chinese hamster ovary celI and the expression quantity of adenovirus vector do not influence really, but prior art is directed to CHO
Cell suspension cultures technique is rarely reported.
The content of the invention
The purpose of the present invention is to overcome prior art defect, there is provided a kind of Chinese hamster ovary celI cultural method.
To realize above technical purpose, the present invention uses following technical scheme：
A kind of Chinese hamster ovary celI cultural method, comprises the following steps：
1) with stirring-type Tissue Culture Flask culture Chinese hamster ovary celI and it is made cell suspension；
2) by step 1) cell suspension move into small-scale reactor carry out serum free suspension culture；
3) by step 2) cell suspension move into commercial scale reactor carry out serum free suspension culture；
Above-mentioned preparation method, in step 1) in, Chinese hamster ovary celI is to have tamed suitable for the full culture that suspends
Cell line.With recovery Chinese hamster ovary celI in liquid nitrogen storehouse from cell work, passed in present T-shaped tissue culture flasks
Generation amplification, is transferred in small-scale reactor in culture to stirring-type Tissue Culture Flask, and then digestion rolling bottle cell
Cultivated, zooblast quantity is amplified in stirring-type Tissue Culture Flask or small-scale reactor, selected form
Healthy, well-grown cell as lower stage reactor seed cell.
Preferably, step 2) in, will select that form is healthy, well-grown rolling bottle production cell pancreas egg
White enzymic digestion liquid digestion, cell suspension is made with serum-free cell culture medium suspension culture, is pressed
0.5~1.5 × 106The density reaction of inoculation device of cells/ml, by the corresponding synchronization system on bioreactor, leads to
Quantitatively control adds air, oxygen, nitrogen and the carbon dioxide four in bioreactor in an orderly manner to cross microcomputer
The flow of kind of gas, makes it keep optimal ratio to control the temperature of cell culture fluid, nutrient solution pH, melt
Oxygen level, makes cell growth carry out cell culture in optimum environment.The condition of serum free suspension culture is：Rise
Beginning inoculating cell density 0.6 × 106cells/ml；Cultivation temperature is at 36~38 DEG C；Ph is maintained between 7.0~7.4；
Oxyty is between 40%~50%；Mixing speed between 100~135rpm, culture the 1st~3 day
Mixing speed is set to 100~120rpm, since culture the 4th day progressively carried in the range of 110~135rpm
Mixing speed high.
Preferably, the bioreactor suspension culture method that the method is used is filling type training method, often
24 hours cell culture mediums of 20% (v/v) of supplement.
The technical scheme that the present invention is provided has filled up the technological gap of Chinese hamster ovary celI suspension culture techniques, selected training
Foster condition is suitable to Chinese hamster ovary celI growth, realizes the maximization of cell culture density, reduces cell culture
Volume, reduces follow-up labour intensity, and quality stable homogeneous, production process linking is tight, easily manipulation.This
Invention carries out fluid infusion in cell culture and virus propagation process, it is ensured that the nutriment needed for cell propagation,
The utilization rate of nutrient solution is improve again, it is cost-effective.
Specific embodiment
Embodiment 1
A kind of Chinese hamster ovary celI cultural method, comprises the following steps：
1) with stirring-type Tissue Culture Flask culture Chinese hamster ovary celI and it is made cell suspension；
2) by step 1) cell suspension move into small-scale reactor carry out serum free suspension culture；
3) by step 2) cell suspension move into commercial scale reactor carry out serum free suspension culture；
Above-mentioned preparation method, in step 1) in, Chinese hamster ovary celI is to have tamed suitable for the full culture that suspends
Cell line.With recovery Chinese hamster ovary celI in liquid nitrogen storehouse from cell work, passed in present T-shaped tissue culture flasks
Generation amplification, is transferred in small-scale reactor in culture to stirring-type Tissue Culture Flask, and then digestion rolling bottle cell
Cultivated, zooblast quantity is amplified in stirring-type Tissue Culture Flask or small-scale reactor, selected form
Healthy, well-grown cell as lower stage reactor seed cell.
Step 2) in, will select that form is healthy, well-grown rolling bottle produces cells trypsinised liquid and disappears
Change, cell suspension is made with serum-free cell culture medium suspension culture, by 0.5~1.5 × 106The density of cells/ml
Reaction of inoculation device, by the corresponding synchronization system on bioreactor, by microcomputer, quantitatively control adds in an orderly manner
Enter air, oxygen, the four kinds of flows of gas of nitrogen and carbon dioxide in bioreactor, it is kept most
Good ratio controls the temperature of cell culture fluid, nutrient solution pH, melts oxygen level, makes cell growth in most suitable
Suitable environment carries out cell culture.The condition of serum free suspension culture is：Initial inoculation cell density
0.6×106cells/ml；Cultivation temperature is at 36~38 DEG C；Ph is maintained between 7.0~7.4；Oxyty is 40%~50%
Between；Mixing speed is set between 100~135rpm in the 1st~3 day mixing speed of culture
100~120rpm, since culture the 4th day mixing speed is stepped up in the range of 110~135rpm.
The bioreactor suspension culture method that the method is used is filling type training method, is supplemented within every 24 hours
The cell culture medium of 20% (v/v).
Embodiment 2
A kind of Chinese hamster ovary celI cultural method, comprises the following steps：
1) with stirring-type Tissue Culture Flask culture Chinese hamster ovary celI and it is made cell suspension；
2) by step 1) cell suspension move into small-scale reactor carry out serum free suspension culture；
3) by step 2) cell suspension move into commercial scale reactor carry out serum free suspension culture；
Above-mentioned preparation method, in step 1) in, Chinese hamster ovary celI is to have tamed suitable for the full culture that suspends
Cell line.With recovery Chinese hamster ovary celI in liquid nitrogen storehouse from cell work, passed in present T-shaped tissue culture flasks
Generation amplification, is transferred in small-scale reactor in culture to stirring-type Tissue Culture Flask, and then digestion rolling bottle cell
Cultivated, zooblast quantity is amplified in stirring-type Tissue Culture Flask or small-scale reactor, selected form
Healthy, well-grown cell as lower stage reactor seed cell.
Embodiments of the invention have been described in detail above, but the content is only preferable implementation of the invention
Example, is not intended to limit the invention.All any modification, equivalents made in application range of the invention
With improve etc., should be included within the scope of the present invention.
Claims (3)
1. a kind of Chinese hamster ovary celI cultural method, it is characterised in that comprise the following steps：
1) with stirring-type Tissue Culture Flask culture Chinese hamster ovary celI and it is made cell suspension；
2) by step 1) cell suspension move into small-scale reactor carry out serum free suspension culture；
3) by step 2) cell suspension move into commercial scale reactor carry out serum free suspension culture；
Above-mentioned preparation method, in step 1) in, Chinese hamster ovary celI is to have tamed suitable for the full culture that suspends
Cell line.With recovery Chinese hamster ovary celI in liquid nitrogen storehouse from cell work, passed in present T-shaped tissue culture flasks
Generation amplification, is transferred in small-scale reactor in culture to stirring-type Tissue Culture Flask, and then digestion rolling bottle cell
Cultivated, zooblast quantity is amplified in stirring-type Tissue Culture Flask or small-scale reactor, selected form
Healthy, well-grown cell as lower stage reactor seed cell.
2. Chinese hamster ovary celI cultural method according to claim 1, it is characterised in that step 2) in, will
Select that form is healthy, well-grown rolling bottle produces cells trypsinised liquid digestion, use serum-free cell
Culture medium suspension culture is made cell suspension, by 0.5~1.5 × 106The density reaction of inoculation device of cells/ml, passes through
Corresponding synchronization system on bioreactor, by microcomputer, quantitatively control is added in bioreactor in an orderly manner
Air, oxygen, four kinds of flows of gas of nitrogen and carbon dioxide, make the optimal ratio of its holding thin to control
The temperature of born of the same parents' nutrient solution, nutrient solution pH, melt oxygen level, cell growth is carried out cell training in optimum environment
Support.The condition of serum free suspension culture is：Initial inoculation cell density 0.6 × 106cells/ml；Cultivation temperature exists
36~38 DEG C；Ph is maintained between 7.0~7.4；Oxyty is between 40%~50%；Mixing speed exists
Between 100~135rpm, 100~120rpm is set in the 1st~3 day mixing speed of culture, from culture the 4th
Its beginning steps up mixing speed in the range of 110~135rpm.
3. Chinese hamster ovary celI cultural method according to claim 1, it is characterised in that what the method was used
Bioreactor suspension culture method is filling type training method, the every 24 hours cells of 20% (v/v) of supplement
Culture medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510809964.5A CN106754635A (en) | 2015-11-19 | 2015-11-19 | A kind of Chinese hamster ovary celI cultural method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510809964.5A CN106754635A (en) | 2015-11-19 | 2015-11-19 | A kind of Chinese hamster ovary celI cultural method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106754635A true CN106754635A (en) | 2017-05-31 |
Family
ID=58885956
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510809964.5A Pending CN106754635A (en) | 2015-11-19 | 2015-11-19 | A kind of Chinese hamster ovary celI cultural method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106754635A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055621A (en) * | 2018-08-28 | 2018-12-21 | 上海景峰制药有限公司 | Optimization method, device, equipment and the medium of Chinese hamster ovary celI culture process |
-
2015
- 2015-11-19 CN CN201510809964.5A patent/CN106754635A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109055621A (en) * | 2018-08-28 | 2018-12-21 | 上海景峰制药有限公司 | Optimization method, device, equipment and the medium of Chinese hamster ovary celI culture process |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Fahim et al. | Impact of energy supply and oxygen transfer on selective lipopeptide production by Bacillus subtilis BBG21 | |
| CN102391995B (en) | Serum-free high density suspension perfusion culture technology of hybridoma cells | |
| CN204490886U (en) | A kind of biology cellulose fermentation system | |
| JP2008545441A5 (en) | ||
| EP3167042B1 (en) | Bioreactor with interruptible gas supply | |
| CN103756886A (en) | High-density continuous culture method and device for microalgae | |
| CN105112352A (en) | ST cell adapting to full-suspension culture, and application thereof, and vaccine virus culturing method | |
| Khan et al. | Bioreactors for hairy roots culture: a review | |
| Hu et al. | Improvement of panaxnotoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors | |
| CN102382794A (en) | Perfusion culture method of mammal cell | |
| Liu et al. | Production of artemisinin by hairy rot cultures of Artemisia annua L in bioreactor | |
| Snape et al. | How suspension cultures of Catharanthus roseus respond to oxygen limitation: Small‐scale tests with applications to large‐scale cultures | |
| Toepel et al. | Photosynthesis driven continuous hydrogen production by diazotrophic cyanobacteria in high cell density capillary photobiofilm reactors | |
| CN106754635A (en) | A kind of Chinese hamster ovary celI cultural method | |
| CN109053867A (en) | A kind of cultural method of high density fermentation expression recombinant rotavirus Δ VP8* antigen | |
| Pörtner et al. | Fixed bed reactors for the cultivation of mammalian cells: design, performance and scale-up | |
| Palabikyan et al. | Scale-up of biomass production by Methanococcus maripaludis | |
| CN203683528U (en) | High-density continuous culture device of microalgae | |
| CN108384748A (en) | A method of automation culture diploid cell | |
| Lee | Microalgae Cultivation Fundamentals | |
| Morris et al. | A fluidised bed vessel for the culture of immobilised plant cells and its application for the continuous production of fine cell suspensions | |
| KR20160080543A (en) | Cell culture apparatus | |
| CN102327609B (en) | Production method of encephalitis B vaccine | |
| GHOSH | Bioreactor | |
| CN105296407A (en) | Method for culturing avibacterium paragallinarum bacterial solution |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170531 |
|
| WD01 | Invention patent application deemed withdrawn after publication |