CN109280636A - A method of suspend domestication 293T cell - Google Patents
A method of suspend domestication 293T cell Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 27
- 210000002966 serum Anatomy 0.000 claims abstract description 38
- 239000000725 suspension Substances 0.000 claims abstract description 18
- 239000012679 serum free medium Substances 0.000 claims abstract description 13
- 238000004114 suspension culture Methods 0.000 claims abstract description 11
- 230000003247 decreasing effect Effects 0.000 claims abstract description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 15
- 239000012894 fetal calf serum Substances 0.000 claims description 15
- 239000002356 single layer Substances 0.000 claims description 8
- 239000001963 growth medium Substances 0.000 claims description 7
- 241000283690 Bos taurus Species 0.000 claims description 6
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- 238000005070 sampling Methods 0.000 claims description 4
- 241000700605 Viruses Species 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 230000012010 growth Effects 0.000 claims description 3
- 241000204031 Mycoplasma Species 0.000 claims description 2
- 230000010261 cell growth Effects 0.000 claims description 2
- 239000006285 cell suspension Substances 0.000 claims description 2
- 210000000981 epithelium Anatomy 0.000 claims 1
- 239000010410 layer Substances 0.000 claims 1
- 239000007788 liquid Substances 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 61
- 210000004962 mammalian cell Anatomy 0.000 abstract description 7
- 230000003321 amplification Effects 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract description 3
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 3
- 108010019160 Pancreatin Proteins 0.000 abstract description 2
- 238000009434 installation Methods 0.000 abstract description 2
- 229940055695 pancreatin Drugs 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 230000006978 adaptation Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- 101710128836 Large T antigen Proteins 0.000 description 1
- 208000008771 Lymphadenopathy Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 208000026487 Triploidy Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000013228 adenopathy Diseases 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 239000012914 anti-clumping agent Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003237 epithelioid cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000007667 floating Methods 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
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- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- 238000007086 side reaction Methods 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
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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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- C12N5/0603—Embryonic cells ; Embryoid bodies
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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
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
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Abstract
The present invention provides a kind of methods of suspension 293T cell line for preparing low serum free culture system to obtain the 293T cell of adhere-wall culture, be digested with pancreatin this method comprises: (1) recovers and cultivates mammalian cell；(2) with the 293T cell of the serum free medium culture of a certain amount of serum adhere-wall culture is added to, the 293T cell is continuously cultivated to adapting to the serum free medium for being added to a certain amount of serum；And (3) repeat the continuous cultivation program in step (2), serum addition to the serum-concentration gradually decreased in the serum free medium is 0, obtains the 293T cell of the suspension culture.Operation of the present invention is simple, is easy to use, expense is low, and safety is good.The amplification in vitro cultural method of 293T cell suspension cultures is not necessarily to special installation, and operation is simple and feasible.There to be extensive prospect in the basic research and clinical application of 293T cell suspension cultures.
Description
Technical field
The present invention relates to field of pharmaceutical biology, and in particular to a kind of method of suspended culture cell.
Background technique
The cultural method of cell in vitro includes adhere-wall culture and the culture that suspends, and adhere-wall culture (refers to cell attachment certain
Solid phase surface carry out culture；Culture suspend by vibrating or rotating device makes cell be in dispersion always to be suspended in culture solution
Interior cultural method；The culture that wherein suspends can be divided into microcarrier suspension culture and the full culture that suspends again.Compared with adhere-wall culture, hang
Floating culture has the advantages that (1) can continuous expanding production amount；(2) be conducive to the nutriment and gas in cell culture medium
It comes into full contact with, and easily controllable condition of culture (temperature, pH, partial pressure of oxygen and CO2 etc.)；(3) condition of culture is stablized, and tends to be equal
One, it is convenient for quantitative study；(4) it is easy to carry out in continuous closed system, reduces the chance of operating procedure and pollution；
(5) it can continuously cultivate for a long time, can not only save manpower, but also cell is enable persistently to maintain logarithmic growth phase；(6) suspend culture
Cell still keeps the originally sensibility and biological characteristics to virus.
293 cells are converted with 5 type adenovirus, 75 strain, and the human embryo kidney (HEK) hypo-triploid cell line containing the area Ad5E1, is one
The kind area E1 defect complementary cell system.It is Canadian McMaster University F.L.Graham and J.S.Miley in
1976 built-up with DNA rotaring dyeing technology.293 cells are anchorage dependence types in epithelioid cell, show typical adenopathy
The phenotype of malicious transformed cells, cell allow Ad5 and other serotype adenovirus to be proliferated on it.293 cells belong to attached cell
System, can equally grow in no Ca2+ or culture medium containing Ca2+, can also be grown in the culture medium of serum-concentration reduction, existing skill
In art, experiment confirms that the growth of cell under the excessively multipair 293 cell monolayer training method of cell passage number produces apparent shadow
It rings.293T cell is derived from by 293 cells, while expressing SV40 large T antigen, the matter containing SV40 replication origin and promoter region
Grain can replicate.It may be up to 50% with 2 transfection efficiency of Ca3 (PO4).Protein expression level is high, uses alkaline phosphatase within 2-3 days after transfection
Enzyme analyzes the albumen that can relatively easily detect expression.Transiently transfecting 293T cell is to be overexpressed albumen and obtain intracellular and thin
The easy way of extracellular (secretion or film) albumen.
When industrializing monolayer cultivation 293T cell at this stage, in culture medium plus a certain proportion of cow's serum (including tire ox
Serum or newborn bovine serum) cell could normal growth, cow's serum not only price, but also derived therefrom is in natural biology
Body, ox source itself carries and acquisition process has the possibility for polluting inoculating microbe and virulence factor, and use can have biology
Security risk.In addition, cow's serum is a kind of natural mixture, specific composition is clear there is presently no analyzing completely, biology system
Difficulty is caused to downstream purification technique after use in product production, if there is residual would generally cause the side reaction of inoculator and is influenced
Product quality.As can using the full suspension process of serum-free, culture scale is easy for linearly putting for cell culture in biological products production
Greatly, it avoids cell adhere-wall culture and uses cow's serum bring defective workmanship, viral yield and product quality can be improved, still
Domestication serum-free full suspension cultivation type cell strain be current most critical one of technical bottleneck.
Summary of the invention
For the above reason, the present invention provides a kind of method that attached cell carries out suspension culture, this method by by
Grade reduce fetal calf serum concentration, take full advantage of suspension culture the advantages of, allow attached cell carry out simplify, efficiently,
A large amount of culture.
The present invention provides a kind of methods of suspension mammalian cell for preparing free serum culture, this method comprises: (1)
It recovers and cultivates mammalian cell, obtain the mammalian cell of adhere-wall culture, digested with pancreatin；(2) certain with being added to
The mammalian cell for measuring the serum free medium culture of the serum adhere-wall culture continuously cultivates the mammalian cell to adaptation
This is added to the serum free medium of a certain amount of serum；And (3) repeat the continuous cultivation program in step (2), gradually drop
Serum addition in the low serum free medium is finally put into suspension cell on shaking table until last serum content is 0 and shakes
Dynamic culture.
Preferably, the mammal in the step (1) is 293T cell.
Preferably, the serum free medium in the step (2) and (3) includes Free style.
Preferably, serum addition described in the step (2) is 10%；It is gradually decreased described in the step (3)
Serum addition is respectively 5%, 2%, 1%, 0.5%, 0.2%, 0.
Preferably, one is added to described in the continuously culture mammalian cell in the step (2) and (3) to adaptation
The program of the serum free medium of quantitative serum is 3 generations of continuous culture.
The invention has the following advantages:
1) operation of the present invention is simple, is easy to use, expense is low, and safety is good.
2) the amplification in vitro cultural method of 293T cell suspension cultures is not necessarily to special installation, and operation is simple and feasible.
3) there will be extensive prospect in the basic research and clinical application of 293T cell suspension cultures.
4) suspension medium of the invention supports 293T cell long-period secondary culture, without long-term and complex adaptation
Journey.
5) suspension medium of the invention is free of serum, and component is clear, is conducive to isolating and purifying for product, improves product product
Matter.
Detailed description of the invention
Fig. 1 be microscopically observation 293T cell adhere-wall culture of the present invention and suspend cultivate picture, 100 times of amplification factor.
Specific embodiment
Specific embodiment
Reagent used in 1.
1) cell strain: 293T cell is bought from ATCC cell bank；
2) DMEM culture solution, Corning, article No.: 10-013-CVR；
3) Free style culture solution, Gibco, article No.: 12338-018；
4) trypsase, Gibco, article No.: 27250, it is configured to 0.25% trypsin solution (EDTA-Na0.3g/L)；
5) Anti clumping agent, Invitrogen, article No.: 0010057AE.
2. instrument equipment and vessel
1) shaking table, KUHNER, model: ISF1-XC；
2) CO2 incubator, Thermo Fisher Scientific Corporation, model: 3111；
3) centrifuge, Eppendorf, model: 5810R；
4) inverted microscope, OLYMPUS, OLYMPUS CKX41 are differed；
5) Tissue Culture Flask (Corning, T25 article No.: 430639, T75 article No.s: 430641)；
6) pyramidal cells bottle 150ml, Jiangsu Haimen, specification: 150ml.
The culture of 1. adhere-wall culture type 293T cell line of embodiment
It is chosen in subsidiary company cell bank and is examined and determine sterile, mycoplasma, exogenous virus is negative adhere-wall culture type 293T
One recovery culture of cell, culture solution are the DMEM of 10% fetal calf serum (following serum-concentration is volumn concentration), training
The condition of supporting: the incubator culture of 37 DEG C of 5%CO2.Vigor is 98.6% after cell recovery, and cell culture 48h forms fine and close single
Layer, in 1: 4 ratio sub-bottle secondary culture in 48h also at fine and close single layer, cell edges are clear, and form is flat, are in epitheliated type.
The domestication of the low serum adhere-wall culture type 293T cell line of embodiment 2.
A) by step (1) the normal adhere-wall culture type 293T cell of growth respectively with the DMEM containing fetal calf serum 10%
Culture solution respectively cultivates three generations；The standard passed on every time is to cultivate in 1:4 ratio sub-bottle in 37 DEG C of 5%CO2, thin in 48-72h
Born of the same parents form fine and close single layer；
B) 10% fetal calf serum is added to continue to cultivate three generations after changing culture solution into Free style culture medium, passage standard is same
Step a)；
C) fetal calf serum is reduced to 5% in culture solution, and by the method in b step, passage ratio is reduced to 1:3 and is further cultured for three generations；
D) tire ox cow's serum is reduced to 2% in culture solution, adds volumn concentration 20% previous generation times training again in culture solution
The culture solution for having supported cell 48-72h continues to cultivate three generations；
It is rapid c) to pass on standard synchronisation；Cell forms fine and close single layer in 48h-72h, and domestication has obtained low serum adhere-wall culture
Type 293T cell line；
The domestication of the low serum of embodiment 3. suspension cultivation type 293T cell line entirely
A) the low serum adhere-wall culture type 293T cell for taming step (2), is pressed with Free style serum free medium
After the mixing of volume ratio 1:1 ratio, adds 2% fetal calf serum that culture solution is made, adjusted cell density to 2-5 × 10 with culture solution5/
Ml, 125r/min, 37 DEG C of 5%CO2 shaking table cultures；
B) repeat step a), every culture three generations reduction serum-concentration, fetal calf serum concentration successively from 2% to 1% to
0.5%；
C) every culture 48h sampling counts, and cell density is not up to 1 × 106When/ml, used instead after 800-1000r/min centrifugation
Fresh medium suspends culture again；Cell density reaches 1 × 106When/ml, 800-1000r/min is centrifuged in 1:2 ratio sub-bottle
Culture；Reach 2 × 10 to cell density6When/ml, 800-1000r/min is centrifuged in 1:3 ratio sub-bottle culture；In continuous three generations
In 1:3 ratio culture 48h cell density up to 2 × 106When/ml, domestication obtains low serum suspension cultivation type 293T cell line entirely.
The domestication of 4. serum-free of embodiment suspension cultivation type 293T cell line entirely
A) in serum free medium plus 0.2% fetal calf serum, low serum obtained in step (3) is suspended cultivation type entirely
293T cell is directly diluted to density 5 × 105/ ml sets 125r/min, 37 DEG C of 5%CO2 shaking table cultures；
B) sampling counts for 24 hours for every culture, and cell density reaches 3 × 106When/ml, cell suspension is directly diluted to 5 ×
105/ ml inoculated and cultured, serum are reduced to 0, completely with free serum culture culture, cultivate 48h cell density up to 3 in continuous three generations
×106When/ml, domestication obtains serum-free suspension cultivation type 293T cell line entirely.
1. 293T cell of table, which suspends, tames culture medium prescription table in operating process
Claims (7)
1. the acclimation method of full suspension cultivation type 293T cell line, it is characterised in that: steps are as follows:
(1) culture of adhere-wall culture type 293T cell
Selecting through sterile, mycoplasma, exogenous virus calibrating is feminine gender, and adhere-wall culture type 293T cell is grown fine and close single to cell
It is passed on the DMEM culture solution containing 10% fetal calf serum in 1:3 ratio sub-bottle after layer, sets the incubator culture of 37 DEG C of 5%CO2,
48-72h inner cell forms fine and close single layer, is that can be used in sharp-edged squamous epithelium type cell and the normal situation of cell growth
In domestication；
(2) domestication of low serum adhere-wall culture type 293T cell line
A) step (1) the normal adhere-wall culture type 293T cell of growth is cultivated with the DMEM containing fetal calf serum 10% respectively
Liquid respectively cultivates three generations；The standard passed on every time is to cultivate in 37 DEG C of 5%CO2, in 1:4 ratio sub-bottle in 48-72h inner cell shape
At fine and close single layer；
B) 10% fetal calf serum is added to continue to cultivate three generations after changing culture solution into Free style culture medium, passage standard synchronisation is rapid
a)；
C) fetal calf serum is reduced to 5% in culture solution, and by the method in b step, passage ratio is reduced to 1:3 and is further cultured for three generations；
D) tire ox cow's serum is reduced to 2% in culture solution, adds volumn concentration 20% previous generation times culture again in culture solution
The culture solution of cell 48-72h continues to cultivate three generations；
It is rapid c) to pass on standard synchronisation；Cell forms fine and close single layer in 48h-72h, and domestication has obtained low serum adhere-wall culture type
293T cell line；
(3) domestication of low serum suspension cultivation type 293T cell entirely
A) the low serum adhere-wall culture type 293T cell for taming step (2) presses volume with Free style serum free medium
After the mixing of 1:1 ratio, adds 2% fetal calf serum that culture solution is made, adjusted cell density to 2-5 × 10 with culture solution5/ ml,
125r/min, 37 DEG C of 5%CO2 shaking table cultures；
B) step a) is repeated, every culture three generations reduces serum-concentration, and fetal calf serum concentration is successively to 0.5% from 2% to 1%；
B) every culture 48h sampling counts, and cell density is not up to 1 × 106When/ml, used instead after 800-1000r/min centrifugation fresh
Culture solution suspends culture again；Cell density reaches 1 × 106When/ml, 800-1000r/min centrifugation is trained in 1:2 ratio sub-bottle
It supports；Reach 2 × 10 to cell density6When/ml, 800-1000r/min is centrifuged in 1:3 ratio sub-bottle culture；It is pressed in continuous three generations
1:3 ratio culture 48h cell density is up to 2 × 106When/ml, domestication obtains low serum suspension cultivation type 293T cell line entirely；
(4) domestication of serum-free suspension cultivation type 293T cell entirely
A) in serum free medium plus 0.2% fetal calf serum, by the suspension cultivation type 293T entirely of low serum obtained in step (3)
Cell is directly diluted to density 5 × 105/ ml sets 125r/min, 37 DEG C of 5%CO2 shaking table cultures；
B) sampling counts for 24 hours for every culture, and cell density reaches 3 × 106When/ml, cell suspension is directly diluted to 5 × 105/ml
Inoculated and cultured, serum are reduced to 0, completely with free serum culture culture, cultivate 48h cell density up to 3 × 106/ in continuous three generations
When ml, domestication obtains serum-free suspension cultivation type 293T cell line entirely.
2. the full suspension cultivation type 293T cell obtained using method described in claim 1.
3. according to the method described in claim 1, wherein, the serum free medium in the step (2) and (3) includes
Free style culture medium.
4. according to the method described in claim 1, wherein, serum addition described in the step (2) is 10%；The step
(3) serum addition gradually decreased described in is respectively 5%, 2%, 1%, 0.5%, 0.2%, 0.
5. a kind of extensive suspension culture method of 293T cell according to claim 1, it is characterised in that step 2) institute
The cultivation temperature stated is 37 DEG C.
6. a kind of extensive suspension culture method of 293 cell according to claim 1, it is characterised in that step 2) and 3)
The pH value is 7.2.
7. a kind of extensive suspension culture method of 293 cell according to claim 1, it is characterised in that step 3) is described
Shaking table speed be set as 125rpm.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110438066A (en) * | 2019-08-19 | 2019-11-12 | 杭州百凌生物科技有限公司 | A kind of 293 C18P of mammal cell line of the culture that suspends that stablizing passage and its preparation method and application |
| CN110713971A (en) * | 2019-11-06 | 2020-01-21 | 深圳市菲鹏生物治疗股份有限公司 | Serum-free suspension culture type 293T cell, preparation method thereof and virus packaging method |
| CN112779226A (en) * | 2021-02-03 | 2021-05-11 | 苏州博腾生物制药有限公司 | Culture process of lentivirus adherent cells |
| CN113717927A (en) * | 2021-08-31 | 2021-11-30 | 宜明(北京)细胞生物科技有限公司 | Preparation method and application of HEK-293 cell serum-free suspension culture |
| CN114591885A (en) * | 2022-03-05 | 2022-06-07 | 河南普华基因科技有限公司 | LLC-PK1Sa cell domestication method adapted to serum-free suspension culture and application |
| CN114591889A (en) * | 2022-04-06 | 2022-06-07 | 湖南远泰生物技术有限公司 | Suspension domestication method of HEK293T cells and application of suspension domestication method in lentivirus production |
| CN115505559A (en) * | 2022-09-30 | 2022-12-23 | 苏州博腾生物制药有限公司 | Development method of HEK293 suspension cell line for virus vector production |
| WO2024051022A1 (en) * | 2022-11-07 | 2024-03-14 | 青岛万明赛伯药业有限公司 | Hek293 monoclonal cell population culturing method |
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Cited By (10)
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| CN110438066A (en) * | 2019-08-19 | 2019-11-12 | 杭州百凌生物科技有限公司 | A kind of 293 C18P of mammal cell line of the culture that suspends that stablizing passage and its preparation method and application |
| CN110713971A (en) * | 2019-11-06 | 2020-01-21 | 深圳市菲鹏生物治疗股份有限公司 | Serum-free suspension culture type 293T cell, preparation method thereof and virus packaging method |
| CN110713971B (en) * | 2019-11-06 | 2021-08-27 | 深圳市菲鹏生物治疗股份有限公司 | Serum-free suspension culture type 293T cell, preparation method thereof and virus packaging method |
| CN112779226A (en) * | 2021-02-03 | 2021-05-11 | 苏州博腾生物制药有限公司 | Culture process of lentivirus adherent cells |
| CN113717927A (en) * | 2021-08-31 | 2021-11-30 | 宜明(北京)细胞生物科技有限公司 | Preparation method and application of HEK-293 cell serum-free suspension culture |
| CN114591885A (en) * | 2022-03-05 | 2022-06-07 | 河南普华基因科技有限公司 | LLC-PK1Sa cell domestication method adapted to serum-free suspension culture and application |
| CN114591885B (en) * | 2022-03-05 | 2024-02-23 | 河南普华基因科技有限公司 | LLC-PK1Sa cell domestication method suitable for serum-free suspension culture and application |
| CN114591889A (en) * | 2022-04-06 | 2022-06-07 | 湖南远泰生物技术有限公司 | Suspension domestication method of HEK293T cells and application of suspension domestication method in lentivirus production |
| CN115505559A (en) * | 2022-09-30 | 2022-12-23 | 苏州博腾生物制药有限公司 | Development method of HEK293 suspension cell line for virus vector production |
| WO2024051022A1 (en) * | 2022-11-07 | 2024-03-14 | 青岛万明赛伯药业有限公司 | Hek293 monoclonal cell population culturing method |
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