CN102154196A - Method for synchronous suspension culture of mammalian cells in animal cell reactor - Google Patents
Method for synchronous suspension culture of mammalian cells in animal cell reactor Download PDFInfo
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- CN102154196A CN102154196A CN 201010617364 CN201010617364A CN102154196A CN 102154196 A CN102154196 A CN 102154196A CN 201010617364 CN201010617364 CN 201010617364 CN 201010617364 A CN201010617364 A CN 201010617364A CN 102154196 A CN102154196 A CN 102154196A
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Abstract
The invention relates to a method for synchronous suspension culture of mammalian cells in an animal cell reactor, which comprises: culturing mammalian cells in a culture medium containing glycerol in a reactor; and after each generation grows for 24 to 30 hours, cooling the reactor quickly, performing low-temperature stress culture of mammalian cells at 4 DEG C for 0.5 to 1 hour, raising the temperature of the reactor, culturing the mammalian cells subjected to low-temperature stress culture at 37 DEG C for 16 to 18 hours, and obtaining animal cells which are basically in synchronous growth state in a cell period. The method is simple in operation, safe, convenient, large in scale and has a great application value for study on the synchronous growth of cells, colony metabolism, virus multiplication and protein expression.
Description
Technical field
The invention belongs to zooblast engineering field, be specifically related to a kind of in the zooblast reactor method of synchronization suspension culture mammalian cell.
Background technology
The large scale culturing of zooblast has been widely used in the production of biological medical product.In recent years, metabolic engineering and metabolic analysis development are rapid, but metabolic analysis is all cultivated resulting data based on cell colony basically, because cell growth and metabolic asynchronous, part has been covered some metabolism behaviors of cell.If using individual cells studies, not only there is technical difficulty, and because quantity is difficult to carry out quantitative analysis very little, feasible method makes cell colony synchronous growth exactly, reflects the real metabolism behavior of cell with colony's cytokinetics characteristic of synchronization growth.
The control method of zooblast synchronization growth mainly contains chemical process and physical method.Use the chemical process blocking-up cell cycle; can obtain higher synchronous efficient; simultaneously also there is number of chemical reagent available; but this class chemical reagent price is very expensive; be not suitable on bio-reactor, mass-producing cultured animals cell being used in a large number; and such chemical reagent all has certain toxicity, and there is potential safety hazard in operator ' s health.Carry out cell cycle synchronization control by physical method and also have himself drawback, because need to use some special experimental installations, as gradient centrifugation equipment etc., physical method prepares synchronization cell length consuming time in addition, easy pollution of operating process and preparation amount are too little, often can not satisfy the needs of research.
Summary of the invention
The purpose of this invention is to provide a kind of in the zooblast reactor method of synchronization suspension culture mammalian cell, can control the synchronization growth of zooblast safely, simply, easily, to adapt to the needs that animal cell large-scale is cultivated.
The invention provides a kind of in the zooblast reactor method of synchronization suspension culture mammalian cell, comprise the steps:
(1) in reactor, with the culture medium culturing mammalian cell that contains glycerine, after every generation grows to 24～30h, reactor is lowered the temperature rapidly, make described mammalian cell under 4 ℃, carry out low temperature stress and cultivate 0.5～1h;
(2) reactor is heated up, make the mammalian cell after low temperature stress is cultivated cultivate 16～18h, obtain to be in substantially the zooblast of cell cycle synchronization growth conditions at 37 ℃.
Described mammalian cell is HEK293 cell or Marc145 cell.
The low temperature stress incubation time is 1h in the step (1).
The concentration of glycerine is 5～10g/L in the described substratum, preferred 8g/L.
Show that after deliberation HEK293 cell and Marc145 cell are under 4 ℃ of low temperature, cellular metabolism is close to and stops, and damage does not appear in cytolemma, but cell survival rate obviously descends along with the prolongation of 4 ℃ of low temperature times; For effectively slowing down even stop the eubolism activity of cell, the basic vigor that can keep cell simultaneously again is constant, and the present invention adopted 4 ℃ of following low temperature stress 1 hour, and the survival rate of two kinds of cells can maintain more than 92% substantially.Then temperature of reactor directly is raised to 37 ℃, continues culturing cell, cell progressively begins to recover growth metabolism, but the HEK293 cellular-restoring grows to 18 hours, the retardance of G2/M phase progressively appears in cell, and the Marc145 cellular-restoring grows to 16 hours, and the retardance of G2/M phase progressively appears in cell.Cell promptly begins the synchronization growth subsequently.
The present invention is simple to operate, safety, convenient, scale is big, can be applied to extensive suspension cultured cells on the bio-reactor, and research cell synchronization growth, colony's metabolism, virus multiplication, protein expression are had great application value.
Below in conjunction with drawings and Examples, the invention will be further described.
Description of drawings
The different time of 4 ℃ of low temperature stress of Fig. 1 (1) expression is to the influence of HEK293 cell survival rate.
The different time of 4 ℃ of low temperature stress of Fig. 1 (2) expression is to the influence of Marc145 cell survival rate.
4 ℃ of low temperature stress of Fig. 2 (1)～Fig. 2 (6) expression are after 1 hour, and 37 ℃ of differences of recovering incubation times are to the influence of the cell cycle distribution of HEK293 cell; Wherein:
The normal culturing cell contrast of Fig. 2 (1) expression;
Fig. 2 (2)～Fig. 2 (6) represents 4 ℃ of low temperature stress 1 hour, and 37 ℃ are recovered the cell cycle distribution that incubation times are respectively 0 hour, 6 hours, 12 hours, 18 hours, 24 hours HEK293 cell.
Fig. 3 (1) expression HEK293 cellular form, arrow represents to be in the cell of G2/M phase among the figure;
Fig. 3 (2) expression Marc145 cellular form, microcarrier is cultivated among the figure be in G2/M phase Marc145 cell present cell individual increase, from the state of microcarrier upper process.
Fig. 3 (1) represents after 4 ℃ of low temperature stress are handled 1h, the cellular form figure of the HEK293 cell of suspension culture after 18h is cultivated in 37 ℃ of recoveries, and observation condition is under the inverted microscope, 20 times of object lens are observed also shooting;
Fig. 3 (2) represents after 4 ℃ of low temperature stress are handled 1h, the cellular form figure of the Marc145 cell of microcarrier suspension culture after 16h is cultivated in 37 ℃ of recoveries, and observation condition is under the inverted microscope, 20 times of object lens are observed also shooting.
Embodiment
1.4 ℃ of low temperature stress different times of embodiment are to the influence of HEK293 cell and Marc145 cell survival rate
Cultivate the HEK293 cell with Maitland culture, cultivate the Marc145 cell, concrete side with the microcarrier suspension culture method
Method is as follows:
1. suspension culture HEK293 cell
With 3 * 10
5The initial density of individual cells/ml is inoculated in 250ml and shakes in the bottle, and the medium component of suspension culture is as shown in the table:
Substratum uses volume to be 60ml, shakes bottle and places 37 ℃, 5%CO
2Incubator in shaking table on, the cultivation rotating speed is 60～100rpm.Drew a certain amount of HEK293 cell inoculation in 4 days and shake in the bottle in newly and go down to posterity, the ratio of going down to posterity is 1 to pass 3, supplies nutrient solution and continues the vibration suspension culture to 60ml.
2. microcarrier suspension culture Marc145 cell
Shaking in the bottle of 250ml, the Marc145 cell is with 2 * 10
5The initial density of individual cells/ml is inoculated on the cytodex1 microcarrier of 3g/L, add 250ml DMEM (wherein having added volumetric concentration and be 10% new-born calf serum and mass concentration is the glycerine of 5～10g/L (preferred 8g/L), in 35rpm, 37 ℃, 5%CO
2Incubator in cultivate.The Marc145 cell is adherent growth on the cytodex1 microcarrier, roughly can cover with the surface of microcarrier substantially in 3 days, with add fresh microcarrier directly go down to posterity or after trysinization, go down to posterity all can, supply fresh medium and continue microcarrier suspension culture.
HEK293 cell and microcarrier suspension culture Marc145 cell by the aforesaid method suspension culture grow into 24～30h in every generation, carry out 4 ℃ of low temperature stress and handle.Investigate the influence of subzero treatment different time pair cell survival rate.From subzero treatment sampling respectively in 0.5 hour to 8 hours, use the trypan blue dyeing counting, determine survival rate.The result is shown in Fig. 1 (1) and Fig. 1 (2).As seen, along with the prolongation of 4 ℃ of subzero treatment times, cell survival rate obviously reduces among the figure.Handled 0.5,1 hour for 4 ℃, two kinds of cells are not had influence substantially, cell survival rate all is higher than 92%.
2.4 ℃ of low temperature stress of embodiment 1 hour are to the influence of the cell cycle distribution of the HEK293 cell of suspension culture and induce the synchronization growth
The HEK293 cell of suspension culture was handled 1 hour through 4 ℃ of low temperature stress, recovered 37 ℃ then and cultivated 0h, 6h, 12h, 18h and 24h, and The cell cycle is carried out in sampling on time respectively, and method is as follows:
1) collects 2～4 * 10 with the centrifugal 10min of 1000rpm
6Individual HEK293 cell adds a certain amount of phosphoric acid buffer (PBS damping fluid: 8g/L NaCl, 0.2g/L KCl, 1.44g/LNa in the aseptic eppendorf centrifuge tube of 1.5ml
2HPO
4With 0.24g/L KH
2PO
4, use behind ℃ sterilization 20min of pH7.2～7.4,121) and resuspended HEK293 cell, cell is cleaned in piping and druming gently, again with the centrifugal 10min of 1000rpm, abandoning supernatant;
2) 70% ethanol that adds the 1ml precooling in cell precipitation fixedly spends the night in 4 ℃, and this fixed cell can be preserved a week in 4 ℃;
3) the fixed cell 10min that obtains with 1000rpm centrifugation step (2), discard ethanol, and add the resuspended HEK293 cell of a certain amount of PBS, cell is cleaned in piping and druming gently, with the centrifugal 10min of 1000rpm, supernatant discarded adds 1ml iodate third ingot (Propidium Iodide again, PI) working fluid, lucifuge dyeing 30～40min under the room temperature;
4) dilute with PBS the dyeing back that finishes, and dispel cell and be single cell suspension, sample introduction 2 * 10
4(Becton Dickinson, San Jose measure dna content in CA) to individual cell, and the result of generation is that ordinate zou and X-coordinate are made the cell cycle distribution figure with Number and FL2-Area respectively by accompanying software in flow cytometer.
The result is shown in table 1 and Fig. 2 (1)-Fig. 2 (6).Table 1 is the raw value that distributes in the different cell cycles of cell as can be seen.Fig. 2 (1)-Fig. 2 (6) is according to the drawn cell cycle figure of the numerical value of cell cycle distribution, ordinate zou Number is meant cell quantity among the figure, X-coordinate channels (FL2-A) is meant the qualification door in the cells were tested by flow cytometry, this use be the integral area that is similar to the DNA fluorescent value of diploid cell, be used for representing cell DNA content by detector.
The result shows, handles 1 hour through 4 ℃ of low temperature stress, recovers 37 ℃ then and cultivates 18h, can cause 52.43% HEK293 cell to be in the G2/M phase and block.Recover to cultivate 24h subsequently, as seen have 32.88% cell to be in the G0/G1 phase, 56.26% cell is in the S phase, have only 10.86% cell to be in the G2/M phase, illustrate that most of cell (89.14%) all is in mitotic early stage, be in the state of synchronization growth substantially.
Table 14 ℃ is coerced and to be cultivated after 1 hour, and 37 ℃ are recovered to cultivate different times, the cell cycle distribution of HEK293 cell
34 ℃ of low temperature stress of embodiment 1 hour are to the influence of the cell cycle of the Marc145 cell of the HEK293 cell of suspension culture and microcarrier suspension culture
The Marc145 cell of the HEK293 cell of suspension culture and microcarrier suspension culture in 7L zooblast reactor respectively, after cultivating 24h culture temperature is made as 4 ℃, the temperature control unit processing that begins to lower the temperature this moment, use ice bag to lower the temperature simultaneously as water coolant unit and tank body, culture temperature is reduced to 4 ℃ fast, and keeps 4 ℃ to handle 1 hour.Culture temperature is set at 37 ℃ and returns to normal culture temperature (37 ℃) continuation cultivation 18h (HEK293 cell) and 16h (Marc145 cell) rapidly subsequently.Can obtain effectively will to be controlled at the cell cycle effect that the G2/M phase blocks thus, shown in Fig. 3 (1) and Fig. 3 (2), can observe a large amount of HEK293 cells this moment and be in the G2/M phase (among the figure shown in the arrow), the Marc145 cell then on microcarrier, present cell individual increase, from the state of microcarrier surface upper process, this is the representative configuration that cell is in the G2/M phase in the microcarrier cell cultivation process.Along with the further prolongation at 37 ℃ of incubation times, cell is progressively unified to begin the new round cell cycle from the G2/M phase, enters the G1 phase simultaneously, reaches the purpose that realizes the growth of at least one wheel cells cycle synchronisation.
Claims (5)
1. the method for a synchronization suspension culture mammalian cell in the zooblast reactor comprises the steps:
(1) in reactor, with the culture medium culturing mammalian cell that contains glycerine, after every generation grows to 24～30h, reactor is lowered the temperature rapidly, make described mammalian cell under 4 ℃, carry out low temperature stress and cultivate 0.5～1h;
(2) reactor is heated up, make the mammalian cell after low temperature stress is cultivated cultivate 16～18h, obtain to be in substantially the zooblast of cell cycle synchronization growth conditions at 37 ℃.
According to claim 1 described in the zooblast reactor method of synchronization suspension culture mammalian cell, it is characterized in that described mammalian cell is HEK293 cell or Marc145 cell.
According to claim 1 or 2 described in the zooblast reactor method of synchronization suspension culture mammalian cell, it is characterized in that the low temperature stress incubation time is 1h in the step (1).
According to claim 1 described in the zooblast reactor method of synchronization suspension culture mammalian cell, the concentration that it is characterized in that glycerine in the described substratum is 5～10g/L.
According to claim 4 described in the zooblast reactor method of synchronization suspension culture mammalian cell, the concentration that it is characterized in that glycerine in the described substratum is 8g/L.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114990053A (en) * | 2022-07-06 | 2022-09-02 | 华兰生物疫苗股份有限公司 | Large-scale low-temperature preservation method for adherent animal cells |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1556203A (en) * | 2003-12-31 | 2004-12-22 | 中国人民解放军军事医学科学院生物工 | Aimal cell multipore micro carrier immobilized high efficiency culturing method and its culturing medium |
| CN101037666A (en) * | 2007-02-09 | 2007-09-19 | 浙江大学 | Low temperature preservation solution for animal cells and preserving method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1556203A (en) * | 2003-12-31 | 2004-12-22 | 中国人民解放军军事医学科学院生物工 | Aimal cell multipore micro carrier immobilized high efficiency culturing method and its culturing medium |
| CN101037666A (en) * | 2007-02-09 | 2007-09-19 | 浙江大学 | Low temperature preservation solution for animal cells and preserving method |
Non-Patent Citations (4)
| Title |
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| 《Cell Cycle》 20020630 Conly L.Rieder，Richard W.Cole Cold-Shock and the Mammalian Cell Cycle 第1卷, 第3期 * |
| 《吉林农业大学学报》 20070430 刘轶, 朱国强 动物细胞培养及微载体技术研究进展 203-206 第29卷, 第2期 * |
| 《细胞工程学》 20030701 王蒂主编， 第十章 动物细胞培养 中国农业出版社，2003年7月第1版 , * |
| 《遗传》 20050528 侯颖,谭立新,李文蓉,牛志刚,郭志勤 一种提高体外培养细胞中期分裂相的新方法 457-460 1-5 第27卷, 第3期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114990053A (en) * | 2022-07-06 | 2022-09-02 | 华兰生物疫苗股份有限公司 | Large-scale low-temperature preservation method for adherent animal cells |
| CN114990053B (en) * | 2022-07-06 | 2024-04-19 | 华兰生物疫苗股份有限公司 | Large-scale low-temperature preservation method of adherent animal cells |
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