CN1112442C - Cell retaining and separating method and device for cell depositing culture - Google Patents
Cell retaining and separating method and device for cell depositing culture Download PDFInfo
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- CN1112442C CN1112442C CN00116518A CN00116518A CN1112442C CN 1112442 C CN1112442 C CN 1112442C CN 00116518 A CN00116518 A CN 00116518A CN 00116518 A CN00116518 A CN 00116518A CN 1112442 C CN1112442 C CN 1112442C
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- 210000004027 cell Anatomy 0.000 claims description 139
- 238000004113 cell culture Methods 0.000 claims description 12
- 210000004408 hybridoma Anatomy 0.000 claims description 9
- 230000014759 maintenance of location Effects 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 239000012679 serum free medium Substances 0.000 claims description 4
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- 210000004102 animal cell Anatomy 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 2
- 238000004114 suspension culture Methods 0.000 claims description 2
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- 230000002262 irrigation Effects 0.000 description 7
- 230000008676 import Effects 0.000 description 5
- 230000002572 peristaltic effect Effects 0.000 description 5
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Abstract
本发明公开了一种细胞灌注培养过程中的细胞截留分离方法及其装置。本发明将含有细胞的培养液由倾斜式细胞重力沉降器的上端口进入，绝大部分活细胞和少部分死细胞返回反应器，部分死细胞和少量活细胞随上清液流出，从根本上解决了沉积细胞逆流滑动受阻的问题，保证了操作的连续性，降低了设备制造成本和操作复杂程度，并具有较大的操作弹性。The invention discloses a method and a device for retaining and separating cells in the process of cell perfusion culture. In the present invention, the culture solution containing cells enters from the upper port of the inclined cell gravity settler, most of the living cells and a small part of dead cells are returned to the reactor, and some of the dead cells and a small amount of living cells flow out with the supernatant, fundamentally It solves the problem that the deposition cells are hindered by countercurrent sliding, ensures the continuity of operation, reduces the equipment manufacturing cost and operation complexity, and has greater operation flexibility.
Description
The present invention relates to a kind of cell perfusion culture method and device thereof, relate in particular to cell retention method and a kind of tilting cell gravitational settler in a kind of cell perfusion culture process.
The cultivation of zooblast generally is divided into batch cultivation and continous pouring is cultivated two kinds of methods, and continous pouring is cultivated the serialization production that more helps suspension cell.In the continous pouring culturing process, it is a crucial link that the holding back of viable cell and dead cell separated.At present, existing many Patent publish in the perfusion culture process cell hold back separation method and device, as: United States Patent (USP) 5817505 discloses a kind of tilting settling vessel, be used for the separation of holding back of cell, this settling vessel is a tilting sealing rectangular parallelepiped groove, come the nutrient solution that autoreactor contains cell to be flowed into by the lower end, most of cell is deposited on the lower surface under action of gravity.The supernatant liquor that contains small amounts of cells is flowed out by the upper end.The cell that is deposited in lower surface then slides to lower end outlet, Returning reactor.Problem and shortcoming that this patent mainly exists are as follows:
(1) continuity of operation is bad.When using this device, the nutrient solution that contains cell is flowed to the upper end by the lower end, the cell that is deposited on the lower shoe from up to down slides with Returning reactor, the sedimentation of cell is opposite with the liquid flow movement direction, the liquid flow path direction is opposite with cell return movement direction, has increased the residence time of cell in settling vessel.This device inventor provides two kinds of solutions, and the one, regularly close peristaltic pump to settling vessel lower end import transfusion, the liquid in the settling vessel stops to flow, and allows the cell that is collected at lower plate slide into the cell collection device of lower end naturally, makes its Returning reactor.The 2nd, settling vessel is the peristaltic pump counter-rotating simultaneously regularly at two ends up and down, and the liquid in the settling vessel is flowed to the lower end from the upper end, and flow direction is consistent with the cell slip direction, promotes that cell reclaims fast.These two kinds of methods all are to be cost to destroy the continuity subsiding movement of cell in settling vessel, have all broken the balance of system process.And gather through sedimentation after a while, base plate has deposited than many cells, allows it freely glide again, and sliding velocity is quite slowly.Even if adopt the method for recoil, effect neither be very big.Cell residence time in settling vessel, long cytoactive and the product ability to express of must causing descended, and the metabolic waste accumulation increases the culture environment in the deteriorative reaction device.
(2) carry the peristaltic pump of liquid because certain interval of time will be closed or reverse, must control automatically by the time of setting-up control device.The assembling of time-controlling arrangement has increased the complicacy of design and operation on the one hand, has also increased the manufacturing cost of equipment on the other hand.
(3) the settling vessel turndown ratio is little.In culturing process, suitably adjust irrigation rate according to the cell growing state, thereby require settling vessel that the elasticity of working is preferably arranged, promptly in certain scope, behind the liquid flow rate of change settling vessel, the treatment effect of settling vessel will be stablized.This settling vessel must influence its job stability after changing irrigation rate significantly, effect of settling and separating effect instability.
(4) in live cell trapping, also held back dead cell.The a large amount of accumulation of dead cell in reactor, severe exacerbation the viable cell culture environment.
Document Selective Recycle of Viable Animal Cells by Coupling ofAirlift Reactor and Cell Settler.Biotechnology and Bioengineering.1992, Vol.39,442-446. reported a kind of gravitational settler, the nutrient solution that contains cell is entered by settling vessel top, the back is by the lower port Returning reactor on the inclined-plane for cell settlement, and supernatant liquor is taken away by another outlet of top.Some problems and shortcoming that this settling vessel is had in having above-mentioned patent, a topmost defective is that settling area and settling vessel volume ratio are significantly less than the tilting settling vessel.The problem of bringing thus is that for finishing same sedimentation task, the settling vessel volume improves greatly with the cell culture reactor volume ratio.A large amount of relatively nutrient solutions and cell are trapped in the outer settling vessel of reactor, and it is very unfavorable that the normal growth of this pair cell and product are expressed, and the efficient of its separation dead cell is lower simultaneously.The effect of this settling vessel is lower than the tilting settling vessel of describing in the above-mentioned patent.Document Inclined Sedimentationfor Selective Retention of Viable Hybridomas in a ContinuousSuspension Bioreactor.Biotechnol.Prog.1990,6,458-464. and ViableCell Recycle with an Inclined Settler in the Perfusion Culture ofSuspended Recombinant Chinese Hamster Ovary Cells.Biotechnol.Prog.1994,10, also there be the defective identical with above-mentioned prior art in working method that 198-208. reported and settling vessel.
One of purpose of the present invention is to provide a kind of live cell trapping of zooblast continous pouring culturing process and method that dead cell is removed of being used for;
Two of purpose of the present invention is to provide a kind of live cell trapping of zooblast continous pouring culturing process and device that dead cell is removed of being used for.
Design of the present invention is such:
Because the difference of the dead zooblast size of living, both settling velocity in gravity field are also different, therefore, the nutrient solution that the contains cell upper port by tilting cell gravitational settler can be entered, and by regulating the position and the angle of inclination of upper port, make the cell in the nutrient solution under the different irrigation rates obtain best hold back and separating, most viable cell and small part dead cell Returning reactor, part dead cell and a small amount of viable cell flow out with supernatant liquor.
According to above-mentioned design, the contriver proposes to realize the technical scheme of the object of the invention by a large amount of experiments, and is specific as follows described:
The upper port that is had the tilting cell gravitational settler of a plurality of inlets by the next nutrient solution that contains cell of cell culture reactor from the upper end enters, in settling vessel, because the settling velocity of viable cell is bigger, overwhelming majority viable cell and part dead cell at first are deposited on the settling vessel lower surface, fluent with main body liquid to the bottom, flow out settling vessel by the viable cell outlet; Part is not finished settled dead cell and is then flowed out settling vessel by the dead cell outlet, thereby reach live, the holding back and separate of dead cell, the nutrient solution that contains the high density viable cell is flowed out by the viable cell outlet of settling vessel bottom, return cell culture reactor by peristaltic pump, dead cell stream then flows into the supernatant liquor collection container by the lower end outlet.
Irrigation rate is 0.2-10/ days; " 1/ day " expression every day perfusion is equivalent to the fresh medium of 1 reactor working volume, is the fresh medium that pours into 0.2 reactor working volume every day in 0.2/ day.
The position that the nutrient solution that contains cell enters tilting cell gravitational settler will change with irrigation rate, and flow is big more, and the position is high more, and generally speaking, irrigation rate doubles, and entrance location also doubles apart from the distance of bottom viable cell outlet.
Said cell is general suspension zooblast, as hybridoma;
Said nutrient solution be suitable for the zooblast suspension culture serum or serum free medium arranged, as the Hybridoma-SFM (1X) (Hybridoma Cell Culture serum free medium) of GIBCO company, the commodity sequence number is 12045-035.
This is held back sepn process and can carry out at normal temperatures.
Above-mentioned method is to finish in following tilting cell gravitational settler:
Said settling vessel is an inclination rectangular parallelepiped groove, and its top is provided with a plurality of nutrient solution inlets, and the bottom is provided with viable cell outlet and dead cell outlet, and said viable cell outlet is positioned at the upside of dead cell outlet, and spacing between the two is 1-10 centimetre;
Obliquity α is the 30-60 degree, length with inlet part is the 1/10-9/10 of total length, the equivalent diameter of rectangular parallelepiped and length ratio are 1/20-1/2, the cross section depth-width ratio is 1/10-1/2, equivalent diameter can define with following formula: equivalent diameter=∏/π, ∏ is the tetragon girth, and π is a pi;
Said cell gravitational settler is operation like this:
The nutrient solution that contains cell enters settling vessel by a certain inlet of the upper end of settling vessel, and in settling vessel, because the settling velocity of viable cell is bigger, most viable cell at first are deposited on the lower surface of settling vessel, flows out settling vessel by the viable cell outlet; Isolated dead cell then flows out settling vessel by dead cell outlet, thus reach live, the holding back and separate of dead cell
Fig. 1 is the structural representation of cell gravitational settler.
As seen from Figure 1, said settler is an inclination cuboid groove, and its top is provided with many Spacing between 1, two entrance 1 of individual nutrient solution entrance is 1-10 centimetre, and the bottom is provided with Living cells outlet 2 and dead cell outlet 3, said living cells outlet 2 is positioned at dead cell and goes out Mouthfuls 3 upside, the spacing of living cells outlet 2 and dead cell outlet 3 take 1-10 centimetre as Good.
Gradient α is the 30-60 degree, and the length L 1 with intake section is for total length L 1/10-9/10, the cross section depth-width ratio is 1/10-1/2, the equivalent diameter of cuboid and length Ratio be 1/20-1/2.
The nutrient solution that contains cell enters settler by a certain entrance 1 of the upper end of settler, In settler, living cells at first is deposited on the lower surface of settler, goes out by living cells Mouth 2 flows out settlers; Isolated dead cell then flows out settler by dead cell outlet 3, Thereby reach live, the holding back and separate of dead cell.
The said cell gravitational settler of the present invention has very significant advantage:
(1). the nutrient solution that contains cell is entered by the upper end of tilting settler, and it is from all On solved the problem that the deposition of cells adverse current is slided and is obstructed since in the settler liquid flow path direction and The deposition of cells glide direction is consistent, can promote cell to leave fast settler, thereby guarantees The operation continuity.
(2). owing to need not regularly stop or the peristaltic pump that reverse, reduced the device fabrication cost with The complicated operation degree.
(3). settler is provided with a plurality of nutrient solution imports, therefore except inclining by adjustment The angle is come outside the Adaptive change, can also be by selecting a conduct in numerous nutrient solution imports Feed pathway changes settling area, so this settler has bigger operating flexibility.
(4) efficiently solve the problem that dead cell constantly accumulates from cell culture reactor. Logical Overregulate the inclination angle and change entrance location, can find suitable operating point, hold back in assurance Under the prerequisite of living cells, constantly remove to greatest extent dead cell.
This settler can be by increasing area, the parallel connection of many monomers or the multiple-unit group of sedimentation face The mode that is integrated realizes amplifying, and is used for the cell retention of large volume cell culture reactor Separate with dead cell.
This settler is the behaviour according to the molecule characteristics of motion in the liquid and tilting settler Design for theoretical foundation as theoretical formula, be not limited only to move so it is held back with separating effect The thing cell culture system. It can be applied to other and the cultivation liquid phase that contains zooblast Similarly system is used for from the liquid-solid mixture separate solid particles, and can be with size Different particles separately.
Below will be further described content of the present invention by embodiment; but embodiment only is the supplementary notes of technical scheme; do not limit protection scope of the present invention; relevant technologies personnel fully can be according to design disclosed by the invention and technical scheme, is used for holding back and separating of all kinds ground suspension cell with drawing inferences about other cases from one instance.
Embodiment 1
This settling vessel makes up the perfusion culture that is used for hybridoma with 1.5 liters of stirring-type cell culture reactors.
The settling vessel structural parameter:
Settling vessel length: 30 centimetres, 4 centimetres of width, 3 centimetres of height have 12 imports, and each import spacing is 2 centimetres;
Cell: cell strain is hybridoma HB-58, and this cell strain is deposited in ATCC (U.S. histocyte storehouse), and preserving number is Hybridoma Cell Line 187.1 (ATCC HB58).Cell is kept cultivation in the 100ml square vase, square vase places 37 ℃ to contain 5%CO
2In the incubator, went down to posterity once in general per two days.Be inoculated in the reactor at the logarithm incubation period.
Substratum is the Hybridoma-SFM (1X) (Hybridoma Cell Culture serum free medium) of GIBCO company, and the commodity sequence number is 12,045 one 035.
Continous pouring was cultivated 50 days.Use is without spissated conventional substratum, and when irrigation rate was 1.5/ day, maximal cell concn reached 2.5 * 10
7Cells/ml, the live cell trapping rate is 99%, and the dead cell rejection is 68%, and reactor inner cell activity is 90%.
Cell density detection method: calculate viable cell density and cytoactive by Hematocyte Counter and the blue staining of platform dish.In the experiment every detected in 12 hours a cell culture reactor neutralization by the effusive supernatant liquor of dead cell spout in cell density and activity.
Claims (7)
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| CN00116518A CN1112442C (en) | 2000-06-14 | 2000-06-14 | Cell retaining and separating method and device for cell depositing culture |
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| CN00116518A CN1112442C (en) | 2000-06-14 | 2000-06-14 | Cell retaining and separating method and device for cell depositing culture |
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| CN1274752A CN1274752A (en) | 2000-11-29 |
| CN1112442C true CN1112442C (en) | 2003-06-25 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297659C (en) * | 2004-02-11 | 2007-01-31 | 上海伯瑞生物技术发展有限公司 | Method and device for perfusion and culture of hematopoietic cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CA2861270C (en) * | 2012-01-18 | 2021-08-03 | Bayer Healthcare Llc | Perfusion bioreactor systems comprising a cell aggregate trap and methods of operating the same |
| CN114599775A (en) * | 2019-11-07 | 2022-06-07 | 默克专利股份公司 | Methods and systems for performing perfusion cell culture |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5817505A (en) * | 1993-05-07 | 1998-10-06 | Bioscot Limited | Particle settler for use in cell culture |
| CN1249000A (en) * | 1997-01-24 | 2000-03-29 | 旭医学株式会社 | Method for separating cells |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5817505A (en) * | 1993-05-07 | 1998-10-06 | Bioscot Limited | Particle settler for use in cell culture |
| CN1249000A (en) * | 1997-01-24 | 2000-03-29 | 旭医学株式会社 | Method for separating cells |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1297659C (en) * | 2004-02-11 | 2007-01-31 | 上海伯瑞生物技术发展有限公司 | Method and device for perfusion and culture of hematopoietic cell |
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| CN1274752A (en) | 2000-11-29 |
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