CN100469863C - Inclined Gravity Settling Live Cell Retention Device - Google Patents
Inclined Gravity Settling Live Cell Retention Device Download PDFInfo
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- CN100469863C CN100469863C CNB2006101184050A CN200610118405A CN100469863C CN 100469863 C CN100469863 C CN 100469863C CN B2006101184050 A CNB2006101184050 A CN B2006101184050A CN 200610118405 A CN200610118405 A CN 200610118405A CN 100469863 C CN100469863 C CN 100469863C
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Abstract
本发明公开了一种倾斜式重力沉降活细胞截留装置，包括斜置的沉降器主体(1)、进料口(2)、收液口(3)和回流口(4)，其特征在于，还包括肋条(6)；所说的沉降器主体(1)的横截面为椭圆形；进料口(2)设置在主体(1)的上端，收液口(3)设置在主体(1)中段的背部；回流口(4)设置在主体(1)的下端；所说的肋条(6)设置在主体(1)内表面(5)的底部。本发明的截留装置适用于不同反应器，灌注速率为范围很广。本发明减少了细胞在沉降器底面的沉积，有效缩短了细胞在反应器外的平均停留时间，缓解了因沉积细胞引起的整体活性下降，反应器环境恶化的问题。
The invention discloses an inclined gravity sedimentation live cell retaining device, which comprises an inclined settler main body (1), a feed port (2), a liquid collection port (3) and a return port (4), and is characterized in that, It also includes ribs (6); the cross section of the settler main body (1) is elliptical; the feed inlet (2) is arranged on the upper end of the main body (1), and the liquid receiving port (3) is arranged on the main body (1) The back of the middle section; the return port (4) is arranged at the lower end of the main body (1); the said rib (6) is arranged at the bottom of the inner surface (5) of the main body (1). The trapping device of the present invention is suitable for use in different reactors with a wide range of perfusion rates. The invention reduces the deposition of cells on the bottom surface of the settler, effectively shortens the average residence time of cells outside the reactor, and alleviates the problems of decreased overall activity and deterioration of the reactor environment caused by deposited cells.
Description
Technical field
The present invention relates to a kind of inclined type gravity precipitation live cell cut-off equipment, relate in particular to a kind of inclined type gravity precipitation device that is used for holding back viable cell in cell continous pouring culturing process selectivity.
Background technology
The perfusion culture mode is to improve the effective ways of cell density.The superiority of this training method is constantly to mend fresh culture under the prerequisite of nutrient solution constancy of volume, discharges metabolite, makes cell growth and maintenance high reactivity fast, thereby realizes the high expression level and the high yield of product.Exploitation cell cut-off equipment, efficient entrapped cell are to implement the critical technical problems that continous pouring is cultivated.
In numerous cell retention devices, the inclined type gravity precipitation device is simple in structure because of it, and cost is low, does not have and stops up, and pair cell is sheared little, and advantages such as easy amplification are considered to a kind of method of cell retention preferably.At present, existing many patent reports be used for the gravity settling cell retention separation method and the device of cell continous pouring culturing process, as: Wang Zhaowei, a kind of dirty tilting cell gravitational settler that Tan Wensong (ZL00116518.6) proposes, this settling vessel is one to contain the rectangular parallelepiped groove of a plurality of inlets, the nutrient solution that contains cell enters settling vessel by a certain inlet of the upper end of settling vessel, wherein most of cell at first is deposited on the lower surface of settling vessel, flow out settling vessel by refluxing opening, Returning reactor, the supernatant liquor that contains small amounts of cells fragment and dead cell is flowed out by liquid receiving mouth.The problem that this patent mainly exists is as follows:
(1) settling vessel to hold back treatment capacity little.When using this device, because refluxing opening is opened in the middle of the lower surface of settling vessel, in order to make viable cell as much as possible flow back to reactor, most of viable cell must be deposited to the lower surface of settling vessel before refluxing opening, otherwise will flow into the supernatant liquor collection container by the lower end liquid receiving mouth, and cell settlement is to the time of lower surface, be that the vertical movement of cell in settling vessel is chronic, equal according to cell tangential movement time in settling vessel with the vertical movement time, must cause the inlet flow rate of settling vessel slower, and the flow of liquid receiving mouth is less than inlet flow rate, and promptly settling vessel holds back treatment capacity and will be restricted.Mention settling vessel in this patent and be provided with a plurality of nutrient solution imports, reach the purpose that treatment capacity is held back in increase by the import that promptly extends of the position of adjusting import to the distance of refluxing opening, this also reflects its limitation from another angle, the length of holding back treatment capacity and settling vessel is directly proportional, and this obviously is a method of curing the symptoms, not the disease.
(2) residence time of cell in settling vessel long, cause the reactor environmental degradation.In order to make most of viable cell from the refluxing opening Returning reactor, can only allow cell settlement to bottom surface, slide to refluxing opening with main body liquid again by the inlet flow rate of the settling vessel that slows down.This has just brought two problems: the one, behind the inlet flow rate of the settling vessel that slowed down, cell flows out by settling vessel from reactor and is trapped again through the just prolongation of the time of refluxing opening Returning reactor, and the temperature remains within the normal range in the settling vessel, and too high or too low envrionment temperature causes cell injury easily; The 2nd, cell is settled down to lower surface earlier, slide to refluxing opening with main body liquid then, in order to guarantee that most of viable cell can both be deposited to the bottom surface, mainbody flow speed must slow down, the cell that is deposited to lower surface so can only be lentamente with sliding to refluxing opening under the main body liquid, even the part cell can be deposited on the settling vessel bottom surface, is difficult to be back to reactor, accumulated a large amount of cells in the final settlement device.These two problems make and cause cytoactive and product ability to express to descend by cell overstand in settling vessel that the metabolic waste accumulation increases the culture environment in the deteriorative reaction device.
(3) shape of cross section of settling vessel is unreasonable.The settling vessel that proposes in this patent is an inclination rectangular parallelepiped groove, the contriver is by a large amount of experiments and computer simulation, discovery adopts the interior flow field of the settling vessel of rectangular cross section unreasonable, because mainbody flow speed is unhappy, there is the dead band, flow field near the wall around the settling vessel, a large amount of cells are outstanding to stagnate in the dead band, causes cell mean residence time outside reactor long, causes cell injury.
Tan Wensong, Jiang Hua, Cai Haibo, occupy (ZL200310109264.2) late and reported a kind of cell gravity settling device for trapping that is used for hematopoietic cell continous pouring culture bioreactors system, tilting cross section is an orthogonal tubulose settling vessel, and the top of one end is provided with opening for feed, settling vessel is provided with discharge port corresponding to the bottom of opening for feed position, its the other end is provided with venting port and leakage fluid dram, is the inclination negative area to be the vertical sedimentation district between opening for feed and the discharge port between opening for feed and the leakage fluid dram.Some problems and shortcoming that this settling vessel is had in having above-mentioned patent, also some defectives of Cun Zaiing are:
(1) continuity of operation is bad.When using this device, settling vessel bottom can deposition part cell, need reclaim with the nutrient solution flushing, and this method is to be cost to destroy the continuity of cell in settling vessel, has broken the balance of system process.
(2) device is complicated, the cost height.There is cell long problem of the residence time in settling vessel equally in this device, propose to adopt the water-bath chuck to make the interior deposition of cells of settling vessel be in metastable environment, thereby alleviate the cell injury that deposition causes, but this method has improved the cost of sedimentation device, increased the complicacy of operation.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of dirty inclined type gravity precipitation live cell trapping device of the continuous long-term perfusion culturing process of zooblast that is used for, it is long to solve viable cell mean residence time in settling vessel, causes the problem of cell injury, reactor environmental degradation.
Technical conceive of the present invention:
The contriver finds that by a large amount of experiments and CFD computer simulation there is certain thickness frictional belt in settling vessel inside, and cell is detained in the frictional belt and the wall adherent cell is the major cause that causes cell overstand in settling vessel.
Solve the problem that cell is detained in the frictional belt, will reduce the sphere of action in frictional belt exactly, mainly carry out from the following aspects:
(1) improves the position of refluxing opening and liquid receiving mouth, reduce the thickness in settling vessel upper part frictional belt, also improved the treatment capacity of sedimentation device simultaneously.In the patent of ZL00116518.6, refluxing opening and liquid receiving mouth are all opened below settling vessel, and cell suspension only flows in lower part of settling vessel, and upper part is the dead band substantially entirely, and a large amount of cells are outstanding to stagnate in this zone.And for major part being entered the live cell trapping Returning reactor of settling vessel, the solution of taking is to allow the lower surface of cell deposition to settling vessel in stream of cells before refluxing opening, otherwise can flow into the supernatant liquor collection container by the lower end liquid receiving mouth, and the settling velocity of cell in gravity field is certain, this has just brought a contradiction: improve the viable cell recurrence rate, just need to reduce mainbody flow speed, the treatment capacity of settling vessel will be restricted; Cell can deposit in a large number at the bottom surface of settling vessel under low mainbody flow speed simultaneously, and the prolongation of cell mean residence time causes cell injury.
Can solve this contradiction by the position that changes refluxing opening and liquid receiving mouth, the liquid receiving mouth position transforms to back, device stage casing by the terminal bottom of device, the refluxing opening position transforms to terminal bottom by bottom, device stage casing, after the nutrient solution that contains cell like this enters settling vessel, cell is through after a while sedimentation, dead cell that settling velocity is low and fragment flow into the supernatant collection container from the liquid receiving mouth at settling vessel back, the high viable cell of settling velocity is from the refluxing opening Returning reactor of bottom, adopt this design, not only solved the blocked up problem in settling vessel upper part frictional belt, and behind the raising mainbody flow speed, even viable cell all is not settled down to lower surface and can returns from refluxing opening yet, when having improved the treatment capacity of sedimentation device, the high recurrence rate of viable cell also can be guaranteed.
(2) improve the shape of cross section of settling vessel.In above-mentioned patent, the cross section of settling vessel all adopts rectangular configuration, the contriver carries out computer simulation by a large amount of experiments and Fluid Mechanics Computation CFD technology, the low-speed region scope is big in the settling vessel flow field of discovery employing rectangular cross section, and after interface structure changes semicircular arc into, main body mobile range of influence increases, and the low regime scope reduces greatly.
(3) the more place of device bottom surface cell adhesion amount adds rib.The effect of rib is to form the quasi-ordering whirlpool near wall, near the cell rolling off wall the wall can be reduced the contact probability of cell and bottom wall.This quasi-ordering whirlpool sphere of action is limited simultaneously, can not influence the efficient of whole device.
Solving the problem of wall adherent cell, is by modification makes cell be difficult for adhering to wall, mainly contains following two methods: one, wall hydrophobization.Because the bottom surface of settling vessel is to be made by wetting ability glass, if being coated with hydrophobic membrane, the surface modifies, can reduce the cell adhesion amount of bottom surface.Two, wall nanometer.As lived particulate matter, cell surface also has electric charge.After the processing of wall nanometer, the wall release electrostatic contacts with the direct of wall to reduce cell.
According to above-mentioned technical conceive, the technical scheme that the present invention proposes is as follows:
A kind of inclined type gravity precipitation cell retention device that is used for continous pouring culture bioreactors system comprises: settling vessel main body, opening for feed, liquid receiving mouth and refluxing opening, it is characterized in that, and also comprise rib;
The cross section of said settling vessel main body is oval;
Opening for feed is arranged on the upper end of main body;
Liquid receiving mouth is arranged on the back in main body stage casing;
Refluxing opening is arranged on the lower end of main body;
Said rib is arranged on the body inner surface bottom;
Inclined type gravity precipitation live cell trapping device of the present invention is to use like this:
The cell suspension that bio-reactor comes enters settling vessel from opening for feed, because the settling velocity of viable cell is bigger than the settling velocity of dead cell and cell debris, overwhelming majority viable cell can be enriched in lower part of settling vessel gradually, mainly be dead cell and cell debris in upper part of settling vessel, can flow out by liquid receiving mouth; All the other viable cell return bio-reactor from refluxing opening, reach the purpose of holding back viable cell.
The invention has the advantages that:
(1) settling vessel is applied widely.Owing to do not exist most of viable cell must be settled down to could reflux the back problem of reactor of lower surface, so the adjustable wide range of the inlet flow rate of settling vessel, the corresponding adjustable scope of liquid receiving mouth flow (being the fresh culture groundwater increment of bio-reactor) is also very wide.
(2) the cell deposition amount in the settling vessel is few.One, make the cross section of settling vessel into ellipse after, improved the flow field situation in the settling vessel, the dead band significantly reduces; Two, changed the position of refluxing opening and liquid receiving mouth after, can strengthen the stagnation that mainbody flow speed reduces cell, guarantee the high viable cell recurrence rate of settling vessel; Three, the settling vessel bottom surface applies rib, causes fluid to form the quasi-ordering whirlpool, and this quasi-ordering whirlpool can be with near the cell rolling off wall the wall; Four, wall being carried out hydrophobic treatment and nanometer handles to reduce the contact probability of cell and wall.These methods have reduced deposition and the adhesion of cell in the settling vessel bottom surface, have effectively shortened the mean residence time of cell outside reactor, have alleviated the overall activity that causes because of deposition of cells and have descended the problem of reactor environmental degradation.
(3) life cycle is long.By structure parameter optimizing, operation parameter optimization, wall hydrophobization and nanometer remodeling, apply bottom surface rib etc., avoid the delay of cell in sedimentation device, can guarantee the long-term stability operation of whole continous pouring system.
Description of drawings
Fig. 1 is a structural representation of the present invention.
Fig. 2 is oval settling vessel cross-sectional view.
Embodiment
Referring to Fig. 1 and Fig. 2, inclined type gravity precipitation cell retention device of the present invention comprises:
Comprise: settling vessel main body 1, opening for feed 2, liquid receiving mouth 3 and refluxing opening 4, it is characterized in that, also comprise rib 6;
The cross section of said settling vessel main body 1 is oval;
Opening for feed 2 is arranged on the upper end of main body 1, and quantity is one;
Liquid receiving mouth 3 is arranged on the back in main body 1 stage casing;
Refluxing opening 4 is arranged on the lower end of main body 1;
Said rib 6 is arranged on the bottom of main body 1 internal surface 5;
Liquid receiving mouth 3 is 40～50 ° with the angle α of main body 1 bottom surface, preferred 45 °;
During use, main body 1 is tilting, is 45-60 ° with the angle β of horizontal direction.
The material of said main body is preferably the low toxicity bio-vitric, and the trade mark of producing as Jiadingtaihe Glass Factory, Shanghai is the product of 95 material (B-40), and glass surface is handled through hydrophobic treatment and nanometer.
The method of glass surface hydrophobic treatment can adopt Zhang Qingyong, and Wang Haozai (sol-gel processing prepares the research of organic/inorganic composite hydrophobic film) document disclosed method comprises the steps:
Ptfe emulsion, distilled water and dispersion agent, tensio-active agent are mixed magnetic agitation become white emulsion, added the silicon sol magnetic agitation 3 hours, adding ammoniacal liquor magnetic agitation seals ageing and obtains colloidal sol after 24 hours after the emulsion that is white in color after half an hour.The glass surface plated film is adopted crystal pulling method, and speed is 24cm/min.Then glass is put into baking oven thermal treatment, till surperficial colloidal sol solidifies.
The method that the nanometer of glass surface is handled can adopt Li Ling in (research of nanometer SiO2/ polyacrylic ester composite membrane antistatic property) document disclosed method, comprises the steps:
Take by weighing nanometer SiO
2Powder, the massfraction by 25% mixes with polyacrylate dispersion, adds a little defoamer, put into the ball mill ball milling after 5 minutes, be poured on the sheet glass (sheet glass cleans the back oven dry through liquid detergent and handles), coating or hydrostomia film forming, oven dry or dry naturally after get final product.
Embodiment 1
This settling vessel makes up the perfusion culture that is used for hybridoma with 1L stirring-type cell culture reactor.
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 300ml rolling bottle, rolling bottle places 37 ℃ to contain 5% CO
2In the incubator, 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 12045-035.
Liquid receiving mouth 3 is 45 ° with the angle α of main body 1 bottom surface;
During use, main body 1 is tilting, with the angle β of horizontal direction be 50 °
Continous pouring was cultivated 60 days.Use the substratum (glucose concn 7mM, glutamine 8.5mM) of glucose limitation, when irrigation rate was 1/ day, maximal cell concn reached 1.3 * 10
7Cells/ml, the live cell trapping rate is 99%, and the dead cell rejection is 50%, and reactor inner cell activity is 95%.
Cell density detection method: calculate viable cell density and cytoactive by Hematocyte Counter and the blue staining of platform dish.Detected a cell culture reactor neutralization by effusive cell density of liquid receiving mouth and activity every 12 hours in the experiment.
Claims (5)
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006101184050A CN100469863C (en) | 2006-11-16 | 2006-11-16 | Inclined Gravity Settling Live Cell Retention Device |
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| Application Number | Priority Date | Filing Date | Title |
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| CNB2006101184050A CN100469863C (en) | 2006-11-16 | 2006-11-16 | Inclined Gravity Settling Live Cell Retention Device |
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| CN1966659A CN1966659A (en) | 2007-05-23 |
| CN100469863C true CN100469863C (en) | 2009-03-18 |
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| AU2013224132B2 (en) * | 2012-02-20 | 2017-10-12 | Bayer Aktiengesellschaft | One-way separator for retaining and recirculating cells |
| CN103436491B (en) * | 2013-09-05 | 2015-10-28 | 东南大学 | A kind of based on the cell density at the bottom of insect wing base and the method arranging controlled cultivation |
| CN105602825B (en) * | 2014-11-21 | 2018-12-07 | 上海保兴生物设备工程有限公司 | Cell culture sedimentation device |
| EP3561038A1 (en) * | 2018-04-24 | 2019-10-30 | Bayer AG | Cell separator |
| CN112391279A (en) * | 2020-11-30 | 2021-02-23 | 江苏信息职业技术学院 | Device structure for capturing circulating tumor cells and manufacturing method thereof |
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