CN1286966C - Cell depositing interception device of hematopoietic cell continuous perfusion suspended culture - Google Patents
Cell depositing interception device of hematopoietic cell continuous perfusion suspended culture Download PDFInfo
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- CN1286966C CN1286966C CN 200310109264 CN200310109264A CN1286966C CN 1286966 C CN1286966 C CN 1286966C CN 200310109264 CN200310109264 CN 200310109264 CN 200310109264 A CN200310109264 A CN 200310109264A CN 1286966 C CN1286966 C CN 1286966C
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Abstract
The present invention relates to a cell settlement trapping device used for a continuous perfusion culture bioreactor system of hematopoietic cells, which comprises an inclined tubular settlement device of which the cross section is rectangular, wherein the upper part of one end of the inclined tubular settlement device is provided with a feed port, the bottom of the settlement device corresponding to the position of the feed port is provided with a discharge port, and the upper bottom surface of the other end of the inclined tubular settlement device is provided with a liquid discharge port and an exhaust port. The cell settlement trapping device has the advantages that the problem that cells of a flat plate filling cavity or a fixed bed cultivation reactor are difficult to be recovered is avoided, the cells can be conveniently recovered, the failure problems of rotary silk screen filtration and membrane filtration blockage are avoided, and the cell settlement trapping device also solves the problem of poor biocompatibility of material utilized by other trapping devices. The inner part of the device comprises a perpendicular settling zone and an oblique settling zone and has the advantages both of the perpendicular settling zone and the oblique settling zone, the deposit of the cells on the bottom surface of the settlement device is reduced, and the average residence time of the cells outside the reactor in the trapping process is effectively shortened.
Description
Technical field
The present invention relates to a kind of cell retention device that is used for hematopoietic cell continous pouring culture bioreactors system, relate in particular to a kind of settling separation device that the continous pouring culturing process is held back hematopoietic cell that is used for.
Background technology
Umbilical hemopoietic stem cell is implanted in the treatment hematopoietic disorder, and there is good prospects for application the aspects such as recovery of hematopoietic potential after the malignant tumor patient radiotherapy.But because the source limitation of bleeding of the umbilicus, the umbilical hemopoietic stem cell quantity not sufficient has become the bottleneck that umbilical hemopoietic stem cell is transplanted research and clinical application, so that the research of the external extensive amplification of umbilical hemopoietic stem cell becomes is very urgent.The perfusion culture process can be brought in constant renewal in substratum because of it in culturing process, keep good culture environment, helps cell amplification, and is considered to realize the effective way of cell high-density culture.
Dull and stereotyped filling cavity is used to cultivate hematopoietic cell, and Koller etc. have developed the filling type culture system of no stroma cell, and the 17-19 that successfully CFU-GM increased doubly.This culture chamber comprises a plurality of microflutes (vertical with the direction of media flow), and it can provide the environment of homogeneous, allows nutraceutical diffusion, but cell is retained in the cavity, is difficult for reclaiming.Fixed-bed bioreactor also is a kind of design that realizes that the hematopoietic cell concern is cultivated, and generally also needs to adopt certain medium to come entrapped cell, the problem that exists cell to be difficult to reclaim equally.
The hematopoietic cell suspension culture has advantage at mass transfer and context of detection, and the effectively catching cell is the key issue of suspension perfusion culture reactor assembly exploitation in culturing process.It is cell retention device commonly used during zooblast (hybridoma, Chinese hamster ovary celI etc.) continous pouring is cultivated that rotary screen filters, this device is by rotating filter silk screen isolated cell and substratum, and the remarkable shortcoming of this method is that silk screen is blocked easily as filtration medium.Hematopoietic cell is compared with general zooblast (hybridoma, Chinese hamster ovary celI etc.), and the homogeneity of cell is relatively poor, so be difficult to determine the rotating speed of rotating filter silk screen.In addition, hematopoietic cell is comparatively responsive to shearing, and the high-shear meeting causes bigger cell injury, holds back so should not adopt rotary screen to filter.Membrane filtration, particularly crossflow membrane filtration also are used to isolated cell and substratum, but not only there is the problem of shearing than big and easily blocked equally in membrane filtration, and the biocompatibility of mould material and hematopoietic cell is relatively poor, so can cause bigger cell injury.
Settling process is not owing to exist the obstruction Problem of Failure, and also being applied to becoming is holding back of zooblast.Vertical cell settlement device often needs bigger cross-sectional area, thereby makes the vertical sedimentation device volume huge because of the flow velocity that must guarantee the settling vessel cross section and the make progress subsidence rate less than cell.In the cell retention process, a large amount of cell suspensions leave the culture space of reactor, add that the cell settlement subsidence rate is less, cause the mean residence time of cell outside reactor longer, are unfavorable for the normal growth of cell.Tilting cell settlement device utilizes the sedimentation effect of cell in slit, can make cell comparatively fast be deposited to the bottom surface, but the direction that cell refluxes in the operation of tilting settling vessel is opposite with main body mobile direction in the settling vessel, cause a large amount of cell depositions in the settling vessel bottom surface, be difficult to be back to reactor, have the long problem of the mean residence time of cell outside reactor equally.The accumulation of a large amount of cells can worsen the culture environment in the settling vessel, and the pair cell amplification is unfavorable.
Wang Zhaowei, Tan Wensong etc. (ZL 00116518.6) are though the settling vessel design that proposes can carry out hybridoma effectively or other one-tenth is holding back of zooblast, but because definite needs of this settling vessel operating parameters are determined critical subsidence rate, and hematopoietic cell is non-homogeneous cell mass, wherein there is significant difference in size between stem cell, progenitor cell and the mature cell, be difficult to determine critical subsidence rate, so operational difficulty.If hematopoietic cell is carried out cell when holding back entirely, because its sedimentation face length degree is big and the flow field is wayward etc., cell easily is deposited on the settling vessel bottom surface and can't refluxes smoothly, causes cell injury again.A series of experiments find that also this method is not suitable for the cell retention of hematopoietic cell continous pouring culturing process.
The size pair cell of cell settlement device is held back effect bigger influence.During the ratio of width to height in settling vessel cross section too little (＜1), cell suspension bigger difference will occur at the flow velocity of different radial heights, cause the overall flow of settling vessel internal flow, be unfavorable for the control of cell settlement process, the back-mixing that causes of whirlpool radially when serious can destroy the stationary state of whole settling process; During the ratio of width to height in settling vessel cross section too big (＞5), a large amount of cells in the suspension easily are deposited on the settling vessel bottom surface, are unfavorable for the recovery of cell.
The determining of settling vessel length designed by Model Calculation according to the average settlement speed of cell, and for hematopoietic cell, the too small cell retention ability that can weaken settling vessel of settling vessel length makes the individual less cell of part overflow with supernatant liquor; And settling vessel length is excessive, not only can strengthen the difficulty of processing and the material cost of settling vessel, and needs the longer system stability time in operation.So this patent, has been determined the ratio of width to height and the settling vessel long-width ratio comparatively optimized by a series of Model Calculation and experimental study.
Experiment shows, according to the sedimentation device of described size design processing, but the effectively catching hematopoietic cell, and obtained higher cell retention efficient.
Summary of the invention
The technical issues that need to address of the present invention provide a kind of cell settlement device for trapping that is used for hematopoietic cell continous pouring culture bioreactors system, energy effectively catching cell in hematopoietic cell perfusion culture system, and do not cause the hematopoietic cell device for trapping that damages, loses than maxicell.
Technical conceive of the present invention:
The particles settling operation often is applied to liquid-solid isolating chemical process.Nineteen twenty, doctor Boycotts has at first reported the sedimentation effect of hemocyte in slanted slots, many afterwards scholars have carried out deep theoretical investigation to the sedimentation phenomenon of tilting settling vessel, have proposed the theoretical calculation formula of upflowing inclination subsider working flow by mathematical model:
S(v)＝v?w(L?sinθ+b?cosθ)
Wherein be S (v) volumetric flow rate, v is a particles settling speed, L, w, b are respectively the lengths of subsider, θ is the inclination angle between subsider and the vertical direction.
Device disclosed by the invention at first makes most cells reflux fast through the vertical sedimentation district, utilize the Boycotts effect of hematopoietic cell again, make and trace back cell settlement to the bottom surface, supernatant liquor flows out from the overflow port of settling vessel upper end, hematopoietic cell in the effectively catching cell suspension, refluxing reactor; Be deposited on cell static cultivation in settling vessel of settling vessel bottom on a small quantity, reclaim with the nutrient solution flushing at last.
Technical scheme of the present invention:
A kind of cell settlement device for trapping that is used for continous pouring culture bioreactors system, comprise that tilting cross section is an orthogonal tubulose settling vessel, 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 leakage fluid dram and venting port, being the inclination negative area between opening for feed and the leakage fluid dram, is the vertical sedimentation district between opening for feed and the discharge port, and the effect of venting port is the air of getting rid of in the settling vessel.
Settling vessel can adopt the low toxicity bio-vitric to make, and settling vessel inside uses silication agent (dimethylsilane 2%/toluene) to carry out siliconizing before use.The width of tubulose settling vessel with the ratio of height is: width: highly=1.5～3: 1; Length is 15～30 times of width.
The cell suspension that bio-reactor comes flows into from opening for feed, and most of viable cell is in the sedimentation of vertical sedimentation district, and flows out from the bottom discharge port, is back to bio-reactor; Small amounts of cells upwards flows, in the inclination negative area; The sedimentation supernatant liquor is discharged from the leakage fluid dram on top, and the air before the work in the settling vessel is discharged from venting port.
The invention has the advantages that and avoided dull and stereotyped filling cavity or fixed bed to cultivate the problem that the reactor cell is difficult to reclaim, cell reclaims easily, avoided the obstruction Problem of Failure of rotary screen filtration and membrane filtration, solved the relatively poor problem of biocompatibility of other device for trapping material therefor, this device is inner to be made up of vertical sedimentation district and inclination negative area, have both advantages concurrently, reduced the deposition of cell, effectively shortened and held back the mean residence time of cell outside reactor in the process in the settling vessel bottom surface.Adopt the water-bath chuck can make the interior deposition of cells of settling vessel be in metastable environment, add that timing refluxes by making cell, can alleviate the cell injury that deposition causes.
Description of drawings
Fig. 1 is a cell settlement device for trapping structural representation.
Fig. 2 is a rectangle settling vessel cross-sectional view.
Fig. 3 be cell density over time.
Fig. 4 holds back effect for hematopoietic cell.
Fig. 5 is the Photomicrograph for supernatant liquor sampling and flushing backflow back settling vessel bottom surface.
Embodiment
A kind of cell settlement device for trapping that is used for continous pouring culture bioreactors system, comprise that tilting cross section is an orthogonal tubulose settling vessel 1, the top of one end is provided with opening for feed 2, settling vessel 1 is provided with discharge port 3 corresponding to the bottom of opening for feed 2 positions, its the other end is provided with the leakage fluid dram 4 and the venting port 5 of supernatant liquor, being the inclination negative area between opening for feed 2 and the leakage fluid dram 4, is the vertical sedimentation district between opening for feed 2 and the discharge port 3.
The width L of said orthogonal tubulose settling vessel 1 with the ratio of height H is:
Width: highly=1.5～3: 1;
The length of tubulose settling vessel 1 is 25～35 times of width;
Opening for feed 2, discharge port 3 is 40～50 ° with the angle of settling vessel 1 bottom surface, constitutes the vertical sedimentation district up and down, constitutes the inclination negative area between opening for feed 2 and the leakage fluid dram 4.
Further, outside settling vessel 1, be provided with chuck 6, be used for feeding insulation fluid (37C °), to alleviate the cell injury that small amounts of cells causes in settling vessel bottom surface deposition.
Cellular segregation and cultivation: separate to obtain mononuclearcell (MNCs) from fresh bleeding of the umbilicus (taking from healthcare hospital for women ﹠ children of China Welfare Institute), with IMDM substratum (the Gibco BRL that contains 20%FBS, USA) suspend again, add SCF, IL-3 and three kinds of cytokines of IL-6, its concentration is respectively: 50ng/ml, 20ng/ml and 40ng/ml, and with 1 * 10
6Cells/ml is seeded in the rolling bottle, places 37 ℃, 5%CO
2, moisture-saturated CO2gas incubator in cultivate standby.
Experimental technique: cell suspension opening for feed 2 from the sedimentation device bottom flows into, and flows out from the discharge port 3 of bottom lower surface, and clear liquid is discharged from the leakage fluid dram 4 that is positioned at the upper end, and the water-bath chuck passes to 37 ℃ of water insulations.
The result: investigated settling process and reached before stable, the change in cell density of leakage fluid dram 4 and discharge port 3 is as Fig. 3.As can be seen, settling process is about 50min steady time.After settling process was stable, it was density stabilized in lower level to overflow mouth cells.
Analyzed the cell retention effect of settling process, after settling process was stable, the situation of holding back of total cell and hold back ratio was as Fig. 4.Inlet flow rate can influence integral body to a certain extent and hold back effect, and when inlet flow rate is big (50mL/h), total cell density rejection is 91.88%, and inlet flow rate is hour (20mL/h), and total cell density rejection reaches 96.47%.
The Photomicrograph of deposition of cells in the settling vessel, the Photomicrograph of settling vessel bottom surface was seen Fig. 5 after supernatant liquor sampling and flushing refluxed.Among the figure, 31 for being deposited on the cell of bottom surface, the cell debris of 32 overflow ports, the settling vessel bottom surface after 33 flushings.This experimental result illustrates that this device can the effectively catching hematopoietic cell under certain experiment condition.
Claims (4)
1. one kind is used for suspend the continuously cell settlement device for trapping of perfusion culture bioreactor system of hematopoietic cell, it is characterized in that, comprise that tilting cross section is an orthogonal tubulose settling vessel (1), the top of one end is provided with opening for feed (2), settling vessel (1) is provided with discharge port (3) corresponding to the bottom of opening for feed (2) position, the upper bottom surface of its other end is provided with the leakage fluid dram (4) and the venting port (5) of supernatant liquor, the length of tubulose settling vessel (1) is 15～30 times of width, and the width of tubulose settling vessel with the ratio of height is: width: highly=1.5～3: 1;
Opening for feed (2), discharge port (3) is 40～50 ° with the angle of settling vessel (1) bottom surface.
2. the cell settlement device for trapping of continous pouring culture bioreactors according to claim 1 system is characterized in that, the outer water-bath chuck (6) that is provided with of settling vessel (1).
3. the cell settlement device for trapping of continous pouring culture bioreactors according to claim 1 system is characterized in that, settling vessel adopts the low toxicity bio-vitric to make, and settling vessel inside is through silication liquid siliconizing.
4. the cell settlement device for trapping of continous pouring culture bioreactors according to claim 1 system is characterized in that the angle of tubulose settling vessel and vertical direction is 40～50 °.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200310109264 CN1286966C (en) | 2003-12-10 | 2003-12-10 | Cell depositing interception device of hematopoietic cell continuous perfusion suspended culture |
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| CN 200310109264 CN1286966C (en) | 2003-12-10 | 2003-12-10 | Cell depositing interception device of hematopoietic cell continuous perfusion suspended culture |
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| CN1286966C true CN1286966C (en) | 2006-11-29 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE102008029307A1 (en) * | 2008-06-20 | 2009-12-24 | Bayer Technology Services Gmbh | Method and device for retention and return of cells |
| CN101805695B (en) * | 2010-01-29 | 2012-06-13 | 福尔生物制药股份有限公司 | Oblique rotational gravity mixed biological reaction equipment |
| CN101805694B (en) * | 2010-01-29 | 2012-07-04 | 福尔生物制药股份有限公司 | Gravity mixing bioreactor obliquely rotating along central shaft of culture vessel |
| DK2817406T3 (en) * | 2012-02-20 | 2019-12-02 | Bayer Ag | Disposable Separator for Retention and Cell Return |
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