CN1261560C - Bioreactor of in vitro stem cell culture - Google Patents
Bioreactor of in vitro stem cell culture Download PDFInfo
- Publication number
- CN1261560C CN1261560C CN 200310109169 CN200310109169A CN1261560C CN 1261560 C CN1261560 C CN 1261560C CN 200310109169 CN200310109169 CN 200310109169 CN 200310109169 A CN200310109169 A CN 200310109169A CN 1261560 C CN1261560 C CN 1261560C
- Authority
- CN
- China
- Prior art keywords
- gas
- reactor
- stem cell
- capping
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The present invention discloses a biologic reactor system for stem cell in-vitro culture, which at least comprises a reactor, a stirring device, a mixed gas and alkali liquid inlet, a mixed gas outlet, a ventilation pipe provided with an upper part gas outlet and a lower part gas outlet, a culture liquid inlet, a culture liquid outlet, a cell back flow inlet, a cell trapped apparatus, a pH electrode, a dissolved oxygen concentration electrode and a control device, wherein the ventilation pipe is communicated with the mixed gas outlet. The biologic reactor of the present invention can simulate in-vivo environment in the stem cell in-vitro culture, the operating flexibility of the reactor is large, the system changes the partial pressure of oxygen in total inlet gas by adjusting nitrogen or the oxygen, and the pH value and the DO level of the environment of the culture are stable.
Description
Technical field
The present invention relates to a kind of bioreactor system, relate in particular to a kind of bioreactor system that is used for the stem cell vitro culture.
Background technology
Vitro culture of stem cell and amplification are one of the emphasis of stem cell biological engineering and difficult point, and to realize that stem cell induces in external growth, amplification and directed differentiation, then must wherein can satisfy stem cell and cultivate the basis that the bioreactor system that requires is realization this purpose for it provides the ideal culture environment.Stem cell is the cell that a class has self and differentiation potential, in a sense, life entity is the renewal and lasting growth of realizing cells in vivo by the division of stem cell, the continuous generation of new noble cells and form and splitted cell or tissue again itself by noble cells, all will be by keeping the quantity of body cell by the cell that stem cell produced with differentiation capability, so stem cell decisive role in life entity.
Discover that the hESC can be in vitro culture, adult stem cell also can be at external cell and the tissue that laterally is divided into other type, and this just provides the foundation for Application of stem cells.Fast development along with various biotechnologys such as genetically engineered, embryo engineering, cell engineerings, become possibility in in-vitro separation, culturing stem cells, and can utilize stem cell further to make up various cells, tissue, organ etc., with this source as transplanted tissue and organ, this is the main direction that stem cell is used, and utilizes the technology of tissues such as stem cell generation or reparation hematopoiesis, skin, nerve, cartilage all to obtain certain success at present.
But the number of stem cell seldom, need breed it external.In vivo, growth of stem cell and propagation are subjected to the influence of number of mechanisms and microenvironment.When vitro culture, need carry out the common cultivation of stem cell and helpers such as all kinds of somatomedin and mesenchymal cell equally; And the stem cell of different tissue sources, its culture condition also is not quite similar, and need study respectively; In addition, before application, need carry out directed differentiation to institute's culturing stem cells according to the type of target tissue and induce, it is broken up to required cell or tissue direction.These processes all require detailed research is carried out in the influence of conditioning signal relevant with development of stem cells and microenvironment, on this basis, simulate microenvironment in its body, provide the comparatively ideal condition of growing, obtain required cell or tissue product with cultivation to stem cell.
The difficult point of stem cell vitro culture is the comparatively small amt of stem cell own, accommodative ability of environment is poor, its regulatory mechanism that grows is also complicated, therefore, need advanced culture apparatus, so that the stem cell that its separation obtains can fast adaptation culture environment, and require in this device, can carry out easily the operations of microenvironment in the analogue body, satisfy the needs that stem cell growth is grown.
Cultivate for stem cell, forefathers had also once developed the bioreactor system of some static cultivation, the flat bio-reactor that hemopoietic stem cell is cultivated that is used for as the exploitation of U.S. Aastrom company, but because culture environment heterogeneity, there are concentration gradient in pH in culture medium, dissolved oxygen, nutrition and metabolite, and this concentration gradient is also can be owing to the amplification degree of distribution, medium component and the culture sample of starting condition such as inoculum density, cell type different and cause very big difference between each batch cultivation.And static system does not have turndown ratio, can not realize dimensional culture, be subjected to the restriction of floorage, cell proliferation during to higher density cell do not have the growth of enough spaces, but mutual superposition is on the bottom surface, therefore cause the restriction of growth, and can not cultivate a large amount of cells.The problem of the cell harvesting difficulty that run in actual application of static cultivation system can not effectively be solved for the moment in addition.
And on the other hand, present existing stirring type bioreactor generally all is to be cell design at one-tenth, and is not suitable for the cultivation of stem cell, and its weak point mainly is:
(1) reaches requirement mixed effect and make the stirring intensity of cell suspension higher, generally at least more than 50rpm, and the deep layer bubbling is a ventilating mode commonly used, but because stem cell likens to being that cell is more responsive to shearing force, so these reactors are applied to the stem cell cultivation and tend to cause cell impaired and can not normal growth;
(2) stem cell will experience the adaptive process from internal milieu to external environment in the vitro culture process, therefore cell itself is higher to the biocompatibility requirement of material, and the used material of general bio-reactor is the cell design at one-tenth just, particular requirement for stem cell considers that cultivation results is unsatisfactory;
(3) volume of general bio-reactor is more than 1.5 liters, and its minimum working volume is at least also wanted more than the 500ml, and its turndown ratio can not satisfy the stem cell culture requirement.One-tenth is to inoculate to reactor after cell generally can be cultivated by seed cell, does not have the very few problem of seed cell quantity.But concerning stem cell, its source and quantity are very limited, once gather and purifying after the cell quantity wretched insufficiency that obtains, if the initial incubation volume is excessive when inoculating, then inoculum density can be low excessively, is unfavorable for the existence and the growth of cell;
It is (4) existing that to be applied to become be the bio-reactor of cell cultures, when control pH and dissolved oxygen (DO) concentration, adopt carbonic acid gas, oxygen, nitrogen independence ram charging to remerge the mode that enters reactor, can not consider phase mutual interference between gas to general animal cell culture process, but because gas flow is less in the stem cell culturing process, phase mutual interference meeting between air inlet causes the big ups and downs of each partial pressure, and then cause fluctuating widely of pH and DO, have a strong impact on the stable and homogeneous of stem cell culture environment.
Summary of the invention
The technical issues that need to address of the present invention are to disclose a kind of bioreactor system that is used for the stem cell vitro culture, to overcome the above-mentioned defective that prior art exists, satisfy the needs of biotechnology development.
Technical scheme of the present invention:
A kind of bioreactor system that is used for the stem cell vitro culture comprises at least:
A cylindric upper end has the cylindrical reactor of capping, and aspect ratio is 2～4: 1;
A whipping appts that is arranged in the described reactor;
A mixed gas and an alkali liquor inlet that is arranged in the capping;
A mixed gas outlet that is arranged in the capping;
One is arranged on the ventpipe that is provided with air outlet, top and air outlet, bottom in the reactor and is connected with said mixed gas outlet;
A nutrient solution inlet that is arranged in the capping;
A nutrient solution outlet that is arranged in the capping;
A cell reflux inlet that is arranged in the capping;
One is provided with the cell retention device that purified liquor outlet and inlet are connected with said nutrient solution outlet, and the outlet of trap is connected with the cell reflux inlet;
One is arranged on pH electrode in the reactor by capping;
One is arranged on dissolved oxygen (DO) concentration electrode in the reactor by capping;
A control device that is connected with the oxyty electrode with pH electrode and is used to control the alkali lye and the gas mixture scale of construction.
Said mixed gas comprises CO
2, O
2, N
2And air.
Further, the agitating vane of said whipping appts can adopt 20～40 ° of oblique leaf hoisting type agitating vanes, and diameter of propeller blade reaches 3～4: 5 with the reactor diameter ratio, and the good mixing effect is arranged when guaranteeing the slow speed of revolution, effectively suspension cell, and reduction fluid shearing.
Reactor of the present invention is operation like this:
Start whipping appts, adjust the range of speed control, make the reactor can steady running between 1-40rpm.Nutrient solution is sent into the reactor that presets cell continuously by the nutrient solution inlet, with the extra-nutrition thing, mixed gas is sent into the upper and lower of the reactor that presets cell continuously by mixed gas and alkali liquor inlet, mixed gas is discharged reactor by mixed gas outlet, the nutrient solution that contains cell enters the cell retention device by the nutrient solution outlet, viable cell and nutrient solution reflux by the cell reflux inlet and enter reactor, metabolic by-prods is discharged by cell retention device purified liquor outlet, pH electrode and the measured signal input control device of oxyty electrode, input and ratio with the control mixed gas, and control the alkali lye input simultaneously, with the pH value of nutrient solution in the conditioned reaction device.
By above-mentioned disclosed technical scheme as seen, bio-reactor of the present invention is when the stem cell vitro culture, the correlation parameter of culturing process such as pH, oxyty adopts the Automatic Detection and Control technology, help realizing vitro culture process optimization and simulated in vivo environment, stem cell replaces the static or adherent culture of tradition in the suspension culture mode, be convenient to cell harvesting and the homogeneous of keeping culture environment, help detection, control and the optimization of parameter; The present invention is directed to the shearing sensibility of stem cell, particular design has been carried out in ventilation and stirring system, wherein the mode that surface ventilation and deep layer bubbling combine is adopted in ventilation, stirring rake adopts 30 ° of oblique leaf hoisting type stirring rakes, and diameter of propeller blade reaches 4/5 with the reactor diameter ratio, the good mixing effect is arranged, effectively suspension cell, and reduction fluid shearing when guaranteeing the slow speed of revolution.At the insufficient problem of source of human stem cell limited quantity, bigger aspect ratio is adopted in reactor tank body design, reaches 3/1, improves turndown ratio greatly, makes the operating restraint of reactor working volume reach 0.1 and rises to 1.2 liters.When initial incubation,, can adopt small volume, make cell at shortest time endoadaptation vitro culture environment to improve cell inoculation density because stem cell population is on the low side.Then along with the raising of cell growth and cell density and nutrition consumption and metabolic by-prods knot tire out, the mode that can adopt medium flow the to add thing that supplements the nutrients, diluting cells density and metabolic by-prods concentration make volume of culture bring up to the reactor working volume simultaneously.The stem cell culturing process can realize the substratum continous pouring in this bio-reactor, and replacement is existing intermittently changes the liquid mode, helps the interior microenvironment of stable and analogue body of cell culture environment;
Aspect pH and the control of dissolved oxygen (DO) concentration, at the stem cell characteristics very responsive to the culture environment parameter fluctuation, not only independent but also relevant Controlling System designs as two with pH and DO control, though they have separately independently controlled target (being pH value and the dissolved oxygen level in the culturing process), owing to the phase mutual interference between four gases (air, nitrogen, oxygen and carbonic acid gas) makes interrelated again between the two.This reactor has adopted the related control design with DO four gases of novel pH for this reason, change the partial pressure ratio of oxygen and carbon dioxide in total air inlet by the charge flow rate of regulating air, nitrogen, oxygen and carbonic acid gas four gases respectively, to realize accurately controlling the purpose of pH and DO.When system changed in total air inlet oxygen partial pressure by regulating nitrogen or oxygen, the partial pressure of carbon dioxide that system can automatic correction causes thus changed, and then the pH value (± 0.02) of stable culture environment; When system changed the dividing potential drop of carbonic acid gas in total air inlet by regulating carbon dioxide, system also can revise the oxygen partial pressure that causes thus automatically and change, and makes culture environment DO level realize stable (± 1%).
Description of drawings
Fig. 1 is the structural representation that is used for the bioreactor system of stem cell vitro culture.
Fig. 2 control device structural representation.
Referring to Fig. 1, the present invention is used for the bioreactor system of stem cell vitro culture, comprising:
A cylindric upper end has the cylindrical reactor 2 of capping 1, and aspect ratio is 2～4: 1, and preferred aspect ratio is 3: 1;
A whipping appts 3 that is arranged in the described reactor 2;
A mixed gas and an alkali liquor inlet 4 that is arranged in the capping 1;
A mixed gas outlet 5 that is arranged in the capping 1;
One is arranged on the ventpipe 8 that is provided with air outlet, top 6 and air outlet, bottom 7 in the reactor 2 and is connected with said mixed gas outlet 5;
A nutrient solution inlet 9 that is arranged in the capping 1;
A nutrient solution outlet 10 that is arranged in the capping 1;
A cell reflux inlet 11 that is arranged in the capping 1;
One be provided with purified liquor outlet 12 and enter the mouth 13 with the said nutrient solution outlet 10 cell retention devices 14 that are connected, the outlet 15 of trap 14 is connected with cell reflux inlet 11, this trap 14 is the device of a kind of this area routine, as adopting the technology of " Wang Zhaowei, Tan Wensong: cell retention separation method and device thereof in the ZL00116518.6 cell perfusion culture process " patent disclosure;
One is arranged on pH electrode 16 in the reactor 2 by capping 1;
One is arranged on oxyty electrode 17 in the reactor 2 by capping 1;
A control device 18 that is connected with oxyty electrode 17 with pH electrode 16, the output terminal of control device 18 respectively with lye pump 22, CO
2CO on the source of the gas 23
2Air flow control valve, N on flowrate control valve, the air source of the gas 24
2N on the gas source of the gas 25
2Airshed control valve and O
2O on the gas source of the gas 26
2The airshed control valve is connected, and is used to control the alkali lye and the gas mixture scale of construction, lye pump 22, C0
2Source of the gas 23, air source of the gas 24, N
2Gas source of the gas 25 and O
2Gas source of the gas 26 is connected with mixed gas and alkali liquor inlet 4.Dotted line is represented to be electrically connected among the figure.
The agitating vane 27 of said whipping appts 2 can adopt 20～40 ° of oblique leaf hoisting type stirring rakes, and blade 27 diameters are 3～4: 5 with reactor 2 diameters ratio.
As seen from Figure 2, said control device 18 comprises pH control instruments 19 and oxyty control instruments 20;
Claims (6)
1. a bioreactor system that is used for the stem cell vitro culture is characterized in that, comprising:
A cylindric upper end has the cylindrical reactor (2) of capping (1);
A whipping appts (3) that is arranged in the described reactor (2);
A mixed gas and an alkali liquor inlet (4) that is arranged in the capping (1);
A mixed gas outlet (5) that is arranged in the capping (1);
One is arranged on the ventpipe (8) that is provided with air outlet, top (6) and air outlet, bottom (7) in the reactor (2) and is connected with said mixed gas outlet (5);
A nutrient solution inlet (9) that is arranged in the capping (1);
A nutrient solution outlet (10) that is arranged in the capping (1);
A cell reflux inlet (11) that is arranged in the capping (1);
One be provided with purified liquor outlet (12) and the inlet (13) export the cell retention device (14) that (10) are connected with said nutrient solution, the outlet (15) of trap (14) is connected with cell reflux inlet (11);
One is arranged on pH electrode (16) in the reactor (2) by capping (1);
One is arranged on oxyty electrode (17) in the reactor (2) by capping (1);
A control device (18) that is connected with oxyty electrode (17) with pH electrode (16), the output terminal of control device (18) respectively with lye pump (22), CO
2CO on the source of the gas (23)
2Air flow control valve, N on flowrate control valve, the air source of the gas (24)
2N on the gas source of the gas (25)
2Airshed control valve and O
2O on the gas source of the gas (26)
2The airshed control valve is connected, lye pump (22), CO
2Source of the gas (23), air source of the gas (24) and N
2Gas source of the gas (25) is connected with mixed gas and alkali liquor inlet (4).
2. the bioreactor system that is used for the stem cell vitro culture according to claim 1 is characterized in that, reactor (2) aspect ratio is 2～4: 1.
3. the bioreactor system that is used for the stem cell vitro culture according to claim 2 is characterized in that, reactor (2) aspect ratio is 3: 1.
4. according to claim 1, the 2 or 3 described bioreactor systems that are used for the stem cell vitro culture, it is characterized in that the agitating vane (27) of said whipping appts (3) adopts 20～40 ° of oblique leaf hoisting type stirring rakes.
5. the bioreactor system that is used for the stem cell vitro culture according to claim 4 is characterized in that, blade (27) diameter and reactor (2) diameter ratio are 3～4: 5.
6. the bioreactor system that is used for the stem cell vitro culture according to claim 1 is characterized in that, said control device (18) comprises pH control instruments (19) and oxyty control instruments (20);
PH control instruments (19) is connected with pH electrode (16), the output terminal of pH control instruments (19) respectively with lye pump (22), CO
2Control valve on the source of the gas (23) is connected;
Oxyty control instruments (20) is connected with oxyty electrode (17), the output terminal of oxyty control instruments (20) respectively with N
2Gas, O
2Gas is connected with control valve on the air source of the gas.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310109169 CN1261560C (en) | 2003-12-08 | 2003-12-08 | Bioreactor of in vitro stem cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200310109169 CN1261560C (en) | 2003-12-08 | 2003-12-08 | Bioreactor of in vitro stem cell culture |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1546641A CN1546641A (en) | 2004-11-17 |
| CN1261560C true CN1261560C (en) | 2006-06-28 |
Family
ID=34335068
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200310109169 Expired - Fee Related CN1261560C (en) | 2003-12-08 | 2003-12-08 | Bioreactor of in vitro stem cell culture |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1261560C (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102071167B (en) * | 2009-11-19 | 2013-08-21 | 上海坤巨科技发展有限公司 | Method and device for culturing cells such as stem cells and the like for therapy in mobile carrier mode |
| CN104977948A (en) * | 2014-04-09 | 2015-10-14 | 上海核创制药系统工程有限公司 | Low-oxygen isolator |
| CN108148749A (en) * | 2016-12-05 | 2018-06-12 | 中国科学院大连化学物理研究所 | Keep the micro-fluidic chip culture systems of culture solution dissolved oxygen concentration and acid-base value |
| CN109810900B (en) * | 2019-03-12 | 2024-01-12 | 华道(上海)生物医药有限公司 | Totally-enclosed cell culture gas control system |
| GB201904083D0 (en) * | 2019-03-25 | 2019-05-08 | Ge Healthcare Bio Sciences Ab | A bioreactor system |
| CN112457986A (en) * | 2021-01-07 | 2021-03-09 | 华润昂德生物药业有限公司 | Bioreactor for human erythropoietin injection CHO cell fermentation |
-
2003
- 2003-12-08 CN CN 200310109169 patent/CN1261560C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1546641A (en) | 2004-11-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4146476B2 (en) | Biological reactor | |
| CN103476245B (en) | For cultivating the method for microorganism and bioreactor | |
| CN102311920B (en) | Culture method for chlorella | |
| CN101265449A (en) | Fast high-density culture method for algae cell | |
| CN102459563A (en) | Bioreactor process for production of hydrogen from biomass | |
| CN104805016B (en) | One kind utilizes CO2The method for cultivating microalgae | |
| CN1261560C (en) | Bioreactor of in vitro stem cell culture | |
| CN102311924A (en) | Method for open-type culture of microalgae | |
| CN101705189A (en) | Spirulina mixed culturing technology and device | |
| CN104250617B (en) | A kind of both culturing microalgae method | |
| CN1724637A (en) | Mend the method that carbon is cultivated little algae by pH value feedback control | |
| Rusch et al. | The hydraulically integrated serial turbidostat algal reactor (HISTAR) for microalgal production | |
| CN116179356B (en) | Method for high-density heterotrophic culture of chlamydomonas reinhardtii and application thereof | |
| CN101153294A (en) | Immobilized cell single-tank high-strength continuous fermentation process for succinic acid | |
| CN110128181A (en) | A kind of production method and process units of the phycomycete fertilizer based on biogas slurry | |
| CN107446867B (en) | A kind of Upland Red Soil symbiotic nitrogen fixation bacterium and its cultural method and application | |
| CN1297659C (en) | Method and device for perfusion and culture of hematopoietic cell | |
| CN107699493B (en) | Microalgae cultivation method | |
| CN113136321A (en) | Method and system for heterotrophic-autotrophic co-culture of photosynthetic microorganisms and method for production of biomass and bioenergy | |
| CN113136339A (en) | Method for mixotrophic-autotrophic continuous culture of photosynthetic microorganisms, culture system and application thereof | |
| Kurz et al. | Continuous culture of plant cells | |
| CN2510499Y (en) | Gas-circulating plant cell culture reactor | |
| CN219824209U (en) | Airlift type photo-bioreactor with built-in LED light source for microalgae cultivation | |
| CN109928508A (en) | A method of biogas slurry containing metal is handled using Dan Xingzao biomembrane adhere-wall culture | |
| CN107287249A (en) | Technique and system based on Anammox coupling and intensifying reclaiming organic waste |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |