CN110747106A - Ventilation device and method in biological reaction system and biological reaction system comprising ventilation device - Google Patents
Ventilation device and method in biological reaction system and biological reaction system comprising ventilation device Download PDFInfo
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- 238000009423 ventilation Methods 0.000 title claims abstract description 137
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000005273 aeration Methods 0.000 claims abstract description 130
- 230000010261 cell growth Effects 0.000 claims abstract description 21
- 239000006185 dispersion Substances 0.000 claims description 32
- 239000012528 membrane Substances 0.000 claims description 23
- 239000011148 porous material Substances 0.000 claims description 5
- 239000000758 substrate Substances 0.000 claims description 2
- 238000013022 venting Methods 0.000 claims 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 abstract description 15
- 229910052760 oxygen Inorganic materials 0.000 abstract description 15
- 239000001301 oxygen Substances 0.000 abstract description 15
- 238000010008 shearing Methods 0.000 abstract description 12
- 230000006378 damage Effects 0.000 abstract description 10
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- 230000012010 growth Effects 0.000 description 17
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- 229910052751 metal Inorganic materials 0.000 description 6
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- 230000004083 survival effect Effects 0.000 description 5
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- 229910052755 nonmetal Inorganic materials 0.000 description 4
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- 229920001343 polytetrafluoroethylene Polymers 0.000 description 3
- 229910001220 stainless steel Inorganic materials 0.000 description 3
- 239000010935 stainless steel Substances 0.000 description 3
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
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- 238000002360 preparation method Methods 0.000 description 2
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- 238000009827 uniform distribution Methods 0.000 description 2
- 241000699802 Cricetulus griseus Species 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
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- 208000014674 injury Diseases 0.000 description 1
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Abstract
The invention provides a ventilation device in a biological reaction system, which is provided with a first ventilation cavity and a second ventilation cavity which are respectively connected with a first ventilation inlet and a second ventilation inlet and are distributed with holes, wherein the hole diameter distributed on the first ventilation cavity is different from the hole diameter distributed on the second ventilation cavity, so that the bubble specification and the ventilation quantity provided for the biological reaction system can be accurately controlled, and the device is simple, easy to assemble and disassemble and convenient to operate; the aeration method in the biological reaction system using the device can independently and uniformly generate micro bubbles, effectively avoid the damage of shearing force generated by the rupture of the micro bubbles to cells, can meet the requirement of oxygen supply in the middle and later stages of cell growth, and has higher application value.
Description
Technical Field
The invention relates to the technical field of biological reaction equipment, in particular to a ventilating device and a ventilating method in a biological reaction system and the biological reaction system comprising the ventilating device and the ventilating method.
Background
With the rise of high-density culture technology, the aeration device of the bioreactor is challenged, the aeration quantity and bubbles generated by aeration have great influence on the biological reaction process, the aeration device commonly used at present is a ring-shaped aeration tube or a sintered metal rod, and the problems that the size of the bubbles cannot be adjusted and the shearing force of the generated bubbles is large exist in the conventional aeration device of the ring-shaped aeration tube; the sintered metal rod can not meet the requirement of high oxygen demand in the middle and later stages of cell growth.
Therefore, the skilled person has made many studies on the development of aeration devices suitable for bioreactors, and much has been directed to the reduction of shearing force of bubbles or uniformity of distribution.
CN202576435U discloses a ventilation device of bioreactor, and the device has avoided the shearing force injury that the bubble brought for the organism of ventilating to a certain extent through setting up shower head connector and hollow cylindrical screen cloth, but the device structure is complicated, is difficult to popularize and apply to, and can't adjust and control to the different growth periods of cell.
CN106834099A discloses a bioreactor low-shear lifting type aeration stirring device, which avoids the damage to cells caused by shear force stirring to a certain extent, but does not consider the requirements of cells in different growth periods, and has a complex structure and difficult preparation.
CN207483732U discloses a gas distributor for a bioreactor, which is provided with a distributor panel below the stirring structure of the reactor, and a plurality of small ventilation holes are distributed on the distributor panel, and the small holes are added to reduce the bubble rupture and the stirring shearing force, but the device is not suitable for cells in different growth periods.
In summary, the existing aeration device in the biological reaction system has a complex structure and does not consider the problems of different growth periods of cells and the like.
Therefore, it is necessary to design a ventilation device which has a simple structure, is easy to prepare and is suitable for biological reaction systems in different growth periods of cells.
Disclosure of Invention
In view of the problems in the prior art, the invention provides the ventilating device in the biological reaction system, the ventilating device is provided with the first ventilating cavity and the second ventilating cavity which are respectively connected with the first ventilating inlet and the second ventilating inlet and are distributed with holes, the hole diameter of the holes distributed on the first ventilating cavity is different from that of the holes distributed on the second ventilating cavity, bubbles with different specifications can be provided for the reaction system through the independent ventilating inlets, the independent ventilating control is more accurate, the device is simple, and the operation is convenient; the aeration method in the biological reaction system using the device can meet the oxygen demand of cells in different growth periods, simultaneously can avoid the adverse effect of the rupture of large bubbles on the growth of the cells, and has higher application value.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an aeration device in a biological reaction system, the aeration device comprising a first aeration chamber and a second aeration chamber disposed at the periphery thereof; the first ventilation cavity is communicated with the first ventilation inlet, the second ventilation cavity is communicated with the second ventilation inlet, holes are distributed in the first ventilation cavity and the second ventilation cavity, and the hole diameter of the holes distributed in the first ventilation cavity is different from that of the holes distributed in the second ventilation cavity.
According to the aeration device in the biological reaction system, the first aeration cavity and the second aeration cavity with different hole diameters are arranged, so that bubbles with different specifications can be generated, and the requirements of cells in different growth periods are met; the aeration device is also provided with a first aeration inlet and a second aeration inlet which are respectively connected with the first aeration cavity and the second aeration cavity, so that the aeration quantity of large bubbles and micro bubbles can be more accurately and independently controlled.
In the present invention, "large bubbles" mean bubbles having a bubble diameter in the range of 50 to 300 μm, and "microbubbles" mean bubbles having a bubble diameter in the range of 1 to 40 μm.
The aperture of the first vent cavity hole is different from that of the second vent cavity hole, wherein the aperture of the first vent cavity hole can be larger than that of the second vent cavity hole or smaller than that of the second vent cavity hole, and the first vent cavity hole and the second vent cavity hole can both better meet the requirements of cells in different growth stages.
Preferably, the aperture of the first vent cavity hole is 5-30 μm, such as 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm or 30 μm, preferably 10-25 μm.
The aperture of the first ventilation cavity hole is 5-30 microns, so that micro bubbles with the diameter of 5-30 microns can be generated, the micro bubbles with the diameter range can meet the oxygen demand of cells, meanwhile, damage to the cells caused by shearing force generated by bubble breakage can be avoided, and the industrial application value is high.
Preferably, the aperture of the second air cavity hole is 80-200 μm, such as 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm or 200 μm, preferably 120-180 μm.
The aperture of the first ventilation cavity hole is 80-200 mu m, so that the requirement of oxygen in the middle and later stages of cell growth can be met while the uniform distribution of gas is ensured.
Preferably, the first ventilation cavity and the second ventilation cavity are concentrically arranged, for example, they may be concentrically arranged with the same central axis, or they may be concentrically arranged without the same central axis, preferably they are concentrically arranged with the same central axis.
Preferably, the first vent cavity is circular.
Preferably, the second vent cavity is circular.
According to the first aeration cavity and the second aeration cavity, the cavity interfaces can be round, square or triangular, preferably round, and the round shape can be better matched with a conventional bioreactor system, so that bubbles are more uniformly distributed when released.
Preferably, the first ventilation cavity and the second ventilation cavity are fixedly connected through a substrate.
Preferably, the first vent inlet is connected to a first valve.
Preferably, the second vent inlet is connected to a second valve.
According to the invention, the first ventilation inlet and the second ventilation inlet are preferably respectively and independently provided with the first valve and the second valve, so that the ventilation volume in the first ventilation cavity and the second ventilation cavity can be independently controlled, and the control is more accurate.
Preferably, the first ventilation cavity and the second ventilation cavity are a first ventilation pipe and a second ventilation pipe respectively.
The first vent pipe and the second vent pipe in the present invention may be prepared by a method well known to those skilled in the art for preparing porous materials, such as metal or non-metal sintering.
In the present invention, the first vent pipe and the second vent pipe may be made of a material commonly used in the art, and may be made of, for example, metal (a special material such as stainless steel, copper, or titanium), non-metal plastic (PTFE, PE, PP, PVDF, EPDM, or PEs), ceramic, or fiber.
Preferably, holes are uniformly distributed on the periphery of the first vent pipe and the second vent pipe.
Preferably, the aperture of the first vent pipe is 5 to 30 μm, such as 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm or 30 μm, preferably 10 to 25 μm.
Preferably, the aperture of the second vent is 80-200 μm, such as 80-200 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm or 200 μm, preferably 120-180 μm.
Preferably, the first ventilation inlet is provided on one side of the first ventilation pipe.
Preferably, the second vent inlet is provided at one side of the second vent pipe.
Preferably, the first ventilation cavity and the second ventilation cavity are respectively a first ventilation groove and a second ventilation groove.
Preferably, the first ventilation groove and the second ventilation groove are both provided with ventilation dispersion films or ventilation dispersion plates.
The mode of preparation of the aeration-dispersed membrane or aeration-dispersed tablet of the present invention may be a method well known to those skilled in the art for preparing porous materials, and may be, for example, metal or non-metal sintering.
The air-permeable dispersion membrane or air-permeable dispersion sheet of the present invention may be made of a material commonly used in the art, and may be made of, for example, metal (special material such as stainless steel, copper, and titanium), non-metal plastic (PTFE, PE, PP, PVDF, EPDM, or PEs), ceramic, or fiber
Preferably, holes are uniformly distributed on the ventilation dispersion film or the ventilation dispersion sheet.
Preferably, the pore diameter of the holes on the aeration-dispersed membrane or aeration-dispersed tablet of the first aeration tank is 5-30 μm, such as 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm or 30 μm, preferably 10-25 μm.
Preferably, the pore diameter of the holes on the aeration dispersion membrane or aeration dispersion tablet of the second aeration tank is 80-200 μm, such as 80-200 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm or 200 μm, preferably 120-180 μm.
Preferably, the first vent inlet is provided at an upper side of the first vent groove.
Preferably, the second vent inlet is disposed at an upper side of the second vent groove.
In the invention, the first ventilation inlet and the second ventilation inlet are preferably arranged at the positions, which are not provided with the ventilation dispersion film or the ventilation dispersion sheet, on the upper sides of the first ventilation groove and the second ventilation groove respectively, so that the independent ventilation of the first ventilation groove and the second ventilation groove is realized.
In a second aspect, the present invention provides a method of aeration in a bioreactor system, the method being carried out using the aeration device of the bioreactor system of the first aspect.
According to the aeration method in the biological reaction system, the aeration device in the biological reaction system is adopted for aeration, so that bubbles with different specifications can be independently introduced, the proportion of large bubbles and micro bubbles can be adjusted, the requirement of cells on oxygen supply in different growth periods can be better met, the damage of the large bubbles to the cells in the initial growth stage of the cells can be effectively avoided, and the aeration method has high application value.
Preferably, the method comprises: air enters the first ventilation cavity and the second ventilation cavity through the first ventilation inlet and the second ventilation inlet respectively, and bubbles with different specifications are introduced into the biological reaction system through the first ventilation cavity and the second ventilation cavity.
According to the invention, air enters different air-through cavities through different air-through inlets respectively and then bubbles with different specifications are introduced into the biological reaction system, so that the air volume of the bubbles with different specifications can be independently regulated and controlled respectively, and the controllability of the ventilation process is enhanced.
Preferably, the method comprises the steps of:
(1) at the initial stage of cell growth, closing the second valve, opening the first valve, and introducing air into the first ventilation cavity through the first ventilation inlet to ventilate so as to generate micro-bubbles;
(2) in the middle and later stages of cell growth, the second valve is opened, and air enters the second ventilation cavity through the second ventilation inlet to ventilate to generate large bubbles;
in the invention, micro bubbles are introduced at the initial stage of cell growth, so that the damage of shearing force generated by breaking of the large bubbles to the initial cells can be effectively avoided; and large bubbles are introduced in the middle and later cell growth stages because the sensitivity of the cells is reduced at the moment, and the requirement of oxygen needed by the middle and later cell growth stages can be better met by introducing the large bubbles.
Preferably, in the step (2), the first valve and the second valve are opened simultaneously, and the air generates the micro-bubbles and the large bubbles through the first vent inlet and the second vent inlet simultaneously.
The invention simultaneously introduces the micro bubbles and the large bubbles in the middle and later stages of cell growth, and can better meet the requirement of the oxygen in the middle and later stages of cell growth.
Preferably, in the step (2), the first valve and the second valve are adjusted to adjust the ratio of the micro bubbles to the macro bubbles.
The invention can better meet the requirements of different cells and cells in different periods on oxygen by adjusting the proportion of micro bubbles to large bubbles, and can better culture the cells.
Preferably, the diameter of the microbubbles is 5 to 30 μm, for example, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm, 15 μm, 16 μm, 17 μm, 18 μm, 19 μm, 20 μm, 21 μm, 22 μm, 23 μm, 24 μm, 25 μm, 26 μm, 27 μm, 28 μm, 29 μm, or 30 μm, preferably 10 to 25 μm.
The diameter of the micro-bubbles generated by the invention is 5-30 μm, and the micro-bubbles in the diameter range not only can meet the oxygen demand of the cells in the initial growth stage, but also can avoid the damage of shearing force generated by bubble breakage to the cells.
Preferably, the diameter of the large bubbles is 80 to 200 μm, for example, 80 to 200 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm, 150 μm, 160 μm, 170 μm, 180 μm, 190 μm or 200 μm, preferably 120 to 180 μm.
The diameter of the large bubble is 80-200 mu m, so that the requirement of oxygen in the middle and later stages of cell growth can be met while the uniform distribution of gas is ensured.
In a third aspect, the present invention provides a biological reaction system comprising an aeration device according to the first aspect.
The biological reaction system provided by the invention comprises the aeration device in the first aspect, so that the gas distribution is uniform, large bubbles or micro bubbles can be independently aerated, and the requirements of cells in different growth periods are met.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) the aeration device in the biological reaction system can realize independent aeration of large bubbles or micro bubbles into the biological reaction system, the gas distribution is uniform, and the aeration device is provided with an independent aeration inlet, so that the control is more accurate;
(2) the aeration device in the biological reaction system provided by the invention can realize that only micro bubbles are aerated at the initial stage of cell growth, the damage of shearing force generated by breaking the large bubbles to cells is avoided, the cell survival rate is more than or equal to 98.55 percent, and the cell density is more than or equal to 5.57 multiplied by 10 after the cell is cultured for 14 days6Per mL;
(3) the aeration method in the biological reaction system can accurately regulate the proportion of large bubbles to small bubbles and meet the requirements of different stages of cell growth.
Drawings
FIG. 1 is a plan view of an aeration apparatus in a biological reaction system provided in example 1 of the present invention.
FIG. 2 is a side sectional view of an aeration apparatus in a biological reaction system provided in example 1 of the present invention.
FIG. 3 is a plan view of an aeration apparatus in a biological reaction system provided in example 3 of the present invention.
FIG. 4 is a side cross-sectional view of an aeration apparatus in a biological reaction system provided in example 3 of the present invention.
In the figure: 101-a first vent pipe; 102-a second vent; 103-a first vent inlet; 104-a second vent inlet; 201-a first vent channel; 202-a second vent slot; 203-a first vent inlet; 204-a second vent inlet; 205-a first aeration dispersion membrane; 206-a second vent dispersion membrane.
Detailed Description
The technical scheme of the invention is further explained by the specific implementation mode in combination with the attached drawings.
The present invention is described in further detail below. The following examples are merely illustrative of the present invention and do not represent or limit the scope of the claims, which are defined by the claims.
First, an embodiment
Example 1
The embodiment provides a double-layer sleeve type aeration device in a biological reaction system, as shown in fig. 1 and fig. 2, the device comprises a first aeration pipe 101 and a second aeration pipe 102 arranged on the periphery of the first aeration pipe 101, the cross-sectional areas of the first aeration pipe 101 and the second aeration pipe 102 are both circular, the first aeration pipe 101 and the second aeration pipe 102 are PTFE pipes, the first aeration pipe 101 and the second aeration pipe 102 are respectively communicated with a first aeration inlet 103 and a second aeration inlet 104 arranged on one side of the first aeration pipe, the first aeration inlet 103 and the second aeration inlet 104 are respectively arranged at positions opposite to the diameter of the aeration device, the device further comprises a first valve and a second valve respectively connected with the first aeration inlet 103 and the second aeration inlet 104, the first aeration pipe 101 and the second aeration pipe 102 are circular pipes which are concentrically distributed on the same central axis and are uniformly provided with holes on the periphery, the aperture of the holes of the first ventilation pipe 101 is 15 μm, and the aperture of the holes of the second ventilation pipe 102 is 120 μm.
Example 2
The embodiment provides a double-layer sleeve type ventilation device in a biological reaction system, which comprises a first ventilation pipe and a second ventilation pipe arranged on the periphery of the first ventilation pipe, wherein the cross-sectional areas of the first ventilation pipe and the second ventilation pipe are both circular, the first ventilation pipe and the second ventilation pipe are PE pipes, the first ventilation pipe and the second ventilation pipe are respectively communicated with a first ventilation inlet and a second ventilation inlet which are arranged on one side of the first ventilation pipe and the second ventilation inlet, the first ventilation inlet and the second ventilation inlet are respectively arranged at positions opposite to the diameter of the ventilation device, and the device further comprises a first valve and a second valve which are respectively connected with the first ventilation inlet and the second ventilation inlet; the first vent pipe and the second vent pipe are circular pipes which are concentrically distributed on the same central shaft and are uniformly distributed with holes at the periphery, the hole diameter of the first vent pipe is 80 mu m, and the hole diameter of the second vent pipe is 30 mu m.
Example 3
The present embodiment provides a double-layer membrane sheet type aeration device in a biological reaction system, as shown in fig. 3 and 4, the device comprises a first aeration tank 201 and a second aeration tank 202 arranged at the periphery thereof, the first aeration tank 201 and the second aeration tank 202 are respectively communicated with a first aeration inlet 203 and a second aeration inlet 204 arranged above the first aeration tank 201 and the second aeration tank 202, and the device further comprises a first valve and a second valve respectively connected with the first aeration inlet 203 and the second aeration inlet 204; the first aeration tank 201 and the second aeration tank 202 are circular tanks concentrically distributed on the same central axis, a first aeration dispersion membrane 205 and a second aeration dispersion membrane 206 made of stainless steel through sintering are respectively arranged on the first aeration tank 201 and the second aeration tank 202, holes are uniformly distributed on the first aeration dispersion membrane 205 and the second aeration dispersion membrane 206, the hole diameter of the first aeration dispersion membrane 205 is 5 micrometers, and the hole diameter of the second aeration dispersion membrane 206 is 200 micrometers.
Example 4
The embodiment provides a double-layer membrane type aeration device in a biological reaction system, which comprises a first aeration groove and a second aeration groove arranged on the periphery of the first aeration groove, wherein the first aeration groove and the second aeration groove 202 are respectively communicated with a first aeration inlet and a second aeration inlet which are arranged above the first aeration groove, and the device also comprises a first valve and a second valve which are respectively connected with the first aeration inlet and the second aeration inlet; the first ventilation groove and the second ventilation groove are circular grooves which are concentrically distributed on the same central shaft, a first ventilation dispersion membrane and a second ventilation dispersion membrane which are formed by sintering ceramics are respectively arranged on the first ventilation groove and the second ventilation groove, holes are uniformly distributed on the first ventilation dispersion membrane and the second ventilation dispersion membrane, the hole diameter of the first ventilation dispersion membrane is 160 mu m, and the hole diameter of the second ventilation dispersion membrane is 20 mu m.
The aeration devices provided in examples 1 to 4 are used in a biological reaction system, wherein the aeration method using the aeration devices provided in examples 1 and 3 comprises the following steps:
(1) at the initial stage of cell growth, closing the second valve, opening the first valve, and introducing air into the first ventilation cavity through the first ventilation inlet to ventilate so as to generate micro-bubbles;
(2) and in the middle and later stages of cell growth, the second valve is opened, air enters the second vent cavity through the second vent inlet to be ventilated to generate large bubbles, the first valve and the second valve are opened simultaneously according to the oxygen demand of the cells, the air generates micro bubbles and large bubbles through the first vent inlet and the second vent inlet simultaneously, the first valve and the second valve are adjusted, and the proportion of the micro bubbles to the large bubbles is adjusted.
The method for aeration by using the aeration device provided in the embodiment 2 and the embodiment 4 comprises the following steps:
(1) in the initial stage of cell growth, the first valve is closed, the second valve is opened, and air enters the second ventilation cavity through the second ventilation inlet to ventilate to generate micro-bubbles;
(2) and in the middle and later stages of cell growth, opening the first valve, introducing air into the first ventilation cavity through the first ventilation inlet to ventilate to generate large bubbles, simultaneously opening the first valve and the second valve according to the oxygen demand of the cells, simultaneously generating micro bubbles and large bubbles by the air through the first ventilation inlet and the second ventilation inlet, adjusting the first valve and the second valve, and adjusting the proportion of the micro bubbles and the large bubbles.
II, cell culture and results
The aeration device provided in examples 1 to 4 was placed in a biological reaction system, and for example, animal cell culture, CHO (Chinese hamster ovary) cells were cultured by first performing amplification culture on the CHO cells using a culture dish and 14 days later until the cell density reached 10X 105Inoculating the seed solution into a 30L biological reaction system when the Cell survival rate is more than 93 percent, taking EX-Cell 302 serum-free culture medium (manufacturer: Sigma-Aldrich) as a basic culture medium, culturing at the constant temperature of 36.5 ℃, introducing micro bubbles into the reaction system at the initial stage of Cell growth, introducing large bubbles or simultaneously introducing large bubbles and micro bubbles into the reaction system at the middle and later stages of Cell growth, determining the specific gas flow according to different growth stages of the cells, culturing for 14 days, sampling the cells, calculating the Cell density and the Cell survival rate of the Cell culture in the examples 1-4, and obtaining the results shown in Table 1.
TABLE 1
| Sample (I) | Cell density (per mL) | Cell survival Rate% |
| Example 1 | 5.57×106 | 98.55 |
| Example 2 | 5.64×106 | 98.65 |
| Example 3 | 6.01×106 | 98.80 |
| Example 4 | 5.78×106 | 98.70 |
As can be seen from Table 1, when the bioreactor containing the aeration devices provided in examples 1 to 4 is used for cell culture, micro bubbles with the size of 5 to 30 μm can be generated in the early growth stage of cells, the damage of shearing force generated by breaking of large bubbles to the cells is avoided, the cell survival rate is not less than 98.55%, the sensitivity of the cells to the shearing force is reduced and the oxygen demand is increased along with the continuous growth of the cells, the large bubbles are introduced into the biological reaction system through the aeration devices in the middle and later stages of the culture to meet the oxygen demand of the cells, and the cell density is not less than 5.57 multiplied by 10 after the cells are cultured for 14 days6The cell culture medium has the advantages of small volume per mL, good cell culture effect and good application prospect.
In conclusion, the aeration device in the biological reaction system can accurately control the specification and the aeration quantity of the bubbles provided for the biological reaction system, and has the advantages of simple device, easy disassembly and assembly and convenient operation; the aeration method in the biological reaction system by using the device can independently and uniformly generate micro bubbles, effectively avoid the damage of shearing force generated by the rupture of the large bubbles to cells, can meet the requirements of different cells and different growth periods of the cells, and has great practical application value.
The applicant declares that the present invention illustrates the detailed structural features of the present invention through the above embodiments, but the present invention is not limited to the above detailed structural features, that is, it does not mean that the present invention must be implemented depending on the above detailed structural features. It should be understood by those skilled in the art that any modifications of the present invention, equivalent substitutions of selected components of the present invention, additions of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. An aeration device in a biological reaction system, which is characterized by comprising a first aeration cavity and a second aeration cavity arranged at the periphery of the first aeration cavity;
the first ventilation cavity is communicated with the first ventilation inlet, the second ventilation cavity is communicated with the second ventilation inlet, holes are distributed in the first ventilation cavity and the second ventilation cavity, and the hole diameter of the holes distributed in the first ventilation cavity is different from that of the holes distributed in the second ventilation cavity.
2. The device according to claim 1, wherein the first ventilation cavity holes have a pore diameter of 5-30 μm, preferably 10-25 μm;
preferably, the aperture of the second ventilation cavity hole is 80-200 μm, preferably 120-180 μm.
3. The device of claim 1 or 2, wherein the first and second vent chambers are concentrically arranged;
preferably, the first vent cavity is circular;
preferably, the second vent cavity is circular.
4. The device according to any one of claims 1 to 3, wherein the first and second vent cavities are fixedly connected by a substrate;
preferably, the first vent inlet is connected to a first valve;
preferably, the second vent inlet is connected to a second valve.
5. The device according to any one of claims 1 to 4, wherein the first and second ventilation cavities are a first and second ventilation tube, respectively;
preferably, holes are uniformly distributed on the periphery of the first vent pipe and the second vent pipe;
preferably, the aperture of the hole of the first vent pipe is 5-30 μm, preferably 10-25 μm;
preferably, the aperture of the hole of the second vent pipe is 80-200 μm, preferably 120-180 μm;
preferably, the first ventilation inlet is provided on one side of the first ventilation pipe;
preferably, the second vent inlet is provided at one side of the second vent pipe.
6. The device according to any one of claims 1 to 4, wherein the first and second vent cavities are a first vent groove and a second vent groove, respectively;
preferably, the first aeration groove and the second aeration groove are both provided with aeration dispersion membranes or aeration dispersion tablets;
preferably, holes are uniformly distributed on the ventilation dispersion film or the ventilation dispersion sheet;
preferably, the aperture of the holes on the aeration dispersion membrane or the aeration dispersion tablet of the first aeration tank is 5-30 μm, preferably 10-25 μm;
preferably, the aperture of the holes on the aeration dispersion membrane or the aeration dispersion tablet of the second aeration tank is 80-200 μm, preferably 120-180 μm;
preferably, the first vent inlet is disposed at an upper side of the first vent groove;
preferably, the second vent inlet is disposed at an upper side of the second vent groove.
7. A method of aeration in a biological reaction system, which is carried out using the aeration device in the biological reaction system according to any one of claims 1 to 6.
8. The method of venting air according to claim 7, comprising: air enters the first ventilation cavity and the second ventilation cavity through the first ventilation inlet and the second ventilation inlet respectively, and bubbles with different specifications are introduced into the biological reaction system through the first ventilation cavity and the second ventilation cavity.
9. The method according to claim 8, characterized in that it comprises the steps of:
(1) at the initial stage of cell growth, closing the second valve, opening the first valve, and introducing air into the first ventilation cavity through the first ventilation inlet to ventilate so as to generate micro-bubbles;
(2) in the middle and later stages of cell growth, the second valve is opened, and air enters the second ventilation cavity through the second ventilation inlet to ventilate to generate large bubbles;
preferably, in the step (2), the first valve and the second valve are opened simultaneously, and the air generates micro bubbles and large bubbles through the first ventilation inlet and the second ventilation inlet simultaneously;
preferably, in the step (2), the first valve and the second valve are adjusted, and the ratio of micro bubbles to macro bubbles is adjusted;
preferably, the diameter of the micro-bubbles is 5-30 μm, preferably 10-25 μm;
preferably, the diameter of the large bubbles is 80-200 μm, preferably 120-180 μm.
10. A bioreaction system comprising an aeration device according to any one of claims 1 to 6.
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| CN201911195011.9A CN110747106A (en) | 2019-11-28 | 2019-11-28 | Ventilation device and method in biological reaction system and biological reaction system comprising ventilation device |
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| CN201911195011.9A CN110747106A (en) | 2019-11-28 | 2019-11-28 | Ventilation device and method in biological reaction system and biological reaction system comprising ventilation device |
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