CN107988074B - 6000L serum-free full-suspension cell culture reactor - Google Patents
6000L serum-free full-suspension cell culture reactor Download PDFInfo
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- CN107988074B CN107988074B CN201810074430.6A CN201810074430A CN107988074B CN 107988074 B CN107988074 B CN 107988074B CN 201810074430 A CN201810074430 A CN 201810074430A CN 107988074 B CN107988074 B CN 107988074B
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- 238000004113 cell culture Methods 0.000 title claims abstract description 51
- 239000000725 suspension Substances 0.000 title claims abstract description 38
- 238000003756 stirring Methods 0.000 claims abstract description 136
- 239000003513 alkali Substances 0.000 claims abstract description 51
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 42
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 42
- 239000001301 oxygen Substances 0.000 claims abstract description 42
- 238000009423 ventilation Methods 0.000 claims abstract description 34
- 238000005507 spraying Methods 0.000 claims abstract description 21
- 230000001502 supplementing effect Effects 0.000 claims abstract description 11
- 239000002344 surface layer Substances 0.000 claims abstract description 8
- 238000003860 storage Methods 0.000 claims abstract description 5
- 239000007788 liquid Substances 0.000 claims description 36
- 238000005273 aeration Methods 0.000 claims description 15
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 14
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 7
- 239000001569 carbon dioxide Substances 0.000 claims description 7
- 238000001514 detection method Methods 0.000 claims description 4
- 238000003760 magnetic stirring Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 2
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- LHGMHYDJNXEEFG-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]iminocyclohexa-2,5-dien-1-one Chemical compound C1=CC(N(C)C)=CC=C1N=C1C=CC(=O)C=C1 LHGMHYDJNXEEFG-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/18—Flow directing inserts
- C12M27/20—Baffles; Ribs; Ribbons; Auger vanes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
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Abstract
The invention relates to the field of serum-free full-suspension cell culture equipment, in particular to a 6000L serum-free full-suspension cell culture reactor, which comprises a tank body; a microbubble ventilation device and a stirring device are arranged in the tank body; a baffle is arranged inside the tank wall of the tank body; an alkali liquor spraying head is arranged above the tank wall of the tank body and is connected with an alkali storage container through a valve; the upper part of the tank wall of the tank body is also provided with a surface layer air inlet. The cell culture reactor provided by the invention is optimized in many aspects according to the culture characteristics of a large-volume reactor, and particularly optimizes a microbubble ventilation system, a bioreactor alkali supplementing system and an air inlet system, so that the reactor is very suitable for serum-free full-suspension cell culture, can realize high-efficiency oxygen mass transfer under low shear force, uniformly mix cells and nutrient substances, effectively adjust the pH in a tank, and has high cell survival rate.
Description
Technical Field
The invention relates to the field of cell culture equipment, in particular to a 6000L serum-free full-suspension cell culture reactor.
Background
The bioreactor is a core device for large-scale cell suspension culture, can effectively increase the culture density of cell unit volume, and is an important support for large-scale production of the product. Relatively perfect bioreactor research and development and industrialization technology systems are established in the united states, swiss, germany and other biotechnology strong countries, and commercial bioreactors with the advantages of high production efficiency and product quality, low production cost and the like and being suitable for various biological cultures can be developed. Internationally, the research of bioreactors for producing antibodies by full suspension culture is developed towards high performance, large scale and intellectualization. The market scale of animal cell reactors (monomers) of companies such as Abec, GEA GROUP and the like reaches more than 10 hundred million Euro. Companies such as Swiss Longsha (Lonza) and Gelansu Smith (GSK) establish a production line of animal cell reactors with various upgrades, and the utilization rate of the biological reactors in the biopharmaceutical industry is increased to more than 80%.
China is already the largest production country and the largest use country of world vaccine products and is increasing at 15% per year; in recent years, development and market demand for antibody drugs have also increased greatly. In order to improve the production efficiency and the product quality, effectively reduce the production cost, change the laggard production process for culturing cells by rotating bottles, and greatly need to upgrade the animal cell culture reactor with the yield scale above thousands of times. However, the scale of the current domestic bioreactor is mostly below 1000L, the comprehensive performance and the production efficiency are not high, the technical level is far different from that of developed countries, and the production of the bioreactor with the scale of thousands of steps is almost monopolized by foreign enterprises (Switzerland Bio, american NBS, french PG and the like), so that a strict technical barrier is formed, and the scale-up technology and equipment are limited and exported to China. Therefore, development and industrialization of the animal cell suspension culture reactor are developed, and the method has very important practical significance for production of vaccines and antibody medicines in China.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a 6000L serum-free full-suspension cell culture reactor, which aims to solve the problems of low yield, high energy consumption, large labor force requirement, nonuniform product quality, difficult control of pollution and the like caused by generally smaller volume of a cell culture reactor (mainly kept below 3000L at present) in the prior art.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
the 6000L serum-free full-suspension cell culture reactor provided by the invention comprises a tank body;
a microbubble ventilation device and a stirring device are arranged in the tank body;
a baffle is arranged inside the tank wall of the tank body;
an alkali liquor spraying head is arranged above the tank wall of the tank body and is connected with an alkali storage container through a valve;
the upper part of the tank wall of the tank body is also provided with a surface layer air inlet.
Compared with the prior art, the cell culture reactor provided by the invention is optimized in many aspects according to the culture characteristics of a large-volume reactor, and particularly optimizes a microbubble ventilation system, a bioreactor alkali supplementing system and an air inlet system, so that the reactor is very suitable for culturing suspension cells, can realize high-efficiency oxygen mass transfer under low shear force, uniformly mix cells and nutrient substances, effectively adjust the pH in a tank, and has high cell survival rate.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a 6000L serum-free whole suspension cell culture reactor according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of an alkali replenishment system according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a microbubble aeration device according to one embodiment of the present invention;
FIG. 4 is a schematic view of a connecting rod according to an embodiment of the present invention;
FIG. 5 is an enlarged schematic view of the air inlet end of a micro-bubble ventilation device according to an embodiment of the present invention;
FIG. 6 is an enlarged schematic view of a notched end of a micro-bubble ventilation device according to one embodiment of the present invention;
FIG. 7 is a top view of a 6000L serum-free full suspension cell culture reactor according to an embodiment of the present invention;
FIG. 8 is a schematic diagram showing the structure of a 6000L serum-free whole suspension cell culture reactor cover according to an embodiment of the present invention;
FIG. 9 is a schematic diagram of a stepwise amplification culture system according to an embodiment of the present invention.
Reference numerals:
a bioreactor tank A; a liquid preparation tank B; an upper tank C;
a microbubble aeration device 1; an air intake hole 101; micropores 102; a connecting device 103; a locking portion 1031; a protrusion 1032; a connecting rod 104; a plug 105;
a stirring device 2; a stirring shaft 201; a stirring paddle 202;
a power device 3; a baffle 4;
an alkali liquor spraying head 5; a valve 501;
a surface layer air inlet 6; an exhaust port 7; an air supply device 8; a carbon dioxide supply device 9; an oxygen supply device 10;
a jacket temperature control system 11; .
An alkali supplementing port 12; a seed poison inlet 13; a seed cell inlet 14; a first standby port 15; a medium replenishment port 16; CIP 17-1; CIP 17-2; a second standby port 18; a lamp mouth 19; a pressure gauge port 20; a viewing port 21 and a manhole 22.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and detailed description, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative of the present invention only and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the description of the present invention, it should be noted that the directions or positional relationships indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings, are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance.
In the description of the present invention, it should be noted that, unless explicitly specified and limited otherwise, the terms "mounted," "connected," and "connected" are to be construed broadly, and may be either fixedly connected, detachably connected, or integrally connected, for example; can be mechanically or electrically connected; can be directly connected or indirectly connected through an intermediate medium, and can be communication between two elements. The specific meaning of the above terms in the present invention will be understood in specific cases by those of ordinary skill in the art.
The invention relates to a 6000L serum-free full-suspension cell culture reactor, which is shown in figure 1 and comprises a tank body A;
a microbubble ventilation device 1 and a stirring device 2 are arranged in the tank body A;
a baffle 4 is arranged in the tank wall of the tank body A;
as shown in fig. 2, an alkali liquor spraying head 5 is arranged above the tank wall of the tank body A, and the alkali liquor spraying head 5 is connected with an alkali storage container through a valve 501;
a surface layer air inlet 6 is also arranged above the tank wall of the tank body A.
Preferably, the tank wall of the tank body A is in a long cylinder shape, and the bottom of the tank body is arched outwards in an arc shape.
Preferably, a liquid distribution port is arranged at the bottom of the tank body A, and the liquid discharge port is used for discharging and/or discharging sewage.
Preferably, the tank body A is also provided with a temperature control system; more preferably, the temperature control system is a jacket temperature control system 11.
The structure of the 6000L serum-free whole suspension cell culture reactor provided by the invention is respectively described below from several systems respectively arranged in the tanks.
Preferably, a culture medium liquid inlet and/or a seed moving port are arranged above the tank wall of the tank body A;
the culture medium liquid inlet is connected with the liquid preparation tank B, and the seed moving port is connected with the upper tank C.
As shown in fig. 9, the cell culture process of the present invention is a stepwise scale-up process. In the cell culture, the scale-up is preferably carried out from a 5L to 25L to 125L to 600L to 1000L to 3000L to 6000L reactor.
Thus the upper stage tank of the 6000L reactor may preferably be a 3000L tank.
It should be noted that, in the present invention, "6000L serum-free whole-suspension cell culture reactor" is merely to highlight that the reactor of the present invention is suitable for culturing cells in a large volume, particularly suspension cells, but "6000L" does not represent a limitation on the protection range of the reactor. Indeed, the inventive concept and content of the present invention is applicable to bioreactors in the volume range 600L to 8000L, preferably 3000L to 6000L.
A cover is arranged at the top of the cell culture reactor; preferably, the cover is provided with an exhaust port 7;
preferably, as shown in fig. 8, the cover is provided with one or more of the following openings/devices in the sea:
an alkali supplementing port 12, a seed virus inlet port 13 (for virus inoculation), a seed cell inlet port 14, a first standby port 15, a culture medium supplementing port 16, CIP 17-1 and CIP 17-2, a second standby port 18, a lamp mirror port 19, a pressure gauge port 20, a mirror port 21 and a manhole 22.
CIP 17-1 and CIP 17-2 are part of a CIP cleaning system commonly known as a cleaning-in-place system, also known as a cleaning-in-place or location cleaning (cleaning in place). The in-situ cleaning means that the cleaning solution with high temperature and high concentration is adopted to perform strong action on the equipment without disassembling or moving the device, so that the contact surface with the product is cleaned, and the cleaning solution is used for cleaning and purifying production equipment with strict requirements on the sanitary level.
Microbubble ventilation system
The microbubble ventilation system mainly comprises a microbubble ventilation device 1, a stirring device 2 and a baffle 4; preferably, a dissolved oxygen amount detection device is also arranged in the tank body A; preferably, the dissolved oxygen amount detection device has two.
The accuracy of DO in the tank is ensured by adopting a mode of 2 DO electrodes, and the operation of the equipment is not affected even if one dissolved oxygen detecting device has a problem and needs to be overhauled.
The switching of the gas flowing into the micro-bubble ventilation device 1 and the rotating speed of the stirring device 2 can be controlled by an electronic control device, and the control device reads and analyzes the dissolved oxygen amount detection device so as to control the operation of the device.
As shown in fig. 3, the microbubble aeration device 1 is a non-closed circular ring with a notch; an air inlet 101 and a plurality of uniformly distributed micropores 102 are arranged on the annular wall of the microbubble ventilation device 1; the interior of the ring body of the microbubble ventilation device is hollow, and the micropores 102 penetrate through the annular wall and are communicated with the hollow cavity of the ring body.
In use of the microbubble aeration device 1, gas such as oxygen is injected through the gas inlet holes 101, and at a certain gas pressure, the oxygen fills the entire microbubble aeration device through the hollow ring body and is released from the micropores 102. The device has simple structure, thus micropores are not easy to be blocked, and maintenance is more convenient. The annular structure increases the gas distribution area, and can release oxygen into the cell culture fluid more uniformly.
Preferably, the diameter of the micropores 102 is 0.05mm to 0.2mm.
In order to increase the residence time of oxygen in the culture medium when the device is used as much as possible, preferably, the micropores 102 are distributed on one side of the plane of the circular ring, so that when the device is used, the surface of the micropores 102 can be arranged downwards to increase the travel distance of the oxygen floating upwards.
The micropores 102 may be arranged in an S-shape or Z-shape, or may be arranged in a plurality of rows, as desired for ventilation requirements. To reduce process difficulties, it is preferred that the micropores 102 be arranged in a row along the circumferential wall; more preferably, the micropores 102 are all equidistant from the center of the microbubble ventilation device 1.
Preferably, in the microbubble ventilation device as described above, the air inlet 101 is disposed opposite to the notch, and the air inlet 101 is fixed to the bottom of the bioreactor through the connection device 103.
Preferably, in the microbubble aeration device as described above, the air inlet 101 is connected to a hollow connecting rod 104 through the connecting device 103, and is fixed to the bottom of the bioreactor;
preferably, as shown in fig. 4, the connecting rod 104 has an L-shaped structure with a curved middle portion; more preferably, the bending angle of the middle part of the connecting rod 104 is 120-160 degrees; 130 deg. to 150 deg., or 140 deg. may also be selected.
The bending angle of the middle part of the connecting rod 104 is 120-160 degrees, namely when one section of the connecting rod is connected with the microbubble ventilating device, the other end of the connecting rod can tilt upwards at an angle of 20-60 degrees, and the connecting rod has the function of placing the microbubble ventilating device below the stirring paddle without scraping and touching the stirring paddle. The other side of the connecting rod 104 is connected with a reactor through a screw nut, so that the disassembly is convenient.
In order to facilitate cleaning of the micro-bubble ventilation device 1, preferably, the ring body of the micro-bubble ventilation device 1 is detachable into two half rings; the cleaning mode can be ultrasonic cleaning.
In order to simplify the structure, it is preferable that the disassembly point of the ring body of the micro-bubble ventilation device 1, which is disassembled into two half rings, is disposed near the air inlet; more preferably, as shown in fig. 5, the connecting device 103 is a three-way device, one end of the connecting device is communicated with the connecting rod 104, and the other two ends of the connecting device are respectively communicated with the two semi-rings and are movably connected with at least one of the two semi-rings. The movable connection part may be connected by a connecting member, as shown in fig. 5, which is composed of a locking part 1031 and a protruding part 1032; the two can be connected in a lock catch mode or can be connected in a magnetic mode.
Preferably, as described above, the end of the ring body at the notch of the micro-bubble ventilation device 1 is in an openable structure; more preferably, as shown in fig. 6, the end of the ring body at the notch of the micro-bubble ventilation device is provided with a detachable plug 105. In one embodiment of the present invention, the diameter of the ring of the microbubble ventilation device 1 is 570mm, the length of the notch is 80mm, the distance between the micropores 102 is 2.5mm, and the diameter of the micropores 102 is 0.05mm to 0.2mm.
As shown in fig. 1, the air inlet 101 of the micro-bubble ventilation device 1 is respectively connected with the oxygen supply device 10, the carbon dioxide supply device 9 and the air supply device 8 through valves;
among these, the gas is preferably in a sterile compressed gas state when stored in the gas supply device. Oxygen and carbon dioxide are common gases in cell culture.
Preferably, the stirring device 2 comprises a stirring shaft 201 and a stirring paddle 202;
the stirring shaft 201 is perpendicular to the horizontal plane and fixed at the bottom of the tank, the stirring paddle 202 is arranged on the stirring shaft 201, and the bottom of the stirring shaft 201 is connected with the power device 3;
the microbubble aeration device 1 is arranged right below the stirring paddle 202 and surrounds the stirring shaft 201; preferably, the micropores 102 of the microbubble aeration device 1 are disposed downward.
Preferably, the power device 3 is a magnetic stirring device.
The animal cell reactor has very strict requirements on sterility, and operations such as loading, sterilization, inoculation and the like, mechanical sealing devices and the like can bring about the risk of bacteria contamination, and once bacteria contamination inside the reactor is caused, huge economic loss is caused. The invention develops a magnetic transmission stirring system with complete isolation function for a bioreactor for culturing 6000L animal cells, and the power transmission of the magnetic transmission stirring system completely depends on electromagnetic force generated by a magnetic cylinder, so that bacteria contamination caused by sealing problem is thoroughly avoided.
The microbubble aeration device 1 is sleeved around the stirring shaft 201 through a notch thereof.
Preferably, the stirring paddles 202 are three, and a first stirring paddle, a second stirring paddle and a third stirring paddle are sequentially arranged from top to bottom on the stirring shaft, and the second stirring paddle stirs liquid to move from top to bottom; the first stirring paddles and the third stirring paddles stir the liquid to move from bottom to top.
Through the arrangement mode, oxygen released by the micro-bubble ventilation device 1 can be trapped between the first stirring paddle and the third stirring paddle (the spiral liquid flow is formed between the three stirring paddles), so that the residence time of the oxygen micro-bubbles in the culture solution is prolonged, and the utilization rate of the oxygen is increased.
Preferably, the stirring paddle 202 is a turbine type stirring paddle or a propulsion type stirring paddle;
the blades of the propelling type stirring paddle have a certain radian, so that the culture solution can form axial flow, and the stirring and suspending effects are more beneficial.
More preferably, the paddles 202 are four-bladed propeller paddles.
Preferably, the blade inclination angle of the stirring paddle is 40-60 degrees; more preferably 50 °; since each paddle causes the direction of movement of the liquid stream to be different, it is preferable that the deflection angle of the second paddle is mirror symmetrical to the deflection angles of the first and third paddles.
Preferably, the ratio of the diameter of the stirring paddle to the diameter of the tank is 0.2-0.7, more preferably 0.3-0.5.
Preferably, the rotation speed of the stirring paddle is 30-70 rpm.
Preferably, a baffle plate 4 is further arranged inside the tank wall of the tank body A, and the baffle plate 4 is perpendicular to the horizontal plane;
more preferably, the bottom of the baffle 4 is flush with the third stirring paddle, and the top is higher than the liquid level of the cell culture liquid.
Preferably, 3 to 5 baffles 4, more preferably 4 baffles are uniformly distributed on the tank wall of the tank body;
preferably, as shown in fig. 7, the baffles 4 are deviated from the warp line at which the connection point of the baffles and the tank A is positioned by 30-40 degrees, and the deflection directions of the baffles are consistent;
preferably, the width of the baffle plate is 1/13-1/10 of the tank diameter.
More preferably, the deflection direction of each baffle plate is consistent with the rotation direction of the stirring paddle; for example, the rotation direction of the stirring paddle is anticlockwise, and the deflection direction of the baffle 4 is shown in fig. 7, and the flow of water is deflected at an obtuse angle along the anticlockwise direction.
Because the second stirring paddle and the first stirring paddle and the third stirring paddle stir the liquid in opposite movement directions, the flowing direction of the liquid is more disordered than the same direction (upward stirring) of the three stirring paddles when the culture solution is stirred, and therefore, the stirring mode can greatly increase the dissolved oxygen amount, but also is easier to form turbulence due to the disordered impact of the liquid flow, and further cells are broken and dead. For this reason, the baffle plate 4 is arranged, the deflection angle of the baffle plate is consistent with the rotation direction of the stirring paddle, the baffle plate 4 can play a role in guiding flow, and the baffle plate 4 on the tank wall can slow down the flow velocity of the liquid at the position close to the tank wall of the bioreactor and guide the flow direction of the liquid because the flow velocity of the cell culture liquid at the position close to the tank wall of the bioreactor is the largest, so that the generation of turbulent flow is effectively avoided.
For example, in one embodiment, the bioreactor tank is a 6000L tank and the microbubble aeration device used is a circular ring aeration device as shown in fig. 3. As shown in fig. 1, a stirring device is arranged at the bottom of the tank, the stirring device comprises a stirring shaft and a stirring paddle, the stirring shaft is perpendicular to the horizontal plane and is fixed at the bottom of the tank, the stirring paddle is arranged on the stirring shaft, and the bottom of the stirring shaft is connected with a power device; the stirring shaft is provided with three stirring paddles, namely a first stirring paddle, a second stirring paddle and a third stirring paddle from bottom to top. The rotating speeds are 40 revolutions per minute and anticlockwise. The second stirring paddle stirs the liquid to move from top to bottom; the first stirring paddles and the third stirring paddles stir the liquid to move from bottom to top. The blade inclination angle of the stirring paddle is 50 degrees, and the ratio of the paddle diameter to the tank diameter is 0.4. Four baffles are arranged on the wall of the tank, the deflection directions of the baffles are all anticlockwise deviated from the warp line 35 degrees where the baffles are positioned at the connecting point of the baffles and the tank body, and the width of the baffles is 1/12 of the tank diameter. This example was set as an experimental group, on which a comparative example was set;
comparative example 1: the only difference from the experimental group is the removal of the baffle.
Comparative example 2: the only difference from the experimental group is that the baffle is arranged perpendicular to the tank.
Comparative example 3: the only difference from the experimental group is that all three paddles agitate the broth upward and remove the baffles.
Comparative example 4: the only difference from the experimental group is that the microbubble aeration device is a conventional cylinder with a porous network inside.
The cultured cells were MDCK cells, and after 5 days of culture at a cell culture density of 180 ten thousand/ml, the following data were examined:
note that: cell viability = (total number of cells-number of dead cells)/(total number of cells×100%, calculated by the table phenol blue staining method).
As is evident from comparative examples 1 and 2, the baffle plate was not properly arranged or the baffle plate was removed, and although the amount of oxygen was reduced, more severe turbulence was formed, resulting in a significant decrease in the cell viability. As can be seen from comparative example 3, when the culture solution is stirred up by all three stirring paddles, the residence time of oxygen in the culture solution is shortened, so that the oxygen utilization rate is reduced, and the total oxygen consumption is obviously increased; in addition, the change of the liquid stirring mode is also unfavorable for uniform oxygen dispersion, and the local oxygen concentration is possibly reduced, so that the cell activity is reduced. As is clear from comparative example 4, the conventional microbubble aeration device in the prior art also decreases the cell viability due to uneven air distribution, and increases the amount of oxygen due to the decrease in the oxygen dissolution efficiency. In addition, since the conventional micro-bubble ventilation device adopted in the prior art is not easy to clean, micropores are blocked after the micro-bubble ventilation device is used for a long time, if the air source pressure is increased, the air flow flowing out of the vent holes without blocking is too fast, so that the air utilization rate is reduced, and especially when a 6000L reactor is used for culturing cells, the micro-bubble air inlet can only supply 0-20% of dissolved oxygen of the culture under the condition that the maximum stirring speed of the cells is not damaged, and the 30-50% of dissolved oxygen required by the growth of the cells cannot be achieved.
In summary, as the overall volume increases, the difficulty in oxygen distribution by the cells increases steeply. Since the precedent of using a large-capacity bioreactor (6000L) to culture cells has never been used before in China, optimization and adjustment for large-capacity bioreactor equipment are lacking in the prior art. The applicant of the invention realizes uniform gas distribution of the large-volume bioreactor by adjusting the ventilation device and adapting to the transformation of the stirring system, reduces the consumption of oxygen and improves the survival rate of cells.
2. Alkali supplementing system of bioreactor
The alkali supplementing system mainly comprises an alkali liquor spraying head 5, a stirring device 2 and a baffle plate 4. Preferably, a pH meter is further arranged in the tank A; preferably, the pH meter has two.
The accuracy of the pH in the tank is ensured by adopting 2 pH electrodes, and the operation of equipment is not affected even if one pH meter has a problem and needs to be overhauled.
Wherein, the opening and closing of the alkali liquor spraying head 5, the control of the spraying quantity and the rotating speed of the stirring device 2 can be controlled by an electronic control device, and the control device controls the operation of the devices by reading and analyzing the pH meter.
As shown in FIG. 2, the alkali liquor spraying head 5 sprays the alkali liquor into the culture medium of the tank body in a pulse mode, and the culture medium is rapidly stirred and uniformly mixed in the stirring device 2, so that cell damage caused by local overhigh pH can be avoided.
When the alkali supplementing operation is performed by using the equipment as described above, the valve 501 is intermittently opened so that the alkali liquor is sprayed by the alkali liquor spraying head 5 in a pulse manner, and at the same time, the stirring device 2 is opened so that the supplemented alkali liquor is rapidly and completely mixed.
Preferably, the alkali liquor is NaHCO of 0.8mol/L to 1.2mol/L 3 A solution; more preferably 1mol/L NaHCO 3 A solution.
Preferably, the valve 501 is opened once every 4.5 s-5.5 s, and each time is opened for 50 ms-200 ms; more preferably, the valve 501 is opened every 5 seconds, each time for 100ms to 150ms.
Preferably, when the alkali liquor is sprayed, the pressure of the liquid in the alkali liquor spraying head 5 is 0.08MPa to 0.12MPa; more preferably, the liquid pressure in the lye jet head 5 is 0.10MPa.
Preferably, the rotation speed of the stirring device 2 is 30 rpm-70 rpm when the alkali supplementing operation is performed; more preferably, the rotation speed of the stirring device 2 is 50rpm.
The alkali liquor concentration, the opening and closing time of the valve 501, the rotating speed of the stirring device 2 and the pressure in the alkali liquor spraying head 5 (the outlet diameter of the spraying head is matched, and the quantity of the alkali liquor sprayed out each time is practically limited) are specially set by the applicant in combination with the structure of the alkali replenishing equipment of the bioreactor tank, so that the alkali liquor can be quickly mixed, and the damage to cells is reduced.
In one embodiment, the bioreactor tank is a 6000L tank, and the suspension MDCK cells are cultured. An alkali liquor spraying head is arranged above the tank wall and is connected with a storage tank through a valve by 1.0mol/L NaHCO 3 The containers of solution are connected and the valves are opened once every 5s, each time for 120ms. When the alkali liquor is sprayed, the pressure of the liquid in the alkali liquor spraying head is 1.0MPa, and the diameter of the spraying opening of the alkali liquor spraying head is 2mm. The bottom in the tank is provided with a microbubble ventilation device for ventilation of oxygen, the bottom in the tank is also provided with a stirring device, the stirring device comprises a stirring shaft and a stirring paddle, the stirring shaft is perpendicular to the horizontal plane and is fixed at the bottom of the tank, the stirring paddle is arranged on the stirring shaft, and the bottom of the stirring shaft is connected with a power device; the stirring shaft is provided with three stirring paddles, namely a first stirring paddle, a second stirring paddle and a third stirring paddle which are sequentially arranged from top to bottom, and the stirring shafts rotate clockwise; said firstThe two stirring paddles stir the liquid to move from top to bottom; the first stirring paddles and the third stirring paddles stir the liquid to move from bottom to top. The blade inclination angle of the stirring paddle is 50 degrees, and the ratio of the paddle diameter to the tank diameter is 0.4. When the alkali solution is dripped, the rotating speed of the stirring paddle is 50 revolutions per minute; when the cells are normally cultured and oxygen is introduced, the rotating speed of the stirring paddle is 40 revolutions per minute. Four baffles are arranged on the wall of the tank, the deflection directions of the baffles are all 35 degrees away from the warp line where the baffles are positioned at the connecting point of the baffles and the tank body clockwise, and the width of the baffles is 1/12 of the tank diameter. This example was set as an experimental group, on which a comparative example was set;
comparative example 1: the only difference from the experimental group is the removal of the baffle.
Comparative example 2: removing the alkali liquor spraying head, and replacing an alkali adding mode by a side wall dropwise adding mode in the prior art; the rotation speed of the stirring paddle is always 40 revolutions per minute.
Comparative example 3: the only difference from the experimental group is that the baffle is arranged perpendicular to the tank.
Comparative example 4: the only difference from the experimental group is that all three paddles agitate the broth upward and remove the baffles.
The same batch of MDCK cells were cultured for 5 days at a cell culture density of 180 ten thousand/ml, and the pH was maintained constant at 6.8-7.2 by adding alkali, and the following data were examined:
as is clear from comparative examples 1 and 2, the different addition modes of the lye have a large influence on the cell viability. The wall-attached dripping mode is continuous, so that the concentration of alkali liquor at the dripping position is often overlarge, and more cells die. If the rotation speed is increased, the increase of the cell death rate caused by mechanical damage can be caused; in addition, the dosage of the wall-attached dripping is not controllable every time, and the pH cannot be accurately adjusted. As is evident from comparative examples 1 and 3, the baffle plate was not properly arranged or the baffle plate was removed, and although the amount of oxygen was reduced, more severe turbulence was formed, resulting in a significant decrease in the cell viability. As is clear from comparative example 4, when the culture solution is stirred up by all three stirring paddles, the residence time of oxygen in the culture solution is shortened, so that the oxygen utilization rate is reduced, and the total oxygen consumption is obviously increased; in addition, the change of the liquid stirring mode is not beneficial to uniform oxygen dispersion, and the difficulty of uniform mixing of oxygen is increased due to the adoption of the large-volume bioreactor, so that the local oxygen concentration is possibly reduced, and the cell activity is possibly reduced.
In summary, the invention adopts a pulse alkali liquor replenishing mode and is matched with a specially arranged stirring device and a baffle plate, so that turbulent flow generated during stirring can be effectively reduced, alkali liquor can be rapidly mixed, and cell death caused by mechanical damage and overhigh local alkali liquor concentration can be reduced. In addition, unexpectedly, the unique stirring paddle rotating mode can also prolong the residence time of the oxygen, thereby effectively reducing the consumption of the oxygen.
3. Air intake system
The air intake system mainly comprises a surface air intake system and a deep air intake system. As shown in fig. 1, the deep air intake system mainly comprises an air supply device 8, a micro-bubble ventilation device 1 and an air outlet 7; the surface air intake system mainly comprises an air supply device 8, a surface air inlet 6 and an air outlet 7.
The air introduced by the microbubble aeration device 1 forms a deep air inlet system of the cell culture reactor, and the compressed air introduction mode can prolong the flow path of the air in the culture medium and increase the transfer quantity of carbon dioxide and ammonia.
The method of the invention also introduces compressed air into the liquid phase surface layer, which can avoid the bottom compressed air from dissolving in the culture medium in the flowing process and increase the overflow of the bottom compressed air. Preferably, the surface air inlet 6 is arranged at the side of the tank wall and is higher than the liquid level by 40-60 cm. Compressed air is fed laterally, and exhaust is carried out above the opposite surface, so that the distance of the compressed air above the liquid level is prolonged, and more carbon dioxide can be carried; the position 40-60 cm higher than the liquid level can ensure that the surface air inlet is not directly blown to the culture solution, thereby preventing the generation of foam in the cell culture process.
Preferably, the pressure of the compressed air is 0.02-0.06 MPa, more preferably 0.04MPa, when the surface layer air intake and the deep layer air intake are carried out, and the compressed air is purified by two-stage air filters.
The air inlet system adopted by the invention combines the surface air inlet system and the deep air inlet system, can effectively drive ammonia and excessive carbon dioxide in the culture solution, and can maintain a stable culture environment in the cell culture process.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (20)
1. A6000L serum-free full-suspension cell culture reactor is characterized by comprising a tank body;
a microbubble ventilation device and a stirring device are arranged in the tank body;
a baffle is arranged inside the tank wall of the tank body; 3-5 baffles are uniformly distributed on the tank wall of the tank body; the baffles are deviated from warp lines at which connection points of the baffles and the tank body are located by 30-40 degrees, and the deflection directions of the baffles are consistent;
an alkali liquor spraying head is arranged above the tank wall of the tank body and is connected with an alkali storage container through a valve; when the alkali liquor is sprayed, the pressure of the liquid in the alkali liquor spraying head is 0.08-0.12 MPa; the aperture of an inner hole at the front end of the alkali liquor spraying head is 1 mm-3 mm;
a surface layer air inlet is also arranged above the tank wall of the tank body; the air inlet is connected with the oxygen supply device, the carbon dioxide supply device and the air supply device through valves respectively;
the microbubble ventilation device is a non-closed circular ring with a notch, and the annular wall is provided with an air inlet and a plurality of uniformly distributed micropores; the interior of the ring body of the microbubble ventilation device is hollow, and the micropores penetrate through the annular wall and are communicated with the hollow cavity of the ring body;
the stirring device comprises a stirring shaft and a stirring paddle, and the microbubble ventilation device is arranged right below the stirring paddle.
2. The 6000L serum-free total suspension cell culture reactor according to claim 1, wherein said micro-holes are all distributed on one side of the plane of said circular ring.
3. The 6000L serum-free total suspension cell culture reactor according to claim 2, wherein said microwells are arranged in a row along said circumferential wall.
4. The 6000L serum-free full suspension cell culture reactor of claim 2, wherein the microwells are equidistant from the center of the microbubble aeration device.
5. The 6000L serum-free total suspension cell culture reactor according to claim 2, wherein the air inlet is arranged opposite to the notch, and the air inlet is fixed at the bottom of the bioreactor through a connecting device.
6. The 6000L serum-free total suspension cell culture reactor according to claim 2, wherein said air supply means is further connected to said surface air inlet via a valve.
7. The 6000L serum-free full suspension cell culture reactor according to claim 1, wherein the stirring shaft is perpendicular to a horizontal plane and fixed to a tank bottom, the stirring paddle is arranged on the stirring shaft, and the bottom of the stirring shaft is connected with the power device.
8. The 6000L serum-free full suspension cell culture reactor according to claim 7, wherein three stirring paddles are provided, and a first stirring paddle, a second stirring paddle and a third stirring paddle are sequentially provided from top to bottom; the second stirring paddle stirs the liquid to move from top to bottom; the first stirring paddles and the third stirring paddles stir the liquid to move from bottom to top.
9. The 6000L serum-free full suspension cell culture reactor according to claim 7, wherein the stirring paddle is a turbine type stirring paddle or a propeller type stirring paddle.
10. The 6000L serum-free full suspension cell culture reactor of claim 7, wherein the paddles are four-bladed pusher paddles.
11. The 6000L serum-free full suspension cell culture reactor according to claim 7, wherein the bottom of the stirring shaft is connected with a power device;
the power device is a magnetic stirring device.
12. The 6000L serum-free total suspension cell culture reactor according to claim 1, wherein there are 4 baffles.
13. The 6000L serum-free full suspension cell culture reactor according to claim 1, wherein when the 6000L serum-free full suspension cell culture reactor performs an alkaline supplementing operation, the valve is intermittently opened so that the alkaline liquid injection head injects the alkaline liquid in a pulse manner, and simultaneously the stirring device is opened so that the supplemented alkaline liquid is rapidly and completely mixed.
14. The 6000L serum-free full suspension cell culture reactor of claim 13, wherein the alkali solution is 0.8mol/L to 1.2mol/L NaHCO 3 A solution.
15. The 6000L serum-free full suspension cell culture reactor according to claim 13, wherein the valves are opened once every 4.5 s-5.5 s, 50 ms-200 ms each time.
16. The 6000L serum-free full suspension cell culture reactor according to claim 13, wherein the rotation speed of the stirring device is 30 rpm-70 rpm when the alkali supplementing operation is performed.
17. The 6000L serum-free full suspension cell culture reactor according to claim 1, wherein a surface layer air inlet is further arranged above the tank wall of the tank body, and an air outlet is arranged on a cover at the top of the tank body.
18. The 6000L serum-free full suspension cell culture reactor according to claim 1, wherein a pH meter and a dissolved oxygen amount detection device are further arranged in the tank body.
19. The 6000L serum-free total suspension cell culture reactor according to claim 18, wherein there are two pH meters.
20. The 6000L serum-free total suspension cell culture reactor according to claim 18, wherein there are two dissolved oxygen measuring devices.
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| CN108410734A (en) * | 2018-05-17 | 2018-08-17 | 江苏弗洛瑞生物工程设备有限公司 | A kind of biological reactor for cell culture |
| CN110241023B (en) * | 2019-06-20 | 2020-09-04 | 江南大学 | Bioreactor for high-density large-scale animal cell culture and application |
| CN111528103B (en) * | 2020-07-01 | 2021-10-08 | 广州齐志生物工程设备有限公司 | Plant tissue culture reactor and amplification culture method thereof |
| CN114561265A (en) | 2020-11-27 | 2022-05-31 | 财团法人工业技术研究院 | Cell activation reactor and cell activation method |
| CN112892371A (en) * | 2021-01-15 | 2021-06-04 | 新疆河润水业有限责任公司 | Preparation facilities of microbial deodorant |
| CN113999767A (en) * | 2021-11-02 | 2022-02-01 | 安及义实业（上海）有限公司 | Stainless steel bioreactor for mammalian cell culture and use method thereof |
| CN118165806A (en) * | 2024-05-15 | 2024-06-11 | 上海数郜机电有限公司 | Biological fermentation tank |
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