CN108441424B - Microcarrier biological reaction tank and method for culturing porcine circovirus type 2 by same - Google Patents
Microcarrier biological reaction tank and method for culturing porcine circovirus type 2 by same Download PDFInfo
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- CN108441424B CN108441424B CN201810364663.XA CN201810364663A CN108441424B CN 108441424 B CN108441424 B CN 108441424B CN 201810364663 A CN201810364663 A CN 201810364663A CN 108441424 B CN108441424 B CN 108441424B
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- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000012258 culturing Methods 0.000 title claims abstract description 19
- 241001673669 Porcine circovirus 2 Species 0.000 title claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 94
- 238000005070 sampling Methods 0.000 claims abstract description 72
- 239000000427 antigen Substances 0.000 claims abstract description 55
- 102000036639 antigens Human genes 0.000 claims abstract description 55
- 108091007433 antigens Proteins 0.000 claims abstract description 55
- 238000001914 filtration Methods 0.000 claims abstract description 35
- 238000010438 heat treatment Methods 0.000 claims abstract description 28
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 25
- 238000003306 harvesting Methods 0.000 claims abstract description 20
- 239000002699 waste material Substances 0.000 claims abstract description 19
- 238000003756 stirring Methods 0.000 claims description 32
- 238000009423 ventilation Methods 0.000 claims description 21
- 238000004113 cell culture Methods 0.000 claims description 17
- 239000011229 interlayer Substances 0.000 claims description 16
- 230000001502 supplementing effect Effects 0.000 claims description 15
- 230000001105 regulatory effect Effects 0.000 claims description 14
- 239000007789 gas Substances 0.000 claims description 13
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 9
- 239000001301 oxygen Substances 0.000 claims description 9
- 229910052760 oxygen Inorganic materials 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- 230000029087 digestion Effects 0.000 claims description 6
- 238000007599 discharging Methods 0.000 claims description 6
- 239000011344 liquid material Substances 0.000 claims description 6
- 238000012423 maintenance Methods 0.000 claims description 6
- 108010019160 Pancreatin Proteins 0.000 claims description 5
- 238000007865 diluting Methods 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 229940055695 pancreatin Drugs 0.000 claims description 5
- 238000005273 aeration Methods 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 239000012228 culture supernatant Substances 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 230000001079 digestive effect Effects 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- 238000005086 pumping Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims 5
- 238000009434 installation Methods 0.000 claims 2
- 239000000969 carrier Substances 0.000 abstract description 13
- 239000000243 solution Substances 0.000 description 25
- 239000001963 growth medium Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000000903 blocking effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000010410 layer Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000002572 peristaltic effect Effects 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
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- 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
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
- C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
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- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/22—Transparent or translucent parts
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- 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
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
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- 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
- C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/10011—Circoviridae
- C12N2750/10051—Methods of production or purification of viral material
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Abstract
The invention discloses a microcarrier biological reaction tank and a method for culturing porcine circovirus type 2 by the microcarrier biological reaction tank.A sandwich layer is arranged on the side wall of a tank body, and jacket water for heating the sandwich layer by a heating pipe is arranged in the sandwich layer to heat liquid in the tank body; the telescopic tube in the tank body is used for sampling, the sampling position can be adjusted according to the quantity of the culture carrier, and the operation is convenient; the observation with a transparent observation area is additionally arranged and connected with the tank body, and the carrier cells can flow into the transparent tube by matching with the corresponding filter gas head and the switch valve body, so that the state of the cells can be observed at any time under a microscope, and the residual gas can be returned into the tank after observation without waste; a carrier filter screen is arranged on the side surface of the bottom of the reaction tank, and is matched with a vent pipe, so that the filter screen is prevented from being blocked by the carrier when the carrier is removed by final antigen during harvesting. The invention improves the microcarrier biological reaction tank culture device, facilitates the real-time observation of cells in the tank, facilitates the drainage of waste liquid, facilitates the filtration of carriers, improves the efficiency, improves the cell density for 24 hours, and cultures cells with better states.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a microcarrier biological reaction tank and a method for culturing porcine circovirus type 2 by using the same.
Background
The traditional microcarrier biological reaction tank is used for heating the liquid in the tank in a mode of heating an electric blanket, the electric blanket is stuck outside the wall of the reaction tank and is heated to the liquid in the tank through heat conduction, and when the heating is just started, the temperature of the electric blanket is very high and directly contacts the surface of the tank body, so that the temperature on the wall in the tank is too high, and the cell sticking wall and the growth on the carrier in the tank can be influenced.
The length of the sampling tube in the tank is fixed, when the cell in the tank is cultured for 24 hours, the liquid is required to be changed, the nutrient solution in the tank is discharged, then new nutrient solution is added, when the nutrient solution in the tank is discharged, the length of the sampling tube is required to be adjusted before the reaction tank is assembled and sterilized, the adjusted length can only be estimated according to the amount of the carrier used by people, and the adjustment is inaccurate in advance, the liquid cannot be thoroughly discharged or the carrier is discharged, and part of the carrier is lost.
When observing cells on carriers in the tank, the carrier cells are required to be taken out of the tank for observation, the operation is quite vexation and locking, the actual state of the observed cells is influenced, all culture solution including the carriers is required to be discharged out of the tank when the final antigen is obtained, then the obtained mixed solution of the antigen and the carriers is filtered through a filter screen, the time and the labor are wasted, the carrier cells are taken out of the tank for observation, on one hand, the carrier cells are wasted, on the other hand, the cells are taken out to generate some changes, and the observed cell state has deviation.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a microcarrier biological reaction tank and a method for culturing porcine circovirus type 2 by using the same.
The method improves a microcarrier biological reaction tank culture device, facilitates the real-time observation of cells in the tank, facilitates the discharge of waste liquid, facilitates the filtration of carriers, improves the efficiency, improves the cell adherence rate, improves the cell density for 24 hours, and cultures cells with better states.
The technical scheme adopted by the invention is as follows:
a microcarrier biological reaction tank comprises a tank body, wherein a stirrer, a biological reaction detection electrode and a defoaming device are arranged in the tank body, the upper surface of the tank body is correspondingly provided with a stirrer mounting port, an electrode interface and a defoaming device interface, the stirrer is used for stirring liquid in the tank body, the bottom of the tank body is provided with a discharge port, a discharge pipe extends out of the discharge port, a discharge valve is arranged on the discharge pipe, the inner wall of the tank body is provided with a filter screen corresponding to the discharge port,
the upper surface of the tank body is also provided with an air inlet interface and a liquid material interface, the air inlet interface is used for introducing air to different positions of the tank body, the liquid material interface comprises a liquid supplementing port, a sampling port and more than two feeding ports, the liquid supplementing port is used for supplementing culture liquid in the culture process, the feeding ports are used for adding culture materials into the tank body,
an adjusting rod is arranged in the sampling port, one end of the adjusting rod extends into the tank body, the other end of the adjusting rod is sleeved with a limiting sleeve, the limiting sleeve limits the other end of the adjusting rod at the sampling port, the length of the adjusting rod extending into the tank body is changed by adjusting the position of the limiting sleeve, one end of the adjusting rod is sleeved with a telescopic pipe, one end of the telescopic pipe is fixed at one end of the adjusting rod, the other end of the telescopic pipe is connected with the sampling pipe,
the sampling tube is provided with a first filtering gas head and a first switch valve body, the first switch valve body is arranged at one end of the first filtering gas head corresponding to the sampling port, the other end of the sampling tube is respectively connected with one end of the second branch tube and one end of the observation tube, the other end of the second branch tube is provided with a collecting bottle, the collecting bottle is provided with a fourth filtering gas head, and the second branch tube is provided with a fourth switch valve body; the observation tube is horizontally arranged and is provided with a transparent observation area, and the other end of the observation tube is connected with a second filtering gas head through a third switch valve body;
the side wall of the tank body is provided with an interlayer space, jacket water is filled in the interlayer space, a heating pipe is arranged in the interlayer space to heat the jacket water, two through holes communicated with the interlayer space are formed in the outer side face of the side wall of the tank body at intervals, and the two through holes are respectively connected to two ends of a circulating pump.
Further, the air inlet interface comprises a respirator interface, a deep air inlet interface, a surface air inlet interface and a filter screen air inlet interface, and a vent pipe is led into the tank body through the filter screen air inlet interface and is opposite to the filter screen for air outlet.
Further, the stirrer comprises a stirring shaft, a plurality of stirring paddles are distributed on the stirring shaft, a stirrer mounting opening is formed in the center of the upper surface of the tank body, one end of the stirring shaft is fixed to the stirring shaft mounting opening, the stirring shaft and the stirring paddles form the stirrer, and the stirrer is vertically arranged in the tank body.
Further, the other end of observation tube still connects the one end of first branch pipe, is equipped with the second switch valve body on the first branch pipe, and the other end of first branch pipe is equipped with reserve sample reserving bottle, is equipped with the third on the reserve sample reserving bottle and filters the air head.
Further, the sampling tube, the first branch tube and the second branch tube are all flexible tubes, and the first switch valve body, the second switch valve body, the third switch valve body and the fourth switch valve body are all air clamps; or the sampling tube, the first branch tube and the second branch tube are hard pipelines, and the first switch valve body, the second switch valve body, the third switch valve body and the fourth switch valve body are all air valves.
The invention also discloses a method for culturing porcine circovirus type 2 by using the microcarrier biological reaction tank, which is characterized by comprising the following steps of: which comprises the following steps:
step 1, a biological reaction tank is started to preheat:
connecting a PH electrode, a temperature electrode and an oxygen dissolving electrode of a biological reaction tank well, adding jacket water into an interlayer space of a tank body, switching on a heating pipe and starting a power supply for heating, and starting a circulating pump to circulate the jacket water in the interlayer space so as to ensure that the temperature of the tank body is uniform;
step 2, cell digestion of the bottle rotating:
selecting a bottle-rotating PK-15 cell with good cell state, digesting the cell by using pancreatin digestive juice, and fully shaking the digested cell;
step 3, cell count: diluting a proper amount of digested cells with a culture solution, adding a proper amount of diluted cells to a cell counting plate for cell counting after diluting, and calculating the cell density of the cell solution;
step 4, cell loading and culturing: adding microcarrier into a biological reaction tank according to 3-5 g/L, and adding cells according to 30-50 cells/microcarrier; cell culture parameters of corresponding devices of the biological reaction tank are regulated: starting an aeration system to carry out aeration culture on the biological reaction tank through an air inlet interface;
step 5, liquid replacement is carried out for 24 hours: after the cells in the biological reaction tank are cultured for 24 hours, stopping stirring, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic tube to ensure that the sampling height of the lower port of the telescopic tube is 0.5cm above the liquid level of the carrier, discharging the cell culture solution, and then adding an equal amount of cell maintenance solution through a fluid supplementing port for continuous culture;
step 6, cell observation: after the cells are cultured for 48 hours, the first switch valve body and the third switch valve body are opened while stirring is maintained, the second switch valve body and the fourth switch valve body are closed, the air suction is stopped when the filter air head is used for sucking air in the injector, the carrier and the cells of the biological reaction tank body flow through the transparent observation area, the first switch valve body is closed, the cell observation area is placed under a microscope for observation, after observation, the first switch valve body K1 is opened, the injector is used for pumping air in the filter air head, and the carrier and the cells of the transparent observation area and the related pipeline part are pumped into the tank body through the original route sampling opening;
step 7, harvesting antigen for the first time: stopping stirring after the cells are cultured for 72 hours, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic tube to the position 0.5cm above the carrier in the tank, collecting the first antigen, placing the harvested antigen liquid at-15 ℃ for freezing preservation in a freezer, and then adding an equal amount of cell maintenance liquid through a liquid supplementing port for continuous culture;
step 8, harvesting the antigen for the second time and culturing in a liquid change mode: stopping stirring after the cells of the biological reaction tank are cultured for 96 hours, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic tube to the position 0.5cm above the carrier in the tank, harvesting the antigen liquid of the culture supernatant again through a sampling port, and freezing and preserving the harvested second antigen liquid in a freezer below minus 15 ℃;
step 9, harvesting antigen for the third time: after the cells in the biological reaction tank are cultured for 96-120h, the revolution is regulated to 100-150 revolutions for 3-5 minutes, then stirring is stopped, standing is carried out for 3-5 minutes, ventilation and pressurization are carried out on the tank body from an air inlet interface through a ventilation system, a discharge valve on a discharge pipe is opened to harvest filtered antigens, meanwhile, a ventilation pipe above a filter screen is opened for air inlet, and finally the harvested third antigen liquid is placed in a freezer below minus 15 ℃ for freezing preservation.
Further, in step 2, digestion of the cells was performed using 0.25% pancreatin digest.
Further, in step 4, the specific method for adjusting the cell culture parameters of the corresponding device comprises: the temperature in the tank is regulated to be 36.5-37.5 ℃ by a temperature electrode, the dissolved oxygen is regulated to be 50-60% by an oxygen dissolving electrode, the PH is regulated to be 7.2-7.4 by a PH electrode, and the revolution of the stirrer is regulated to be 40-60r/min.
Further, the specific method for draining the cell culture fluid in the step 5 comprises: the first switch valve body and the fourth switch valve body are opened, the second switch valve body and the third switch valve body are closed, the ventilation system ventilates and pressurizes the tank body through the air inlet interface, the waste liquid bottle is used as a collecting bottle, and the culture solution above 0.5cm above the liquid level of the carrier continuously flows into the collecting bottle through the telescopic pipe through the sampling pipe under the action of the pressure because the pressure in the tank body is higher than the pressure outside the tank body until the liquid level of the culture solution is lower than the sampling height of the lower port of the telescopic pipe; at this time, the first switch valve body is closed, and simultaneously, the air filtering head and the air filtering head are inflated by using the injector respectively, and the cell culture solution in the transparent observation area and the relevant pipeline part is pumped into the collecting bottle.
Further, specific methods for collecting the first antigen and the first antigen: the first switch valve body and the fourth switch valve body are opened, the second switch valve body and the third switch valve body are closed, the ventilation system ventilates and pressurizes the tank body through the air inlet interface, the antigen bottle is used as a collecting bottle, and the culture solution with the pressure higher than 0.5cm above the liquid level of the carrier flows through the sampling tube continuously under the action of the pressure through the telescopic tube and flows into the collecting bottle until the liquid level of the culture solution is lower than the sampling height of the lower port of the telescopic tube; at this time, the first switch valve body is closed, and simultaneously, the air filtering head and the air filtering head are inflated by using the injector respectively, and the cell culture solution in the transparent observation area and the relevant pipeline part is pumped into the collecting bottle.
According to the technical scheme, the heating mode of liquid in the biological reaction tank is designed to be that the reaction tank is two layers, a jacket is arranged in the middle of the reaction tank, a heating pipe is arranged on the jacket, water of the jacket is circulated continuously through a peristaltic pump, and the liquid in the reaction tank is heated by the water of the jacket. The sampling tube is partially used as a retractable tube, and when the amount of the culture medium is large, the tube is lifted to a proper position when the waste liquid is discharged, and when the amount of the culture medium is large, the tube is lowered to a proper position when the waste liquid is discharged, so that the medium is not discharged. The sampling tube in the outer part of the canister is connected to a device which is stopped when the carrier cells flow through the device and the device is placed on a microscope to observe the cell status. Meanwhile, the residual liquid can be taken back into the tank after observation, so that waste is avoided. A carrier filter screen is arranged on the side face of the bottom of the reaction tank, a vent pipe is arranged above the filter screen, when the carrier is removed by final antigen during harvesting, the gas above the filter screen is started, the filter screen is not blocked by the carrier during filtering, and the filtering is smooth. The invention has the following advantages: 1. the sampling port in the tank is designed to be liftable and adjustable, and the height of the sampling port can be adjusted according to the liquid level of the carrier quantity when waste liquid is discharged, so that the operation is convenient, and meanwhile, the liquid can be discharged more thoroughly. 2. The heating pipe is used for heating the jacket water in the heating tank, the jacket water circularly flows through the pump, and the jacket water is used for heating the liquid in the heating tank in a water bath, so that the temperature of the liquid in the heating tank is uniform and the liquid in the heating tank cannot be locally too high. 3. On-line observation cells are connected with the inside of the tank through a pipeline arranged outside the tank, a customized transparent glass tube is additionally arranged on the pipeline arranged outside the tank, carrier cells in the tank can flow into the transparent tube through suction, the state of the cells can be observed at any time under a microscope, and residual cells can be returned to the tank after observation without waste. 4. A filtering port is arranged on the side face of the bottom of the tank, a carrier filter screen is arranged on the filtering port, carriers can be removed through the filter screen in the process of harvesting antigen liquid, the filter screen is easy to be blocked by the carriers in the filtering process, an air inlet pipe is additionally arranged above the filter screen, ventilation is started in the filtering process, the carriers of the filter screen can be discharged without blocking the filter screen, and the antigen can be harvested smoothly.
In the prior art, 2-3 persons are required for operation after the carrier and the antigen are harvested, and only 1 person is required for operation when the antigen is harvested by adopting the invention, so that the efficiency is improved, and the labor force is saved. The invention improves the microcarrier biological reaction tank culture device, facilitates the real-time observation of cells in the tank, facilitates the drainage of waste liquid, facilitates the filtration of carriers, improves the efficiency, improves the cell adherence rate, improves the cell density for 24 hours, and cultures cells with better states.
Drawings
The invention is described in further detail below with reference to the drawings and detailed description;
FIG. 1 is a schematic diagram of a microcarrier bioreactor tank according to the present invention;
FIG. 2 is a schematic cross-sectional view of a microcarrier bioreactor tank according to the present invention.
Detailed Description
As shown in fig. 1 or 2, the invention discloses a microcarrier biological reaction tank, which comprises a tank body 1, wherein a stirrer, a biological reaction detection electrode and a defoaming device are arranged in the tank body 1, the upper surface of the tank body 1 is correspondingly provided with a stirrer mounting port 11, an electrode port and a defoaming device port 12, the stirrer is used for stirring liquid in the tank body 1, the bottom of the tank body 1 is provided with a discharge port 13, the discharge port 13 is extended with a discharge pipe 2, a discharge valve 21 is arranged on the discharge pipe 2, the inner wall of the tank body 1 is correspondingly provided with a filter screen 22 corresponding to the discharge port 13,
the upper surface of the tank body 1 is also provided with an air inlet interface and a liquid material interface, the air inlet interface is used for introducing air to different positions of the tank body 1, the liquid material interface comprises a liquid supplementing port 14, a sampling port 15 and more than two feeding ports 10, the liquid supplementing port 14 is used for supplementing culture liquid in the culture process, the feeding ports 10 are used for adding culture materials into the tank body 1,
the sampling port 15 is internally provided with an adjusting rod 3, one end of the adjusting rod 3 extends into the tank body 1, the other end of the adjusting rod 3 is sleeved with a limiting sleeve 31, the limiting sleeve 31 limits the other end of the adjusting rod 3 at the sampling port 15, the length of the adjusting rod 3 extending into the tank body 1 is changed by adjusting the position of the limiting sleeve 31, one end of the adjusting rod 3 is sleeved with a telescopic pipe 4, one end of the telescopic pipe 4 is fixed at one end of the adjusting rod 3, the other end of the telescopic pipe 4 is connected with the sampling pipe 5,
the sampling tube 5 is provided with a first filter gas head F1 and a first switch valve body K1, the first switch valve body K1 is arranged at one end of the first filter gas head F1 corresponding to the sampling port 15, the other end of the sampling tube 5 is respectively connected with one end of the second branch tube 30 and one end of the observation tube 6, the other end of the second branch tube 30 is provided with a collecting bottle 40, the collecting bottle 40 is provided with a fourth filter gas head F4, and the second branch tube 30 is provided with a fourth switch valve body K4; the observation tube 6 is horizontally arranged, the observation tube 6 is provided with a transparent observation area 61, and the other end of the observation tube 6 is connected with a second filter air head F2 through a third switch valve body K3;
the side wall of the tank body 1 is provided with an interlayer space, jacket water is filled in the interlayer space, a heating pipe 60 is arranged in the interlayer space to heat the jacket water, two through holes 22 communicated with the interlayer space are formed in the outer side face of the side wall of the tank body 1 at intervals, and the two through holes 22 are respectively connected to two ends of a circulating pump 23.
Further, the biological reaction detecting electrode comprises a PH electrode, a temperature electrode and an oxygen dissolving electrode, and the electrode interface comprises a PH electrode interface 17, a temperature electrode interface 18 and an oxygen dissolving electrode interface 19.
Further, the air intake interfaces include a respirator interface 24, a deep air intake interface 25, and a surface air intake interface 26.
Further, the air inlet port further comprises a filter inlet port 27, and a vent pipe 50 is led into the tank 1 through the filter inlet port 27 and is discharged against the filter screen 22.
Further, the stirrer comprises a stirring shaft 28, a plurality of stirring paddles 29 are distributed on the stirring shaft 28, a stirrer mounting opening 11 is formed in the center of the upper surface of the tank body 1, one end of the stirring shaft 28 is fixed to the stirring shaft 28 mounting opening, the stirring shaft 28 and the stirring paddles 29 form a stirrer, and the stirrer is vertically arranged in the tank body 1.
Further, the other end of the collecting bottle observation tube 6 is further connected with one end of a first branch tube 7, a second switch valve body K2 is arranged on the first branch tube, a standby sample reserving bottle 9 is arranged at the other end of the first branch tube 7, and a third filtering air head F3 is arranged on the standby sample reserving bottle 9. When the standby sample reserving bottle 9 is arranged and a small batch of tests are needed, the standby sample reserving bottle 9 is used for collecting samples.
Further, the sampling tube 5, the first branch tube 7 and the second branch tube 30 are all flexible tubes, and the first switch valve body K1, the second switch valve body K2, the third switch valve body K3 and the fourth switch valve body K4 are all air clamps.
Further, the sampling tube 5, the first branch tube 7 and the second branch tube 30 are hard pipelines, and the first switch valve body K1, the second switch valve body K2, the third switch valve body K3 and the fourth switch valve body K4 are all air valves.
Further, the invention further discloses a method for culturing porcine circovirus type 2 by using the microcarrier biological reaction tank, which comprises the following steps:
step 1, a biological reaction tank is started to preheat: the PH, temperature and dissolved oxygen electrodes of the biological reaction tank are connected, the water of the jacket of the reaction tank is added, the heating pipe 60 is connected, the power supply is started for heating, and the circulating pump 23 is started to circulate the water in the tank to make the temperature uniform.
Step 2, cell digestion of the bottle rotating: selecting bottle-rotating PK-15 cells with good cell state, using 0.25% pancreatin digestive juice to digest the cells, and fully shaking the digested cells.
Step 3, cell count: diluting the digested cells with a maintaining solution, adding the diluted cells to a cell counting plate for cell counting, and calculating the cell density of the cell solution.
Step 4, cell loading and culturing: adding microcarrier into a biological reaction tank according to 3-5 g/L, and adding cells according to 30-50 cells/microcarrier;
calculating the required cell quantity to be used according to the number of microcarriers in the reaction tank, the number of cells per ball and the cell density after digestion of the rotary bottle, and adding the required cell quantity into the biological reaction tank through a pipeline corresponding to the cell bottle and the feed inlet; cell culture parameters of the biological reaction tank related devices are regulated: temperature: 36.5-37.5 ℃, DO:50% -60%, PH:7.2-7.4, revolutions: and (3) selecting a ventilation system mode (Air/O2/CO 2) at 40-60r/min, and starting the ventilation system to perform ventilation culture on the biological reaction tank through the Air inlet interface.
Step 5, liquid replacement is carried out for 24 hours: after the cells in the biological reaction tank are cultured for 24 hours, stopping stirring, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic pipe 4, enabling the sampling height of the lower port of the telescopic pipe 4 to be 0.5cm above the liquid level of the carrier, discharging the cell culture solution, and then adding an equal amount of cell maintenance solution through the fluid supplementing port 14 for continuous culture.
The specific method for discharging the cell culture solution in the step 5 comprises the following steps: the first switch valve body K1, the third switch valve body K3 and the fourth switch valve body K4 are opened, the second switch valve body K2 is closed, the ventilation system ventilates and pressurizes the tank body through the air inlet interface, the waste liquid bottle is used as a collecting bottle, and the culture solution with the pressure higher than 0.5cm above the liquid level of the carrier flows through the sampling tube under the action of the pressure and continuously flows into the collecting bottle through the telescopic tube until the liquid level of the culture solution is lower than the sampling height of the lower port of the telescopic tube 4; at this time, the first switch valve body K1 is closed, and the cell culture liquid in the transparent observation area 61 and the relevant pipe portion is pumped into the collection bottle by using the injectors for inflation in the filter air head F1 and the filter air head F2, respectively.
Step 6, cell observation: after the cells are cultured for 48 hours, the first switch valve body K1 and the third switch valve body K3 are opened while stirring is maintained, the second switch valve body K2 and the fourth switch valve body K4 are closed, the air suction is stopped when the filter air head F2 is used in an injector, the carrier and the cells of the biological reaction tank body 1 flow through the transparent observation area 61, the first switch valve body K1 is closed, the cell observation area is placed under a microscope for observation, after the observation, the first switch valve body K1 is opened, the injector is used for pumping air in the filter air head F2, and the carrier and the cells of the transparent observation area 61 and the related pipeline part are pumped into the tank body from an original path through a sampling port.
Step 7, harvesting antigen for the first time: after the cells are cultured for 72 hours, stopping stirring, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic tube 4 to the position 0.5cm above the carrier in the tank, collecting the first antigen, placing the harvested antigen liquid at-15 ℃ for freezing preservation by a refrigerator, and then adding an equal amount of cell maintenance liquid through the liquid supplementing port 14 for continuous culture.
Specific methods for collecting first antigen: the first switch valve body K1 and the fourth switch valve body K4 are opened, the second switch valve body K2 and the third switch valve body K3 are closed, the ventilation system ventilates and pressurizes the tank body through the air inlet interface, the antigen bottle is used as a collecting bottle, and the culture solution with the pressure higher than 0.5cm above the liquid level of the carrier flows through the sampling tube continuously into the collecting bottle under the action of the pressure through the telescopic tube due to the fact that the pressure in the tank body is higher than the pressure outside the tank body until the liquid level of the culture solution is lower than the sampling height of the lower port of the telescopic tube 4; at this time, the first switch valve body K1 is closed, and the cell culture liquid in the transparent observation area 61 and the relevant pipe portion is pumped into the collection bottle by using the injectors for inflation in the filter air head F1 and the filter air head F2, respectively.
Step 8, harvesting the antigen for the second time and culturing in a liquid change mode: after the cells in the biological reaction tank are cultured for 96 hours, stopping stirring, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic tube 4 to the position 0.5cm above the carrier in the tank, harvesting the antigen liquid of the culture supernatant again through the sampling port 15, and placing the harvested second antigen liquid at the temperature of minus 15 ℃ in a freezer for freezing and preserving.
Specific methods for collecting the second antigen: the first switch valve body K1 and the fourth switch valve body K4 are opened, the second switch valve body K2 and the third switch valve body K3 are closed, the ventilation system ventilates and pressurizes the tank body through the air inlet interface, the antigen bottle is used as a collecting bottle, and the culture solution with the pressure higher than 0.5cm above the liquid level of the carrier flows through the sampling tube continuously into the collecting bottle under the action of the pressure through the telescopic tube due to the fact that the pressure in the tank body is higher than the pressure outside the tank body until the liquid level of the culture solution is lower than the sampling height of the lower port of the telescopic tube 4; at this time, the first switch valve body K1 is closed, and the cell culture liquid in the transparent observation area 61 and the relevant pipe portion is pumped into the collection bottle by using the injectors for inflation in the filter air head F1 and the filter air head F2, respectively.
Step 9, harvesting antigen for the third time: after the cells in the biological reaction tank are cultured for 96-120h, the revolution is regulated to 100-150 revolutions for 3-5 minutes, then stirring is stopped, standing for 3-5 minutes, ventilation and pressurization are carried out on the tank body from an air inlet interface through a ventilation system, a discharge valve 21 on a discharge pipe 2 is opened to harvest filtered antigens, a ventilation pipe 50 above a filter screen is opened to perform air inlet, the filtered antigens are ensured to be harvested, sample retention is carried out to detect antigen titer, the antigens are smoothly harvested, and finally harvested third antigen liquid is placed at minus 15 ℃ and is frozen and stored in a refrigerator.
The method for culturing the porcine circovirus type 2 by using the microcarrier biological reaction tank has the following advantages:
1. improving the cell adherence efficiency and increasing the cell density after adherence. The novel reaction tank is used for culturing cells, and can be used for extractingThe adherence efficiency of the cells is high, the adherence rate can be maintained to be more than 95%, dead cells of liquid are fewer in the culture process, the cell density is improved after adherence, and the cell density is higher than 2.00 multiplied by 10 in 24 hours 6 And each ml.
Table one shows a comparison of cell attachment rates and cell densities for 24h for 5 experiments using a 10 liter microcarrier bioreactor tank culture volume according to the present invention.
List one
2. The operation is convenient, the time and the material consumption are saved. The height of the sampling port can be directly adjusted when the reaction tank is used for discharging waste liquid, the reaction tank is convenient and quick, the carrier is not easy to discharge, the material is not wasted, the cell state can be observed on line, the carrier cells can flow back into the tank after the observation, the tank does not need to be taken out for observation, and the carrier and the cells are not wasted.
The carrier cell loss rate of different 5 batches of experiments using a 10 liter microcarrier bioreactor tank to culture 5 liter cells was almost zero and negligible.
3. Efficiency is improved, and labor force is saved. In the prior art, 2-3 persons are required for operation after the carrier and the antigen are harvested, and only 1 person is required for operation when the antigen is harvested by adopting the invention, so that the efficiency is improved, the time of 1 hour can be saved, and the labor force is saved.
According to the technical scheme, the heating mode of liquid in the biological reaction tank is designed to be that the reaction tank is two layers, a jacket is arranged in the middle of the reaction tank, a heating pipe 60 is arranged on the jacket, water in the jacket is circulated continuously through a peristaltic pump, and the liquid in the tank is heated by the water in the jacket. The sampling tube is partially used as a retractable tube, and when the amount of the culture medium is large, the tube is lifted to a proper position when the waste liquid is discharged, and when the amount of the culture medium is large, the tube is lowered to a proper position when the waste liquid is discharged, so that the medium is not discharged. The sampling tube 5 in the outer part of the canister is connected to a device which is stopped when the carrier cells flow through the device and the device is placed on a microscope to observe the cell status. Meanwhile, the residual liquid can be taken back into the tank after observation, so that waste is avoided. A carrier filter screen is arranged on the side surface of the bottom of the reaction tank, a vent pipe 50 is arranged above the filter screen, and when the carrier is removed by final antigen during harvesting, the gas above the filter screen is opened, so that the filter screen is not blocked by the carrier during filtering, and the filtering is smooth. The invention has the following advantages: 1. the in-tank sampling port 15 is designed to be capable of being adjusted in a lifting manner, the height of the sampling port 15 can be adjusted according to the liquid level of the carrier amount when waste liquid is discharged, and the operation is convenient, and meanwhile liquid can be discharged more thoroughly. 2. The heating pipe 60 is used for heating jacket water in the tank, the jacket water circularly flows through the pump, and the jacket water is used for heating the liquid in the tank in a water bath, so that the temperature of the liquid in the tank is uniform and the liquid in the tank cannot be locally too high. 3. On-line observation cells are connected with the inside of the tank through a pipeline arranged outside the tank, a customized transparent glass tube is additionally arranged on the pipeline arranged outside the tank, carrier cells in the tank can flow into the transparent tube through suction, the state of the cells can be observed at any time under a microscope, and residual cells can be returned to the tank after observation without waste. 4. A filtering port is arranged on the side face of the bottom of the tank, a carrier filter screen is arranged on the filtering port, carriers can be removed through the filter screen in the process of harvesting antigen liquid, the filter screen is easy to block by the carriers in the filtering process, an air inlet pipe is additionally arranged above the filter screen, ventilation is started in the filtering process, the carriers at the filtering port can be discharged without blocking the filtering port, and the antigen can be harvested smoothly. In the prior art, 2-3 persons are required for operation after the carrier and the antigen are harvested, and only 1 person is required for operation when the antigen is harvested by adopting the invention, so that the efficiency is improved, and the labor force is saved. The invention improves the microcarrier biological reaction tank culture device, facilitates the real-time observation of cells in the tank, facilitates the drainage of waste liquid, facilitates the filtration of carriers, improves the efficiency, improves the cell adherence rate, improves the cell density for 24 hours, and cultures cells with better states.
Claims (9)
1. A microcarrier biological reaction tank, characterized in that: the device comprises a tank body, wherein the tank body is internally provided with a stirrer, a biological reaction detection electrode and a defoaming device, the upper surface of the tank body is correspondingly provided with a stirrer mounting port, an electrode interface and a defoaming device interface, the stirrer is used for stirring liquid in the tank body, the bottom of the tank body is provided with a discharge port, a discharge pipe extends out of the discharge port, a discharge valve is arranged on the discharge pipe, the inner wall of the tank body is correspondingly provided with a filter screen corresponding to the discharge port,
the upper surface of the tank body is also provided with an air inlet interface and a liquid material interface, the air inlet interface is used for introducing air to different positions of the tank body, the liquid material interface comprises a liquid supplementing port, a sampling port and more than two feeding ports, the liquid supplementing port is used for supplementing culture liquid in the culture process, the feeding ports are used for adding culture materials into the tank body,
an adjusting rod is arranged in the sampling port, one end of the adjusting rod extends into the tank body, the other end of the adjusting rod is sleeved with a limiting sleeve, the limiting sleeve limits the other end of the adjusting rod at the sampling port, the length of the adjusting rod extending into the tank body is changed by adjusting the position of the limiting sleeve, one end of the adjusting rod is sleeved with a telescopic pipe, one end of the telescopic pipe is fixed at one end of the adjusting rod, the other end of the telescopic pipe is connected with the sampling pipe,
the sampling tube is provided with a first filtering gas head and a first switch valve body, the first switch valve body is arranged at one end of the first filtering gas head corresponding to the sampling port, the other end of the sampling tube is respectively connected with one end of the second branch tube and one end of the observation tube, the other end of the second branch tube is provided with a collecting bottle, the collecting bottle is provided with a fourth filtering gas head, and the second branch tube is provided with a fourth switch valve body; the observation tube is horizontally arranged and is provided with a transparent observation area, and the other end of the observation tube is connected with a second filtering gas head through a third switch valve body;
the side wall of the tank body is provided with an interlayer space, jacket water is filled in the interlayer space, a heating pipe is arranged in the interlayer space to heat the jacket water, two through holes communicated with the interlayer space are formed in the outer side face of the side wall of the tank body at intervals, and the two through holes are respectively connected to two ends of a circulating pump.
2. A microcarrier bioreactor tank as in claim 1, wherein: the air inlet port comprises a respirator port, a deep air inlet port, a surface air inlet port and a filter screen air inlet port, and a vent pipe is communicated into the tank body through the filter screen air inlet port and is opposite to the filter screen for air outlet.
3. A microcarrier bioreactor tank as in claim 1, wherein: the agitator includes the (mixing) shaft, and the last distribution of (mixing) shaft has a plurality of paddles, and the center department of jar upper surface is located to the agitator installation mouth, and the one end of (mixing) shaft is fixed in the (mixing) shaft installation mouth, and (mixing) shaft and paddle constitute the agitator, and the agitator is vertical setting in jar internal.
4. A microcarrier bioreactor tank as in claim 1, wherein: the other end of observation tube still connects the one end of first branch pipe, is equipped with the second switch valve body on the first branch pipe, and the other end of first branch pipe is equipped with reserve sample reserving bottle, is equipped with the third on the reserve sample reserving bottle and filters the air head.
5. The microcarrier bioreactor tank of claim 4, wherein: the sampling tube, the first branch tube and the second branch tube are all flexible tubes, and the first switch valve body, the second switch valve body, the third switch valve body and the fourth switch valve body are all air clamps; or the sampling tube, the first branch tube and the second branch tube are hard pipelines, and the first switch valve body, the second switch valve body, the third switch valve body and the fourth switch valve body are all air valves.
6. A method of culturing porcine circovirus type 2 using a microcarrier bioreactor according to any one of claims 1 to 5, characterized in that: which comprises the following steps:
step 1, a biological reaction tank is started to preheat:
connecting a PH electrode, a temperature electrode and an oxygen dissolving electrode of a biological reaction tank well, adding jacket water into an interlayer space of a tank body, switching on a heating pipe and starting a power supply for heating, and starting a circulating pump to circulate the jacket water in the interlayer space so as to ensure that the temperature of the tank body is uniform;
step 2, cell digestion of the bottle rotating:
selecting a bottle-rotating PK-15 cell with good cell state, digesting the cell by using pancreatin digestive juice, and fully shaking the digested cell;
step 3, cell count: diluting a proper amount of digested cells with a culture solution, adding a proper amount of diluted cells to a cell counting plate for cell counting after diluting, and calculating the cell density of the cell solution;
step 4, cell loading and culturing: adding microcarrier into a biological reaction tank according to 3-5 g/L, and adding cells according to 30-50 cells/microcarrier; cell culture parameters of corresponding devices of the biological reaction tank are regulated: starting an aeration system to carry out aeration culture on the biological reaction tank through an air inlet interface; specific methods for adjusting cell culture parameters of the corresponding devices: the temperature in the tank is regulated to be 36.5-37.5 ℃ through a temperature electrode, the dissolved oxygen is regulated to be 50-60% through an oxygen dissolving electrode, the PH is regulated to be 7.2-7.4 through a PH electrode, and the revolution of the stirrer is regulated to be 40-60r/min;
step 5, liquid replacement is carried out for 24 hours: after the cells in the biological reaction tank are cultured for 24 hours, stopping stirring, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic tube to ensure that the sampling height of the lower port of the telescopic tube is 0.5cm above the liquid level of the carrier, discharging the cell culture solution, and then adding an equal amount of cell maintenance solution through a fluid supplementing port for continuous culture;
step 6, cell observation: after the cells are cultured for 48 hours, the first switch valve body and the third switch valve body are opened while stirring is maintained, the second switch valve body and the fourth switch valve body are closed, the air suction is stopped when the filter air head is used for sucking air in the injector, the carrier and the cells of the biological reaction tank body flow through the transparent observation area, the first switch valve body is closed, the cell observation area is placed under a microscope for observation, after observation, the first switch valve body K1 is opened, the injector is used for pumping air in the filter air head, and the carrier and the cells of the transparent observation area and the related pipeline part are pumped into the tank body through the original route sampling opening;
step 7, harvesting antigen for the first time: stopping stirring after the cells are cultured for 72 hours, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic tube to the position 0.5cm above the carrier in the tank, collecting the first antigen, placing the harvested antigen liquid at-15 ℃ for freezing preservation in a freezer, and then adding an equal amount of cell maintenance liquid through a liquid supplementing port for continuous culture;
step 8, harvesting the antigen for the second time and culturing in a liquid change mode: stopping stirring after the biological reaction tank cells are cultured for 96 hours, standing for 3-5 minutes, adjusting the sampling height of the lower port of the telescopic tube to a position 0.5cm above the carrier in the tank, harvesting the culture supernatant antigen liquid again through a sampling port, and freezing and preserving the harvested second antigen liquid at the temperature of minus 15 ℃ in a freezer;
step 9, harvesting antigen for the third time: after the cells in the biological reaction tank are cultured for 96-120h, the revolution is regulated to 100-150 revolutions for 3-5 minutes, then stirring is stopped, standing is carried out for 3-5 minutes, ventilation and pressurization are carried out on the tank body from an air inlet interface through a ventilation system, a discharge valve on a discharge pipe is opened to harvest filtered antigens, meanwhile, a ventilation pipe above a filter screen is opened for air inlet, and finally the harvested third antigen liquid is placed at minus 15 ℃ and is frozen and stored in a freezer.
7. The method for culturing porcine circovirus type 2 using a microcarrier bioreactor according to claim 6, wherein: in step 2, digestion of cells was performed using 0.25% pancreatin digest.
8. The method for culturing porcine circovirus type 2 using a microcarrier bioreactor according to claim 6, wherein: the specific method for discharging the cell culture solution in the step 5 comprises the following steps: the first switch valve body and the fourth switch valve body are opened, the second switch valve body and the third switch valve body are closed, the ventilation system ventilates and pressurizes the tank body through the air inlet interface, the waste liquid bottle is used as a collecting bottle, and the culture solution above 0.5cm above the liquid level of the carrier continuously flows into the collecting bottle through the telescopic pipe through the sampling pipe under the action of the pressure because the pressure in the tank body is higher than the pressure outside the tank body until the liquid level of the culture solution is lower than the sampling height of the lower port of the telescopic pipe; at this time, the first switch valve body is closed, and simultaneously, the air filtering head and the air filtering head are inflated by using the injector respectively, and the cell culture solution in the transparent observation area and the relevant pipeline part is pumped into the collecting bottle.
9. The method for culturing porcine circovirus type 2 using a microcarrier bioreactor according to claim 6, wherein: specific methods for collecting first antigen and first antigen: the first switch valve body and the fourth switch valve body are opened, the second switch valve body and the third switch valve body are closed, the ventilation system ventilates and pressurizes the tank body through the air inlet interface, the antigen bottle is used as a collecting bottle, and the culture solution with the pressure higher than 0.5cm above the liquid level of the carrier flows through the sampling tube continuously under the action of the pressure through the telescopic tube and flows into the collecting bottle until the liquid level of the culture solution is lower than the sampling height of the lower port of the telescopic tube; at this time, the first switch valve body is closed, and simultaneously, the air filtering head and the air filtering head are inflated by using the injector respectively, and the cell culture solution in the transparent observation area and the relevant pipeline part is pumped into the collecting bottle.
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Address after: No.110 Yuanzhong village, Gushan Town, Jin'an District, Fuzhou City, Fujian Province Applicant after: Zhaofenghua Biotechnology (Fuzhou) Co.,Ltd. Address before: No.110 Yuanzhong village, Gushan Town, Jin'an District, Fuzhou City, Fujian Province Applicant before: FUZHOU DA BEI NONG BIOTECH Co.,Ltd. |