CN111944692B - Animal cell hollow fiber culture system and cell culture method thereof - Google Patents
Animal cell hollow fiber culture system and cell culture method thereof Download PDFInfo
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Abstract
The invention discloses an animal cell hollow fiber culture system and a cell culture method thereof, wherein the system comprises a hollow fiber cylinder, a liquid storage system, a circulating system (an inner circulating system and an outer circulating system) and a detection control system; the hollow fiber cylinder is used for circulating a culture medium through an internal circulation system; the external circulation system is used for cell inoculation and cell digestion, and the cells are uniformly distributed among the hollow fibers through circulation of a peristaltic pump in the external circulation system, so that the cell inoculation uniformity is improved. The condition similar to in vivo growth is established, and the culture effect is better. The alternative positive pressure circulating system (mode) is adopted to ensure the alternative replacement of nutrient components in the internal and external systems of the mesoporous fiber, and also avoid the problems of the blockage of the hollow fiber and the incapability of replacing the dead volume of the nutrient solution. Meanwhile, the system is designed in a closed manner, has better biological safety, can effectively control pH, dissolved oxygen and temperature in the culture process, and is suitable for the culture of animal cells for producing vaccines and antibodies.
Description
Technical Field
The invention relates to the technical field of animal cell culture, in particular to an animal cell hollow fiber culture system suitable for vaccine and antibody production and a cell culture method thereof.
Background
Common cell culture modes are classified into adherent culture and suspension culture, and for suspension cell culture, the scale-up of a bioreactor can be used for realizing the scale-up culture of suspension cells, but when no proper suspension cell line exists, the adherent cell culture is also the main mode for vaccine production and antibody production. The commonly used industrialized cell adherent culture modes comprise rotating bottle culture and fixed bed reactor culture, the rotating bottle culture is no longer approved for the production of biological medicines at present because of the risks of complex operation, large batch-to-batch difference, easy contamination and the like, and the fixed bed bioreactor for the adherent cell culture needs to be filled with sheet carriers in different forms for culture, so that the problems of large occupied space, low cell density, poor cell distribution uniformity and the like are difficult to realize the enlarged culture.
Disclosure of Invention
Aiming at the problem that the amplification culture is difficult to realize due to the problems of low cell density, poor cell distribution uniformity and the like during the adherent culture of the cells, the invention designs a hollow fiber culture system for the adherent culture of animal cells.
In order to solve the technical problems, the invention provides the following technical scheme:
an animal cell hollow fiber culture system comprises a hollow fiber cylinder, a liquid storage system and a circulating system, wherein the circulating system comprises a hollow fiber cylinder fiber tube inner circulating system and a hollow fiber cylinder fiber tube outer circulating system; the internal circulation system is used for the circulation of the culture medium; the external circulation system is used for cell inoculation and cell digestion, the needle tube can be connected through the three-way luer connector, the seed cells are inoculated to the external circulation system, the cells are uniformly distributed among the hollow fibers through circulation of the peristaltic pump in the external circulation system, and the cell inoculation uniformity is improved.
The hollow fiber cylinder comprises a hollow fiber pipe, a cylinder cover and an outer cylinder; the hollow fiber tube is arranged in the outer cylinder, and the cylinder covers are arranged at two ends of the outer cylinder; the hollow fiber cylinder comprises a fiber tube inner space and a fiber tube outer space, wherein a culture medium is arranged in the fiber tube inner space, and the fiber tube outer space is inoculated with cultured cells; the space outside the fiber tube comprises a space between the hollow fiber tube and the outer tube; the outer cylinder may be made of (but not limited to) plastic of food and pharmaceutical grade such as PP or PE.
The liquid storage system comprises a liquid storage bottle, a fresh culture medium bottle and a harvesting bottle; the fresh culture medium bottle is communicated with the liquid storage bottle through a silicone tube and a peristaltic pump, and the liquid storage bottle is connected with the harvesting bottle through a silicone tube;
the circulating system comprises a hollow fiber cylinder fiber tube internal circulating system and a hollow fiber cylinder fiber tube external circulating system; the inner circulating system is characterized in that a cylinder cover of the space in the fiber tube is communicated with a liquid storage bottle through a silicone tube and a peristaltic pump, and a culture medium in the liquid storage bottle circulates in the space in the fiber tube through the inner circulating system; the external circulation system is formed by connecting a first interface of the space outside the hollow fiber tube and a second interface of the space outside the hollow fiber tube with a peristaltic pump through a silicone tube.
Further, an alkali liquor inlet for feeding alkali liquor, a reserved interface for sampling and analyzing in the culture process, an air inlet, an air outlet, a fiber tube internal circulation interface, an oxygen dissolving electrode interface, a pH electrode interface and a fresh culture medium bottle connecting interface are arranged on the liquid storage bottle, wherein the air inlet is connected with an air pump; preferably, the number of reserved interfaces can be set to 3.
Further, the device also comprises a detection control system, wherein the detection control system is used for detecting and adjusting the pH value, the dissolved oxygen value and the temperature in the liquid storage bottle so as to enable the culture medium to be at the set pH value, dissolved oxygen value and temperature.
Further, detect control system and include pH, temperature control module, pH, temperature control module are by the pH electrode, pH executor module and temperature execution module constitute, the pH electrode includes pH test probe and temperature test probe, the pH executor module comprises carbon dioxide control module and sodium bicarbonate control module, the pH electrode sets up in pH electrode kneck, when the pH test probe of pH electrode detects pH and is higher than the setting value, carbon dioxide control module increases the carbon dioxide ventilation volume, when pH is less than the setting value, the sodium bicarbonate control module who is connected with sodium bicarbonate starts, add proper amount alkali lye, when the temperature test probe of pH electrode detects the temperature and is less than the settlement temperature, temperature execution module starts, heat up the stock solution bottle to the settlement temperature.
Furthermore, the detection control system comprises a dissolved oxygen control module, the dissolved oxygen control module consists of a dissolved oxygen electrode and a dissolved oxygen actuator module, the dissolved oxygen electrode is installed at a dissolved oxygen electrode interface, and the dissolved oxygen actuator module realizes dissolved oxygen control by controlling the flow of air.
Furthermore, the detection lens is installed on the outer barrel through the observation lens seat, a transparent window is arranged at the corresponding position of the outer barrel, the detection lens can be arranged in a plurality of, for example 3, and the cells can be observed in three positions on line. The lens mount may be an annular strip secured to the exterior of the hollow fiber barrel.
Furthermore, a three-way luer connector is arranged on a silicone tube of the external circulation system, the three-way luer connector can be connected with an injector for cell inoculation, the cells are uniformly distributed among the hollow fibers (external space) through circulation of a peristaltic pump, and the cell inoculation uniformity is improved.
Furthermore, the temperature execution module is a heating blanket arranged outside the liquid storage bottle, and the temperature of the liquid in the liquid storage bottle can be regulated and controlled by starting or closing the heating blanket.
Furthermore, after the culture medium in the liquid storage bottle circulates for a certain time, the control system can control the peristaltic pump to pump part of the culture medium in the liquid storage bottle into the harvesting bottle, and then the fresh culture medium in the part of the fresh culture medium bottle is added into the liquid storage bottle.
Further, the hollow fiber cartridge 1 is obliquely arranged on the support, and the control system can set the mode and frequency of the intermittent up-and-down movement of the support. Preferably, the support sets up to 2 supports, and one of them support is scalable, and this support can realize flexible through the eccentric wheel structure, and eccentric wheel one end is connected with the motor for the control rotates, and the other end is connected to the support, thereby rotates through the motor of intermittent type formula, drives the eccentric wheel and rotates and realize the upper and lower flexible of support. The telescopic structure controls the whole intermittent up-and-down movement of the hollow fiber, and the opening and the frequency setting are mainly controlled automatically or manually after the cells are injected into the external circulation system. Therefore, the whole swing of the hollow fiber column is realized through the intermittent up-and-down movement of the support, and the uniformity of cell adherence is further promoted.
Further, the hollow fiber cartridge may be serially connected by a plurality of hollow fiber tubes, so that multistage amplification may be performed.
The cell culture method of the animal cell hollow fiber culture system specifically comprises the following steps:
firstly, a detection control system starts an internal circulation system, and a culture medium in a liquid storage bottle circulates in the space in the fiber tube through the internal circulation system; after the internal circulation system works for a period of time, closing the internal circulation system; connecting an injector containing seed cells with a three-way luer connector for cell inoculation, and starting an external circulation system after inoculation;
under the action of the external circulation system, cells uniformly adhere to the wall and grow outside the hollow fiber tube, and a culture medium, metabolic waste and produced antibodies or viruses of the cells outside the hollow fiber enter the internal circulation system through the hollow fiber;
starting an internal circulation system after the cells are attached, and circulating a culture medium in the liquid storage bottle in the space in the fiber tube through the internal circulation system to promote the exchange of the nutrient solution and the waste liquid after the cells outside the hollow fibers are consumed; after the inner circulation system works for a plurality of times, the inner circulation system stops working, and the outer circulation system is started again, so that a plurality of alternate circulations are carried out;
in the whole process, the detection control system detects and adjusts the pH value, the dissolved oxygen value and the temperature in the liquid storage bottle so as to enable the culture medium to be at the set pH value, dissolved oxygen value and temperature, and the on-line observation of the cell growth state is realized through the detection lens;
the detection control system can enable the support to enable the hollow fiber cylinder support to swing up and down through the control system, uniformity of cell adherence is promoted, after the culture medium in the liquid storage bottle circulates for a certain time, the detection control system controls the peristaltic pump to pump part of the culture medium in the liquid storage bottle into the harvesting bottle, and then the fresh culture medium in part of the fresh culture medium bottle is added into the liquid storage bottle.
In the invention, the hollow fiber cylinder comprises a fiber tube inner space and a fiber tube outer space, wherein a culture medium is arranged in the fiber tube inner space, cultured cells are inoculated in the fiber tube outer space, and the control and observation of the cell growth condition can be realized through a detection lens; the external circulation system can circulate the liquid in the space outside the fiber tube, which is beneficial to uniformly inoculating cells; the internal circulation system is used for medium circulation, nutrient substances pass through the hollow fiber tube to reach the space outside the tube, and metabolic waste and products generated by cells pass through the fiber tube to enter the space inside the fiber tube; the culture medium circulates in the liquid storage bottle and the space in the fiber tube through the circulating system, after a certain period of circulation, the culture medium in the liquid storage bottle flows into the harvesting bottle through the circulating system, and the fresh culture medium in the fresh culture medium bottle is added into the liquid storage bottle through the circulating system.
The system achieves uniformity of cell distribution in hollow fibers and combines the advantages of controllable parameters of a bioreactor system: the double-system circulation of culture medium materials and cells is realized through the double-circulation system, the problems that the bioreactor cannot ensure the inoculation uniformity, the materials are difficult to flow on line and the products are harvested continuously are solved, and the problem that the uniformity of the bioreactor is poor after amplification is also solved; the hollow fiber tube has the effect similar to a micro-blood vessel, can form a semi-permeable membrane barrier between cultured cells and a flowing culture medium, establishes a stable state which is more similar to in-vivo growth conditions, has a better culture effect, has a closed design system, has better biological safety, can effectively control pH, dissolved oxygen and temperature in the culture process, and is suitable for culturing animal cells for producing vaccines and antibodies.
Compared with the prior rotating bottle commonly used for animal cell culture, the system has the advantages of large attachment area, small volume, simple structure and the like, compared with a fixed bed reactor, the system has the characteristics of good amplification uniformity, good reproducibility in the culture process, integrated design, easy operation, easy amplification and good uniformity, can realize high-density culture of animal cells and high-efficiency production of cell products, and can be widely used in the production process of virus vaccines and antibodies.
The method comprises the steps of inoculating cells through a three-way luer connector on a silicone tube of an external circulation system, and enabling the cells to slowly flow between the outer walls of the hollow fibers under the driving of a peristaltic pump, so that the cells can be uniformly attached to the outer walls of the hollow fibers; the uniform distribution of the outer walls of the hollow fibers of the cells can be ensured through the up-and-down movement of the hollow fiber cylinder and the circulation of the peristaltic pump; the internal circulation system simulates the structure of a human body circulation system, ensures the supply of nutrient components for cell culture, and conveys a culture medium into the hollow fibers to realize the exchange of the nutrient components of the hollow fibers, thereby promoting the cells to quickly reach higher cell amount;
when the nutrient solution circulates in the hollow fiber, the nutrient solution is promoted to exchange with the waste liquid after the cells outside the hollow fiber are consumed by depending on the pressure brought to the circulating solution by the operation of the peristaltic pump, but along with the growth of the cells, the metabolic waste is increased and the fragments of the crushed cells are increased, the effective and continuous exchange of the nutrient solution can not be realized only by the internal circulation pressure, and through an alternative positive pressure circulating system (mode), namely the alternative positive pressure circulating system combining an external circulating system and an internal circulating system, the alternative replacement of nutrient components in the hollow fiber is ensured, and the problems that the hollow fiber is blocked and the dead volume of the nutrient solution can not be replaced are also solved. The alternative positive pressure circulation system (mode) is that after the internal circulation system works for a plurality of times (10-20 seconds), the internal circulation system stops working, the external circulation system starts, and under the pressure of a peristaltic pump of the external circulation system, the culture medium, the metabolic waste and the produced antibody or virus which are consumed by the external cells of the hollow fiber enter the internal circulation system through the hollow fiber; the 'alternating positive pressure circulating system' has the greatest advantage that the repeated exchange of liquid in the internal and external circulating systems is promoted by continuously and alternately operating the internal and external circulating systems.
And thirdly, the pH, dissolved oxygen and temperature are controlled by a detection control system, so that the process dilemma that the cell growth condition cannot be observed on line when animal cells such as a basket bioreactor are cultured and amplified adherent to the wall is solved.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 is a schematic structural diagram of an embodiment of the present invention;
FIG. 2 is a schematic structural view of a hollow fiber cartridge of an embodiment of the present invention;
FIG. 3 is a schematic structural view of a plurality of hollow fiber tubes arranged in series in a hollow fiber cartridge in the embodiment;
FIG. 4 is a schematic view of an external circulation system of a hollow fiber cartridge fiber tube according to an embodiment of the present invention;
FIG. 5 is a schematic view of the arrangement of the plurality of ports of the liquid storage bottle in the embodiment of the invention;
FIG. 6 is a schematic structural view of the telescoping structure of the stand of the present invention;
fig. 7 is a block diagram of a detection control system according to an embodiment of the present invention.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Examples
An animal cell hollow fiber culture system comprises a hollow fiber cylinder, a liquid storage system, a circulation system and a detection control system.
As shown in fig. 1-2, the hollow fiber cartridge 1 includes a hollow fiber tube 9, two cartridge covers 8, an outer cartridge 12; the hollow fiber tube 9 is arranged in the outer cylinder 12, and the cylinder cover 8 is arranged at two ends of the outer cylinder 12;
the circulating system comprises a hollow fiber cylinder fiber tube internal circulating system and a hollow fiber cylinder fiber tube external circulating system; the internal circulation system is used for the circulation of the culture medium; the external circulation system is used for cell inoculation and cell digestion, the three-way luer 15 can be connected with the needle tube, so that cells are inoculated into the external circulation system, the cells are uniformly distributed among the hollow fibers through circulation of the peristaltic pump in the external circulation system, and the cell inoculation uniformity is improved.In this example, the peristaltic pump may be selected from the group consisting of Watson-Marlo model 114 peristaltic pump And (4) a pump.
The hollow fiber cylinder comprises a fiber pipe inner space and a fiber pipe outer space, the fiber pipe inner space is a culture medium and comprises a hollow fiber cylinder 1 and a cylinder cover 8, and the cylinder cover 8 is communicated with the hollow fiber cylinder 1; the fiber tube outer space includes a space between the hollow fiber tube 9 and the outer cylinder 12. The outer cylinder can be made of food-medicine grade plastic such as PP or PE. The hollow fiber is made of materials such as (but not limited to) PES (polyether sulfone), PVDF (polyvinylidene fluoride), Cellulose and the like; the pore diameter of the hollow fiber is (but not limited to) 100kD, 300kD, 500kD, 0.1 μm, 0.2 μm and 0.45 μm.
The stock solution system includes stock solution bottle 5, fresh culture medium bottle 4 and results bottle 6, and stock solution bottle 5 outsourcing has heating blanket 27 for adjust the temperature of culture medium.
As shown in fig. 5, the bottle cap of the liquid storage bottle 5 is provided with a plurality of interfaces, which are respectively an alkali liquor inlet 17, an exhaust port 16, a fiber tube inner space liquid circulation interface 18, an oxygen dissolving electrode interface 19, a harvesting bottle connection interface 20, a reserved interface 21, an air inlet 22, a fiber tube inner space circulation interface 24, a pH electrode interface 25 and a fresh culture medium bottle connection interface 26; wherein the air inlet 22 is connected with an air pump, the number of the reserved interfaces 21 is 2-3, and the reserved interfaces are used for sampling and analyzing in the culture process. The alkali liquor inlet 17 is communicated with an alkali liquor bottle, and the alkali liquor can be sodium bicarbonate and other solutions.
Fresh culture medium bottle 4 and harvest bottle 6 all are provided with two interfaces, and fresh culture medium bottle 4 is connected interface 26 intercommunication through silicone tube and fresh culture medium peristaltic pump and the fresh culture medium bottle of stock solution bottle 5, and stock solution bottle 5 is connected through silicone tube and harvest bottle peristaltic pump harvest bottle connection interface 20.
The culture system has the function of automatically replacing the culture medium, namely after the culture medium in the liquid storage bottle 5 circulates for a certain time, the circulation system pumps part of the culture medium in the liquid storage bottle 5 to the harvest bottle 6, and then the circulation system adds part of the fresh culture medium in the fresh culture medium bottle 4 into the liquid storage bottle 5.
The circulating system comprises a hollow fiber cylinder fiber tube internal circulating system and a hollow fiber cylinder fiber tube external circulating system; wherein, the inner circulating system is that two cylinder covers 8 of the space in the fiber tube are respectively communicated with a liquid circulating interface 18 of the space in the fiber tube of the liquid storage bottle 5 and a circulating interface 24 of the space in the fiber tube through a silicone tube and a peristaltic pump of the inner circulating system, and the culture medium in the liquid storage bottle 5 circulates in the space in the fiber tube through the inner circulating system;
the outer circulation system is characterized in that a first connector 11 of the outer space of the hollow fiber tube and a second connector 13 of the outer space of the hollow fiber tube are connected through a silicone tube, and the silicone tube is arranged on an outer circulation peristaltic pump 14, so that circulation of liquid (containing seed cells) in the outer space of the tube is realized, and uniform inoculation of the seed cells is promoted.
As shown in fig. 3, the hollow fiber cartridge may be provided with a plurality of hollow fiber tubes connected in series through a cartridge cover, thereby allowing multi-stage enlargement.
As shown in fig. 4, a T-shaped connector 15 or a three-way luer connector is arranged on a silicone tube connecting the first interface 11 in the outer space of the hollow fiber tube and the peristaltic pump, the T-shaped connector 15 can be connected with an injector to inoculate cells, and the peristaltic pump circulates in the inoculation process to realize uniform distribution of the cells in the hollow fiber tube and improve the uniformity of cell inoculation.
The hollow fiber cylinder 1 is arranged on a first bracket 2 and a second bracket 3, the first bracket 2 and the second bracket 3 are both arranged on a base with steps, the height of the first bracket 2 is higher than that of the second bracket 3, so that the hollow fiber cylinder 1 can be obliquely arranged on the first bracket 2 and the second bracket 3 at a certain angle;
the first support can be adjusted in a height telescopic mode through the support movement actuator, the support movement actuator in the implementation is an eccentric wheel 29 and a motor 30 connected with one end of the eccentric wheel, the motor is used for controlling the rotation of the eccentric wheel, the other end of the eccentric wheel 29 is connected to the support 2, the motor 30 can rotate intermittently, so that the eccentric wheel is driven to rotate, up-and-down telescopic of the support 2 is achieved, and the integral swing of the hollow fiber cylinder can be achieved.
As shown in fig. 6, the detection control system comprises a pH, a temperature control module and a dissolved oxygen control module, the pH, the temperature control module is composed of a pH electrode, a pH actuator module and a temperature execution module, the pH actuator module is composed of a carbon dioxide control module and a sodium bicarbonate control module, the pH electrode is arranged at a pH electrode interface 25 and covers the pH detection probe and the temperature detection probe, when the pH detection probe of the pH electrode detects that the pH is higher than a set value, the carbon dioxide control module increases the carbon dioxide ventilation volume, when the pH is lower than the set value, the sodium bicarbonate control module connected with sodium bicarbonate is started, a proper amount of alkali liquor is added, when the temperature detection probe of the pH electrode detects that the temperature is lower than the set temperature, the temperature execution module is started to heat the liquid storage bottle to the set temperature. In this embodiment, adopt the opening and closing of the heating blanket that the liquid storage bottle outsourcing was established to realize temperature control.
The dissolved oxygen control module consists of a dissolved oxygen electrode and a dissolved oxygen actuator module, the dissolved oxygen electrode is arranged at the interface 19 of the dissolved oxygen electrode, and the dissolved oxygen actuator module realizes the dissolved oxygen control by controlling the flow of air.
The detection lens 28 is installed through the observation lens mount 10, and 3 observation lens mounts 10 are all arranged on the outer cylinder 12; the cells can be observed on line in the culture process at three positions.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. The utility model provides an animal cell hollow fiber culture apparatus, includes a hollow fiber section of thick bamboo, stock solution system and circulation system, the circulation system includes hollow fiber section of thick bamboo fiber tube inner loop system and hollow fiber section of thick bamboo fiber tube outer loop system, its characterized in that:
the hollow fiber cylinder comprises a hollow fiber pipe, a cylinder cover and an outer cylinder; the hollow fiber tube is arranged in the outer cylinder, and the cylinder covers are arranged at two ends of the outer cylinder; the hollow fiber cylinder comprises a fiber tube inner space and a fiber tube outer space, wherein the fiber tube inner space is internally circulated with a culture medium, and the fiber tube outer space is inoculated with cultured cells; the space in the fiber tube comprises a hollow fiber tube space and a tube cover communicated with the hollow fiber tube space, and the space outside the fiber tube comprises a space between the hollow fiber tube and the outer tube;
the liquid storage system comprises a liquid storage bottle, a fresh culture medium bottle and a harvesting bottle; the fresh culture medium bottle is communicated with the liquid storage bottle through a silicone tube and a peristaltic pump, and the liquid storage bottle is connected with the harvesting bottle through the silicone tube and the peristaltic pump;
the circulating system comprises a hollow fiber cylinder fiber tube internal circulating system and a hollow fiber cylinder fiber tube external circulating system; the inner circulating system is characterized in that a cylinder cover of the space in the fiber tube is communicated with a liquid storage bottle through a silicone tube and a peristaltic pump, and a culture medium in the liquid storage bottle circulates in the space in the fiber tube through the inner circulating system; the external circulation system is characterized in that a first interface of the external space of the hollow fiber tube and a second interface of the external space of the hollow fiber tube are connected with a peristaltic pump through a silicone tube;
the system also comprises a detection control system, wherein the detection control system is used for detecting and adjusting the pH value, the dissolved oxygen value and the temperature in the liquid storage bottle so as to enable the culture medium to be at the set pH value, dissolved oxygen value and temperature;
the detection lens is arranged on the outer cylinder through the observation lens seat, a transparent window is arranged at the corresponding position of the outer cylinder, and the on-line observation of the cell growth state is realized through the detection lens;
a three-way luer connector is arranged on a silicone tube of the external circulation system and is connected with an injector for cell inoculation;
after the culture medium in the liquid storage bottle circulates for a certain time, the control system controls the peristaltic pump to pump part of the culture medium in the liquid storage bottle into the harvesting bottle, and then the fresh culture medium in part of the fresh culture medium bottle is added into the liquid storage bottle;
the hollow fiber cylinder is arranged on the first support and the second support, the first support and the second support are both arranged on the base with the ladder, and the height of the first support is higher than that of the second support, so that the hollow fiber cylinder can be obliquely arranged on the first support and the second support at a certain angle;
the first support is used for performing height telescopic adjustment through a support movement actuator to realize the integral swing of the hollow fiber cylinder;
firstly, a detection control system starts an internal circulation system, and a culture medium in a liquid storage bottle circulates in the space in the fiber tube through the internal circulation system; after the internal circulation system works for a period of time, closing the internal circulation system; connecting an injector containing seed cells with a three-way luer connector for cell inoculation, starting an external circulation system after inoculation,
under the action of the external circulation system, cells uniformly adhere to the wall and grow outside the hollow fiber tube, and a culture medium, metabolic waste and produced antibodies or viruses of the cells outside the hollow fiber enter the internal circulation system through the hollow fiber;
starting an internal circulation system after the cells are attached, and circulating a culture medium in the liquid storage bottle in the space in the fiber tube through the internal circulation system to promote the exchange of the nutrient solution and the waste liquid after the cells outside the hollow fibers are consumed; after the inner circulation system works for a plurality of times, the inner circulation system stops working, and the outer circulation system is started again, so that a plurality of alternate circulations are carried out;
in the whole process, the detection control system detects and adjusts the pH value, the dissolved oxygen value and the temperature in the liquid storage bottle so as to enable the culture medium to be at the set pH value, dissolved oxygen value and temperature, and the on-line observation of the cell growth state is realized through the detection lens;
the detection control system enables the support to enable the hollow fiber cylinder support to swing up and down through the control system, uniformity of cell adherence is promoted, after the culture medium in the liquid storage bottle circulates for a certain time, the detection control system controls the peristaltic pump to pump part of the culture medium in the liquid storage bottle into the harvesting bottle, and then the fresh culture medium in part of the fresh culture medium bottle is added into the liquid storage bottle.
2. A hollow fiber culture device for animal cells according to claim 1, wherein the liquid storage bottle is provided with an alkali liquid inlet for feeding alkali liquid, a reserved interface for sampling and analyzing in the culture process, an air inlet, an air outlet, an in-fiber tube circulation interface, an oxygen dissolving electrode interface, a pH electrode interface and a fresh culture medium bottle connection interface, wherein the air inlet is connected with an air pump.
3. The animal cell hollow fiber culture device according to claim 1, wherein the detection control system comprises a pH and temperature control module, the pH and temperature control module comprises a pH electrode, a pH actuator module and a temperature execution module, the pH electrode comprises a pH detection probe and a temperature detection probe, the pH actuator module comprises a carbon dioxide control module and a sodium bicarbonate control module, the pH electrode is installed at a pH electrode interface, when the pH detection probe of the pH electrode detects that the pH is higher than a set value, the carbon dioxide control module increases the carbon dioxide ventilation, when the pH is lower than the set value, the sodium bicarbonate control module connected with sodium bicarbonate is started, a proper amount of alkaline solution is added, when the temperature detection probe of the pH electrode detects that the temperature is lower than the set temperature, the temperature execution module is started to raise the temperature of the liquid storage bottle to the set temperature.
4. A hollow fiber culture apparatus for animal cells according to claim 3, wherein the detection control system comprises a dissolved oxygen control module, the dissolved oxygen control module is composed of a dissolved oxygen electrode and a dissolved oxygen actuator module, the dissolved oxygen electrode is installed at the interface of the dissolved oxygen electrode, and the dissolved oxygen actuator module realizes DO control by controlling the flow of air/oxygen.
5. The animal cell hollow fiber culture apparatus according to claim 1, wherein the hollow fiber cartridge realizes enlargement of culture scale by series connection of a plurality of hollow fiber tubes.
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