CN111334427B - Totally-enclosed cell culture system - Google Patents
Totally-enclosed cell culture system Download PDFInfo
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- CN111334427B CN111334427B CN202010167455.8A CN202010167455A CN111334427B CN 111334427 B CN111334427 B CN 111334427B CN 202010167455 A CN202010167455 A CN 202010167455A CN 111334427 B CN111334427 B CN 111334427B
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
- C12M27/02—Stirrer or mobile mixing elements
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/04—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus by injection or suction, e.g. using pipettes, syringes, needles
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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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
Abstract
The invention provides a totally-enclosed cell culture system, which at least comprises the following parts: incubator, culture tank, air current subassembly, liquid flow subassembly, temperature regulating subassembly and central controller. The system of the invention realizes a constant temperature culture environment, adopts a perfusion mode to culture cells, and realizes a totally-enclosed integrated process from cell activation, infection and amplification to finished product recovery. The radial shearing force is reduced as much as possible by axially stirring the cells and the culture solution in the tank body, so that the cells can be effectively protected, and the cell yield is improved. The system separately inlet air, ensures that the content of each gas component is stable in the culture process, can ensure that the change of external gas in the cell culture process can not directly influence the cell culture, ensures that the content of each gas component is unchanged, controls the culture temperature, the liquid amount and the gas concentration in the culture process, keeps the culture environment stable, reduces manual operation, reduces the cost, reduces the risk of misoperation in the culture process, and improves the culture efficiency.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a totally-enclosed cell culture system.
Background
In recent years, CAR-T cell immunotherapy has been considered as one of the most promising therapies for combating cancer. There are many other incomparable advantages to this therapy, such as CAR-T cells can have multiple targeting sites, improving tumor treatment accuracy, and the course of action is not limited by MHC (major histocompatibility complex); the CAR-T cells have wider tumor killing range and longer effect; strong technical properties, strong replicability, etc. In 2018, two CD19 CAR-T cell drugs (kymeriah and yescata, respectively) were approved by the FDA, which gave good results in the treatment of hematological malignancies. However, CAR-T cell immunotherapy still has many limitations, such as the preparation of CAR-T cells. In the CAR-T treatment process, the T cells subjected to technical transformation are subjected to in vitro culture, so that the number of cells meeting the treatment requirement (hundreds of millions or even billions of CAR-T cells are needed for a patient in general) is achieved, and then the cells are returned to the patient for targeted killing of cancer cells, however, the in vitro culture time of the CAR-T cells is longer due to the current technical means, so that the clinical treatment period is prolonged.
Cell culture (cell culture) is a method of simulating in vitro an in vivo environment (sterility, proper temperature, ph, certain nutritional conditions, etc.) to survive, grow, reproduce and maintain major structures and functions. Cell culture techniques are important and commonly used techniques in cell biology research methods, and a large number of cells can be obtained by culturing cells through the cell culture techniques, and signal transduction, anabolism, growth and proliferation of the cells and the like of the cells can be studied.
At present, most of cell culture is manually operated culture, and when a large amount of cultured cells are required for industrialization, a large amount of labor cost and time cost are required, and meanwhile, as the burden of operators is increased, the risk of error is also greatly increased; in addition, artificial culture cannot accurately control the environment of cell growth, which is unfavorable for cell growth.
Disclosure of Invention
In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a totally enclosed cell culture system.
To achieve the above and other related objects, a first aspect of the present invention provides a totally enclosed cell culture system, comprising at least the following parts:
an incubator;
the culture tank is used for culturing cells and is arranged in the culture box; the culture tank is provided with a stirrer, and the stirrer is arranged in the culture tank;
an air flow assembly in communication with the incubator for regulating the concentration of oxygen and carbon dioxide in the incubator;
the liquid flow component is communicated with the culture tank and is used for regulating the liquid flow in the culture tank, filtering the metabolites produced by cell culture and recovering finished cells;
a temperature regulating assembly for regulating the temperature within the incubator;
And the central controller is connected with and controls the air flow assembly, the liquid flow assembly and the temperature regulating assembly.
In a second aspect, the invention provides the use of the aforementioned totally enclosed cell culture system for totally enclosed cell culture.
In a third aspect, the present invention provides a method for culturing totally enclosed cells using the aforementioned totally enclosed cell culture system, comprising the steps of:
1) The liquid inlet process comprises the following steps: setting the liquid inlet amount, and injecting a culture medium into a culture tank;
2) Building a culture environment: presetting concentration values of oxygen and carbon dioxide, respectively injecting the oxygen and the carbon dioxide into the incubator, measuring gas concentration values of the oxygen and the carbon dioxide in the incubator in real time, comparing the gas concentration values with set values respectively, and injecting the gas in the incubator into the incubator after the oxygen and the carbon dioxide values in the incubator reach standards; setting the temperature of the incubator and preheating the incubator;
3) Continuous culture: injecting cells, injecting factors, starting a stirrer, injecting a culture medium into a culture tank in continuous culture, filtering metabolites and discharging waste liquid;
4) Displacement and concentration: after the cell culture is completed, firstly replacing the culture medium with normal saline, concentrating after the replacement is completed, continuously discharging waste liquid, and reducing the liquid volume in the culture tank;
5) And (3) recovering a finished product: the stirrer was stopped and the finished cells in the culture tank were recovered.
As described above, the totally enclosed cell culture system of the present invention has the following beneficial effects:
the system of the invention realizes a constant temperature culture environment, adopts a perfusion mode to culture cells, and realizes a totally-enclosed integrated process from cell activation, infection and amplification to finished product recovery. The system adopts a perfusion mode, but not a perfusion mode, can discharge waste liquid in the culture process, prevents the accumulation of harmful metabolites, is favorable for achieving higher cell culture density, can reduce subsequent processing steps, can recover finished cells without operations such as centrifugation, simplifies the operation, can improve the culture efficiency, is easy to industrialize, and realizes a totally-enclosed integrated process from cell activation, infection and amplification to finished product recovery. The radial shearing force is reduced as much as possible by axially stirring the cells and the culture solution in the tank body, so that the cells can be effectively protected, and the cell yield is improved. The system is used for separately feeding air, guaranteeing the stable content of each gas component in the culture process, ensuring that the change of external gas in the cell culture process can not directly influence the cell culture, guaranteeing the unchanged content of each gas component, controlling the culture temperature, the liquid amount and the gas concentration in the culture process, keeping the culture environment stable, reducing the manual operation, reducing the cost, reducing the risk of misoperation in the culture process and improving the culture efficiency.
Drawings
FIG. 1 is a signal transmission diagram of a totally enclosed cell culture system according to the present invention;
FIG. 2 is a front view showing the construction of the totally enclosed cell culture system of the present invention;
FIG. 3 is a schematic diagram showing the back side of the whole cell culture system according to the present invention;
FIG. 4 shows a diagram of the components of the incubator layout of the totally enclosed cell culture system of the present invention;
FIG. 5 is a schematic diagram showing the communication between the culture tank and the flow module of the totally enclosed cell culture system according to the present invention.
FIG. 6 shows the internal structure of the culture tank of the totally enclosed cell culture system of the invention.
FIG. 7 is a schematic diagram showing a top view of a culture tank stirrer of the whole-closed cell culture system according to the present invention.
Description of element reference numerals
1. Incubator
2. Culture pot
2.1 Liquid inlet
2.2 First circulation port
2.3 Second circulation port
2.4 Recovery port
2.5 Stirrer
2.51 Center shaft
2.52 Blade
2.53 Closed accommodating cavity
2.6 Culture cover
2.61 Cover body
2.62 Exhaust part
2.63 Air inlet part
2.7 Concave part
3. Airflow assembly
3.1 Air passage
3.1.1 Air filter
3.1.2 Air pipeline
3.1.3 Vent opening
3.2 Carbon dioxide passage
3.2.1 Carbon dioxide storage device
3.2.2 Carbon dioxide relief pressure valve
3.2.3 Carbon dioxide passage switch
3.2.4 Carbon dioxide pipeline
3.2.5 Carbon dioxide passage incubator inlet
3.3 Oxygen passage
3.3.1 Oxygen storage device
3.3.2 Oxygen pressure reducing valve
3.3.3 Oxygen passage switch
3.3.4 Oxygen pipeline
3.3.5 Inlet of oxygen passage incubator
3.4 Mixed gas suction passage
3.4.1 Mixed gas suction pump
3.4.2 Mixed gas suction pipeline
3.4.3 Mixed gas passage incubator outlet
3.5 Exhaust gas discharge passage
3.6 Gas concentration sensing module
3.6.1 Oxygen gas concentration sensor
3.6.2 Carbon dioxide gas concentration sensor
3.7 Gas exhaust passage
3.8 Fan with fan body
4. Liquid flow assembly
4.1 Liquid inlet passage
4.1.1 Liquid storage bag
4.1.2 Liquid inlet pipeline
4.1.3 Liquid inlet pump
4.1.5 Liquid level meter
4.2 Circulation passage
4.2.1 Circulation pipeline
4.2.2 Circulation pump
4.2.3 Filter device
4.3 Waste liquid passage
4.3.1 Waste liquid pump
4.3.2 Waste liquid barrel
4.3.3 Waste liquid pipeline
4.4 Recovery passage
4.4.1 Recovery pipeline
4.4.2 Recovery pump
4.4.3 Recovery bag
4.5 Weighing sensor
5. Temperature adjusting assembly
5.1 Heating device
5.2 Temperature sensor
6. Central controller
7. Sterilizing lamp
8. Stirring driver
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
Please refer to fig. 1-7. It should be understood that the structures, proportions, sizes, etc. shown in the drawings are for illustration purposes only and should not be construed as limiting the invention to the extent that it can be practiced, since modifications, changes in the proportions, or otherwise, used in the practice of the invention, are not intended to be critical to the essential characteristics of the invention, but are otherwise, required to achieve the objective and effect taught by the invention. Also, the terms such as "upper," "lower," "left," "right," "middle," and "a" and the like recited in the present specification are merely for descriptive purposes and are not intended to limit the scope of the invention, but are intended to provide relative positional changes or modifications without materially altering the technical context in which the invention may be practiced.
It should be noted that the central controller according to the present invention may be located at any position on the outer wall of the incubator, or on a console outside the incubator, etc., as long as it can be connected to other components of the totally enclosed cell culture system, and thus the central controller position is not shown in fig. 2-4.
As shown in fig. 1 to 4, the fully-enclosed cell culture system provided by the invention at least comprises the following parts:
A culture tank 2 for culturing cells, provided in the incubator; the culture tank 2 is provided with a stirrer, and the stirrer is arranged in the culture tank;
a gas flow assembly 3, which is communicated with the incubator 1 and is used for adjusting the concentration of oxygen and carbon dioxide in the incubator 1;
a liquid flow component 4 communicated with the culture tank 2 and used for regulating the liquid flow in the culture tank 2, filtering the metabolites produced by cell culture and recovering the finished cells;
a temperature adjusting component 5 for adjusting the temperature in the incubator;
a central controller 6 connected to and controlling the gas flow assembly, the liquid flow assembly and the temperature regulating assembly.
The culture system adopts a perfusion mode, but not a perfusion mode, can discharge waste liquid in the culture process, prevents the accumulation of harmful metabolites, is favorable for achieving higher cell culture density, can reduce subsequent processing steps, can recover finished cells without operations such as centrifugation, simplifies the operation, facilitates the recovery of cells, can improve the culture efficiency, and is easy to industrialize.
Further, the airflow assembly 3 includes:
the separated air passage 3.1, carbon dioxide passage 3.2 and oxygen passage 3.3 are respectively communicated with the incubator 1 for delivering gas into the incubator to form mixed gas.
A mixed gas suction passage 3.4 communicating the incubator 1 and the culture tank 2 for inputting the mixed gas in the incubator 1 into the culture tank 2.
An exhaust gas discharge passage 3.5 communicating with the culture tank 2 for discharging exhaust gas generated during cell culture.
A gas concentration sensing module 3.6 including an oxygen gas concentration sensor 3.6.1 and a carbon dioxide gas concentration sensor 3.6.2; the gas concentration sensing module is used for respectively measuring the real-time oxygen gas concentration value and the real-time carbon dioxide gas concentration value in the incubator and providing detection information for the central controller 6.
Further, the air passage 3.1 comprises an air line 3.1.2; the air line 3.1.2 communicates with the incubator 1.
In one embodiment, the air passage 3.1 further comprises an air filter 3.1.1 for filtering the outside air to clean the air entering the incubator 1.
The carbon dioxide passage 3.2 comprises a carbon dioxide storage device 3.2.1, a carbon dioxide pressure reducing valve 3.2.2, a carbon dioxide passage switch 3.2.3 and a carbon dioxide pipeline 3.2.4; the carbon dioxide storage device 3.2.1 is connected with the carbon dioxide pipeline 3.2.4, the carbon dioxide pipeline 3.2.4 is driven by a carbon dioxide pressure reducing valve, and the carbon dioxide pipeline 3.2.4 is communicated with the incubator 1; the carbon dioxide passage is provided with a carbon dioxide passage switch 3.2.3, and the carbon dioxide passage switch 3.2.3 is controlled by the central controller 6.
In one embodiment, the carbon dioxide passage switch is selected from one or more of a carbon dioxide storage device switch, a carbon dioxide line switch, and a carbon dioxide pressure reducing valve switch. May be a solenoid valve.
The oxygen passage 3.3 comprises an oxygen storage device 3.3.1, an oxygen pressure reducing valve 3.3.2, an oxygen passage switch 3.3.3 and an oxygen pipeline 3.3.4; the oxygen storage device 3.3.1 is connected with the oxygen pipeline 3.3.4, the oxygen pipeline 3.3.4 is driven by the oxygen pressure reducing valve 3.3.2, and the oxygen pipeline 3.3.4 is communicated with the incubator 1; an oxygen passage switch 3.3.3 is arranged on the oxygen passage, and the oxygen passage switch 3.3.3 is controlled by the central controller 6.
In one embodiment, the oxygen passage switch is selected from one or more of an oxygen storage device switch, an oxygen line switch, and an oxygen pressure reducing valve switch. May be a solenoid valve.
The mixed gas suction passage 3.4 includes a mixed gas suction pump 3.4.1 and a mixed gas suction line 3.4.2; the mixed gas suction line 3.4.2 is driven by a mixed gas suction pump 3.4.1, and the mixed gas suction line 3.4.2 is used for injecting mixed gas from the incubator 1 into the culture tank 2; the mixed gas suction pump 3.4.1 is controlled by a central controller 6.
In one embodiment, the mixed gas suction line 3.4.2 is provided with a gas filter. For filtering the gas entering the culture tank.
Further, the exhaust gas discharge channel 3.5 comprises an exhaust gas discharge line. The exhaust gas discharge pipeline is provided with a one-way valve to prevent external air from entering the tank through the exhaust gas discharge pipeline.
The exhaust gas discharge passage directly discharges exhaust gas to the outside of the system, and is not in communication with the incubator 4.
Further, a gas discharge passage 3.7 is arranged in the incubator and used for discharging gas in the incubator and keeping the pressure in the incubator stable.
Further, the gas exhaust passage 3.7 includes a gas exhaust pipe for exhausting the gas in the tank to keep the pressure in the tank stable.
In one embodiment, the gas exhaust passage 3.7 and the air passage 3.1 are the same passage.
In one embodiment, the carbon dioxide pathway 3.2 and the oxygen pathway 3.3 are provided with a carbon dioxide pathway incubator inlet 3.2.5 and an oxygen pathway incubator inlet 3.3.5 on the incubator, and the carbon dioxide pathway incubator inlet 3.2.5 and the oxygen pathway incubator inlet 3.3.5 are both provided at the upper portion of the incubator. The cold air is easy to sink, the hot air rises, and the gas can be mixed at the top more quickly to reach the consistency of the gas concentration in the whole environment.
In one embodiment, the mixed gas passageway is provided with a mixed gas passageway incubator outlet 3.4.3 on the incubator, and the mixed gas passageway incubator outlet 3.4.3, the oxygen gas concentration sensor 3.6.1 and the carbon dioxide gas concentration sensor 3.6.2 are provided in the lower part of the incubator. The concentration of the mixed gas can be accurately reflected, so that the index of the mixed gas entering the culture tank is more real.
The air passage 3.1 and/or the gas exhaust passage 3.7 is provided with a vent 3.1.3 on the incubator, and the vent 3.1.3 is far away from the carbon dioxide passage incubator inlet 3.2.5, the oxygen passage incubator inlet 3.3.5, the mixed gas passage incubator outlet 3.4.3, the oxygen gas concentration sensor 3.6.1 and the carbon dioxide gas concentration sensor 3.6.2. Preventing the gas from escaping too quickly.
In one embodiment, a fan 3.8 is provided in the incubator to agitate the air flow, speed up mixing, make gas mixing more uniform, and speed up heat exchange inside the incubator.
In one embodiment, the fan 3.8 is disposed at an upper portion within the incubator.
Further, the central controller 6 includes the following parts:
the gas concentration comparison unit is used for comparing the real-time oxygen gas concentration value sent by the gas concentration sensing module 
And a real-time carbon dioxide gas concentration value->
And a preset oxygen gas concentration value +.>
And preset carbon dioxide concentrationMetric value->
Respectively, and obtaining the difference of the required concentration according to the formulas (I) and (II), namely the concentration difference +.>
And->
A gas concentration switch control unit for controlling the opening and closing of the oxygen passage, the carbon dioxide passage and the mixed gas suction passage:
according to
The on-off time of the oxygen passage is regulated;
according to
Adjusting the on-off time of the carbon dioxide passage; />
When (when)
Is->
When the gas mixture satisfies the set threshold range, the mixed gas suction passage is opened to suck the gas in the incubator into the incubator.
When (when)
Is->
When at least one of the gas mixture suction passages does not satisfy the set threshold range, the gas mixture suction passage is closed.
Oxygen gas concentration value
Carbon dioxide concentration value->
And the threshold range can be set according to the cell requirements to be cultured. In a preferred manner,/->
Is->
The threshold range may be selected from-0.1% to 0.1%.
In one embodiment, when
Is->
When the gas in the incubator is sucked into the incubator, the mixed gas suction passage is opened by timing control when the gas in the incubator satisfies the set threshold range, and the timing time is determined according to the gas capacity in the incubator and the flow rate of the mixed gas suction pump. Timing control means that when- >
Is->
When the set threshold value ranges are satisfied, the opening and closing of the mixed gas suction passage is controlled according to the time set by the system without immediately opening the mixed gas suction passage.
Further, the opening and closing of the oxygen passage are controlled by controlling the opening and closing of the oxygen passage switch; the opening and closing of the carbon dioxide passage is controlled by controlling the opening and closing of the carbon dioxide passage switch, and the opening and closing of the mixed gas suction passage is controlled by controlling the opening and closing of the mixed gas suction pump.
In one embodiment, a program algorithm may be used to control the opening and closing of the oxygen passage switch, the carbon dioxide passage switch, and the mixture gas intake passage. And according to the current gas concentration measured value, controlling different on-off times of the gas passage switch by a program, so that the actual gas concentration value in the incubator is close to or equal to a set value.
In one embodiment, the opening and closing of the mixed gas suction passage is adjusted by controlling the opening and closing of the mixed gas suction pump.
In one possible embodiment, this may be based on
And (3) adjusting the on-off time of the carbon dioxide passage in a grading regulation mode. For example according to->
Value classification of->
The smaller the two detection compartments the shorter the carbon dioxide passage opening time.
In one possible embodiment, this may be based on
And (3) adjusting the on-off time of the oxygen passage switch in a grading regulation mode. For example according to->
Value classification of->
Smaller, two-pass detection of compartment oxygenThe shorter the path length.
The carbon dioxide passage or the oxygen passage has constant gas flow rate during ventilation, and the ventilation capacity can be adjusted by controlling the on-off time of the passage switch. The longer the open time, the greater the ventilation. The mode is simple and accurate in ventilation control and controllable in accessory cost.
Further, in order to ensure uniform mixing of the gases, the gas concentration sensing module may be used to measure the gas concentration after the oxygen or carbon dioxide passages are disconnected to fully mix the gases. After the gas is injected, the gas concentration sensing module is used for measuring the gas concentration value after the open passage is mixed for a period of time. In general, the shorter the aeration period, the shorter the required break-even time. The gas refers to oxygen or carbon dioxide. The passage means an oxygen passage or a carbon dioxide passage.
Taking incubator sizes 373mm by 330mm by 250mm as an example:
in one embodiment of the present invention, in one embodiment,
the value of (2) is divided into seven stages,
when the ventilation state is maintained, the gas flow rate is constant; when->
When the content is more than or equal to 2%, controlling dioxygenThe carbon dissolving passage switch is turned on for 1.5 seconds and then turned off, then the carbon dissolving passage switch is waited for 12 seconds to uniformly mix the gases, and then the concentration value of the carbon dioxide gas concentration sensor is read and is continuously compared with a set value; when->
When the carbon dioxide channel switch is controlled to be opened for 1 second and then closed, and then after waiting for 9 seconds, the concentration value of the carbon dioxide gas concentration sensor is read and is continuously compared with a set value; when->
When the carbon dioxide channel switch is controlled to be opened for 0.8 seconds and then closed, and then after waiting for 3 seconds, the concentration value of the carbon dioxide gas concentration sensor is read and is continuously compared with a set value; when->
When the carbon dioxide gas concentration sensor is in the state of being opened for 0.6 seconds, the carbon dioxide passage switch is controlled to be closed, the concentration value of the carbon dioxide gas concentration sensor is directly read, and the carbon dioxide gas concentration sensor is continuously compared with a set value; when->
When the carbon dioxide gas concentration sensor is in the state of being opened for 0.5 seconds, the carbon dioxide passage switch is controlled to be closed, the concentration value of the carbon dioxide gas concentration sensor is directly read, and the carbon dioxide gas concentration sensor is continuously compared with a set value; when->
When the carbon dioxide gas concentration sensor is in the state of being opened for 0.3 seconds, the carbon dioxide passage switch is controlled to be closed, the concentration value of the carbon dioxide gas concentration sensor is directly read, and the carbon dioxide gas concentration sensor is continuously compared with a set value; when- >
When the carbon dioxide passage switch is kept in the closed state.
The liquid flow assembly comprises a liquid inlet passage 4.1, a circulating passage 4.2, a waste liquid passage 4.3 and a recovery passage 4.4; the liquid inlet passage 4.1 and the circulating passage 4.2 are respectively communicated with the culture tank 2, the liquid inlet passage 4.1 is used for feeding liquid into the culture tank 2, and the circulating passage 4.2 is used for filtering metabolites produced by cells; the waste liquid passage 4.3 is communicated with the circulating passage 4.2 for discharging the metabolites, and the recovery passage 4.4 is communicated with the culture tank 2 for recovering finished cells; the flow control components of the feed liquid passage 4.1, the circulation passage 4.2, the waste liquid passage 4.3 and the recovery passage 4.4 are controlled by the central controller 6.
The flow control means may be a flow pump or a flow control switch.
The liquid inlet passage 4.1 comprises a liquid storage bag 4.1.1, a liquid inlet pipeline 4.1.2 and a liquid inlet pump 4.1.3, and the liquid storage bag 4.1.1 is communicated with the liquid inlet pipeline 4.1.2; the liquid inlet pipeline 4.1.2 is driven by the liquid inlet pump 4.1.3, and the liquid inlet pipeline 4.1.2 is communicated with the culture tank 2; the liquid inlet pump 4.1.3 is controlled by a central controller 6; the liquid in the liquid storage bag can be replaced according to the requirement, and can be a culture medium or physiological saline, for example. The medium may be a liquid medium.
Further, a cell branch pipe is arranged on the liquid inlet pipeline, and cells can be injected into the culture tank through the branch pipe. After the injection of the cells is completed, the manifold is closed, for example, a manifold cover may be provided to close the manifold.
In one embodiment, the feed channel 4.1 further comprises a level gauge 4.1.5. The liquid level meter 4.1.5 is arranged on the liquid inlet pipeline 4.1.2 and connected with the central controller for detecting the liquid level change of the liquid inlet pipeline. The liquid level information in the liquid inlet pipeline is sent to the central controller 6 in real time.
The circulating passage 4.2 comprises a circulating pipeline 4.2.1, a circulating pump 4.2.2 and a filter 4.2.3, the circulating pipeline 4.2.1 is communicated with the culture tank 2, the filter 4.2.3 is arranged on the circulating pipeline 4.2.1 and is communicated with the circulating pipeline 4.2.1, the circulating pipeline 4.2.1 is driven by the circulating pump 4.2.2, and the circulating pump 4.2.2 is controlled by the central controller 6.
In one embodiment, the filter 4.2.3 may be a hollow fiber column.
In one embodiment, the waste liquid channel 4.3 includes a waste liquid pump 4.3.1, a waste liquid tank 4.3.2 and a waste liquid pipeline 4.3.3, the waste liquid pipeline 4.3.1 is communicated with the circulation channel 4.2, the waste liquid tank 4.3.2 is connected with the waste liquid pipeline 4.3.1, the waste liquid pipeline 4.3.1 is driven by the waste liquid pump 4.3.1, and the waste liquid pump 4.3.1 is controlled by the central controller 6.
In one embodiment, the recovery path 4.4 includes a recovery line 4.4.1, a recovery pump 4.4.2 and a recovery bag 4.4.3, the recovery line 4.4.1 is in communication with the culture tank 2, the recovery line 4.4.1 is driven by the recovery pump 4.4.2, the recovery bag 4.4.3 is in communication with the recovery line 4.4.1, and the recovery pump 4.4.2 is controlled by the central controller 6.
In one embodiment, the culture system is provided with a recovery bag placing tray for placing recovery bags. And the recycling bag placing plate is provided with anti-slip ribs.
Further, as shown in fig. 5, the side wall of the culture tank 2 is provided with four ports:
a liquid inlet 2.1 for communicating with the liquid inlet passage 4.1;
a first circulation port 2.2 and a second circulation port 2.3 for communicating with the circulation passage 4.2;
and a recovery port 2.4 for communicating with the recovery passage 4.4.
As shown in FIG. 5, when the circulation path is in an open state during cell culture, the mixture of the cell culture medium and the cells in the culture tank is discharged from the first circulation port 2.2 into the filter 4.2.3 of the circulation path 4.2 under the drive of the circulation pump, the pore size of the filter 4.2.3 can be 0.2-1 μm, the water and the components of the metabolic waste produced by the cell culture in the culture medium can be permeated, and the cells themselves cannot permeate, so that part of the culture medium and the waste formation waste liquid are filtered, and the rest of the culture medium and the cells enter the culture tank 2 again from the second circulation port 2.3.
During the cultivation, the circulation path may be opened or closed according to the user's need.
Further, a waste liquid cavity is arranged on the filter 4.2.3 and is used for temporarily storing waste liquid. The filter 4.2.3 is provided with a first outlet for re-conveying the remaining part of the culture medium and the cells into the culture tank and a second outlet. The second outlet is used for communicating the waste liquid cavity with the waste liquid pipeline 4.3.1 and is used for discharging waste liquid.
The first circulation port 2.2 and the recovery port 2.4 are arranged at the bottom of the side wall of the culture tank to fully suck out the cells and the liquid in the culture tank 2.
In one embodiment, the flow assembly 4 includes a load cell 4.5, the load cell 4.5 being configured to determine the weight of the culture tank in real time, and to provide a test message to the central controller 6 at the bottom of the culture tank.
The central controller comprises a liquid control module: the central controller can receive the user instruction and control the on-off of the liquid inlet passage, the circulating passage, the waste liquid passage and the recovery passage according to the user instruction.
In the culture process, a user can carry out instructions, and the liquid inlet amount and the liquid waste discharge amount are set according to the needs.
In one embodiment, the central controller 6 may convert the liquid volume into weight according to the liquid density, and then control the liquid inlet amount and the liquid discharge amount according to the load cell. When the culture is not added with liquid initially, the culture tank is peeled, and the weight of the culture tank is the weight of the content of the culture tank in the culture process.
In one embodiment, the central controller 6 comprises:
weight comparison unit for measuring the weight of the culture tank in real time sent by the weighing sensor during liquid feedingQuantity M t With the weight M of the culture tank instructed by the user in0 Comparing, obtaining a weight difference M according to formula (III) in ：
M in ＝M 0 -M t (III)
The on-off control unit of the liquid inlet passage is used for controlling the liquid inlet passage according to M in And controlling the on-off of the liquid inlet passage.
Further, according to M in The on-off of the liquid inlet passage is controlled as follows:
M in greater than 0, open the liquid inlet passage, M in And when the pressure is equal to or less than 0, the liquid inlet passage is disconnected.
The central controller also comprises a waste liquid passage on-off control unit. When liquid is discharged, the weight comparison unit measures the weight M of the culture tank sent by the weighing sensor in real time t With the weight M of the culture tank instructed by the user out0 Comparing to obtain a weight difference M according to a formula (IV) out ：
M out ＝M t- M out0 (Ⅳ)
Waste liquid passage on-off control unit according to M out And controlling the on-off of the waste liquid passage.
M out Greater than 0, open the waste liquid passage, M out And when the pressure is equal to or less than 0, the liquid inlet passage is disconnected.
The temperature regulating assembly 5 comprises a heating device 5.1 and a temperature sensor 5.2; the heating device 5.1 and the temperature sensor 5.2 are arranged in the incubator 1 and are respectively connected with the central controller 6, and the heating device 5.1 is used for heating the inner cavity of the incubator; the temperature sensor 5.2 is used for measuring a real-time temperature value in the incubator and providing detection information for the central controller 6; the heating device 5.1 is controlled by the central control 6. The temperature adjusting component can enable the culture tank in the incubator to be in a constant temperature environment, and ensures that the temperature of gas entering the culture tank is constant.
In one embodiment, the preset temperature value T 0 The temperature value may be a temperature value suitable for cell culture; the temperature sensor is connected with the central controllerProviding a real-time temperature value T in the incubator t And T is equal to 0 Comparing, when T t Less than T 0 When the heating device is started, the central controller controls the heating device to be started; when T is t Greater than or equal to T 0 And when the central controller controls the heating device to stop running.
The heating means 5.1 may be a heating plate. Is attached to the inner wall of the incubator.
The heating plate and the temperature sensor are all commercial products.
As shown in fig. 6 and 7, the stirrer 2.5 includes: center shaft 2.51; and at least two blades 2.52 which are rotationally symmetrical with the axis of the central shaft and are connected with the central shaft, wherein each blade comprises a blade body, the blade body spirally extends, the axial length of the blade body accounts for 20 to 35 percent of the length of the central shaft, the rotation angle of the blade body is 15 to 50 degrees, the maximum radial length is 20 to 54mm, and the radial length from the bottom to the top is gradually reduced. The blade with the shape can reduce the shearing force on the T cells and simultaneously ensure that the T cells and the culture system are uniformly stirred.
Further, the radial length of the blade body gradually decreases in a linear relationship with axial height from the bottom to the top of the blade body.
Further, the spiral shape is formed by a part which is remained inside the conical surface after the conical surface is cut off on the basis of a positive spiral surface, and the half cone angle of the conical surface is 20-45 degrees. The radial vortex space formed by the blades and the side wall of the tank body can be reduced while the technical effect of facilitating axial up-and-down rolling of cells and culture solution is achieved, so that shearing force generated in the stirring process is reduced, T cells are protected, and the yield of the T cells is improved.
The bottom surface of the culture tank comprises a concave part 2.7 positioned at the center of the bottom surface of the culture tank and used for fixing the culture tank.
The culture system is provided with a stirring driver 8 for driving the stirrer. The agitation drive is controlled by the central controller 6.
In one embodiment, the blade further comprises a closed housing cavity 2.53 at the maximum radial length of the blade body for housing a magnet for driving the stirrer in rotation. At this time, the stirring driver 8 is a magnetic driver, and the magnetic driver acts on the magnet to drive the stirrer to rotate. The stirrer is driven in a non-contact mode by adopting the magnetic stirring principle, and compared with a mode of directly driving a rotating shaft of the stirrer by adopting a motor, the method can ensure the cleanness and convenient maintenance of the cell culture environment in the culture tank. The reason is that the motor is adopted to directly drive the rotating shaft of the stirrer, the motor is usually required to be arranged outside the culture tank, and then the rotating shaft of the stirrer must extend out of the culture tank, so that strict sealing between the rotating shaft of the stirrer and the culture tank in a culture environment for a long time is required to be ensured, the complexity of the system is increased, the maintenance is difficult, and the abrasion of the sealing element per se may pollute the culture system.
The blades of the stirrer adopt the design of a spiral surface, specific axial dimension and radial dimension, and the effects of axially stirring cells and culture solution in a tank body, reducing radial shearing force as much as possible, protecting the cells and improving the cell yield are achieved in a culture system.
The culture tank 2 is also provided with a culture cover 2.6 which is in a closed state in the culture process.
Further, as shown in FIG. 6, the culture lid 2.6 of the culture tank 2 comprises a lid body 2.61, an air inlet portion 2.63 and an air outlet portion 2.62 which are arranged on the lid body and used for conveying and discharging air to the space inside the tank body, wherein the length of the air inlet portion 2.63 in the direction vertical to the bottom surface of the lid body is longer than that of the air outlet portion 2.62; when the cell culture is carried out, the air inlet part is used for conveying the air into the cell culture tank, and when the cell culture is carried out, the air inlet part adopts a non-contact conveying mode to convey the air, namely the structure of the air inlet part is not contacted with the liquid level of the culture solution, and the arrangement has the advantages that the air inlet part stretches into the liquid level with the air inlet pipe in the prior art, so that bubbles are generated in a culture system to damage T cells, the non-contact conveying can prevent the generation of the bubbles, and the yield of the T cells is improved. Meanwhile, the length of the air inlet part perpendicular to the bottom surface direction of the cover body is larger than that of the air outlet part, and the air inlet cover has the beneficial effects that compared with the arrangement that the length of air inlet is equal to or shorter than that of air outlet, the concentration of oxygen and carbon dioxide in a culture system can be regulated more rapidly. The gas components of the inlet gas are blended with different stages of culture, the proportion of carbon dioxide of the inlet gas in the initial stage is properly increased, and a certain amount of carbon dioxide is also generated by cell respiration with the progress of culture, so that the proportion of carbon dioxide in the inlet gas can be reduced.
In one embodiment, an ultraviolet sterilizing lamp is arranged in the incubator 4 for sterilizing the incubator.
In one embodiment, the culture system is provided with an alarm module and is driven by the central controller 6. The method comprises the steps of presetting alarm critical values of a liquid flow component, a gas flow component and a temperature regulating component, and controlling the alarm module to give an alarm when the central controller receives detection information of the liquid flow component, the gas flow component and the temperature regulating component to exceed the limit.
The central controller can be a single-chip microcomputer, and the single-chip microcomputer can be an 8-bit minimum system. The central controller can also be selected from different brands and models, or a higher-order controller or a processor. The central controller may be used to install a related control program. After relevant control programs are installed, the central controller can receive signals of the liquid flow component, the air flow component and the temperature regulating component and instructions of a user, and adjust part parameters in the components according to requirements so as to enable the system to run stably.
Further, the culture tank is made of a non-breathable material. .
The culture tank and/or the accessories thereof exchange gas or liquid with the outside through the various passages only.
The incubator further comprises an incubator door, and the incubator is separated from the external environment, so that a relatively independent environment is formed in the incubator.
The fully-closed structure means that in the whole process from activation, infection and amplification of cell culture to recovery of finished products, the whole culture environment (comprising a tank body, a filter, a pipeline and the like) is in a relatively closed state and is communicated with the outside only through a sterile gas or liquid passage, and the incubator is relatively independent of the outside environment, so that the environment in the incubator is in a controllable range.
The totally-enclosed cell culture system provided by the invention can be used for totally-enclosed cell culture.
The method for carrying out the totally-enclosed cell culture by adopting the totally-enclosed cell culture system provided by the invention comprises the following steps:
1) The liquid inlet process comprises the following steps: setting the liquid inlet amount, and injecting a culture medium into a culture tank;
2) Building a culture environment: presetting concentration values of oxygen and carbon dioxide, respectively injecting the oxygen and the carbon dioxide into the incubator, measuring gas concentration values of the oxygen and the carbon dioxide in the incubator in real time, comparing the gas concentration values with set values respectively, and injecting the gas in the incubator into the incubator after the oxygen and the carbon dioxide values in the incubator reach standards; setting the temperature of the incubator and preheating the incubator;
3) Continuous culture: injecting cells, injecting factors, starting a stirrer, injecting a culture medium into a culture tank in continuous culture, filtering metabolites and discharging waste liquid;
4) Displacement and concentration: after the cell culture is completed, firstly replacing the culture medium with normal saline, concentrating after the replacement is completed, continuously discharging waste liquid, and reducing the liquid volume in the culture tank;
5) And (3) recovering a finished product: the stirrer was stopped and the finished cells in the culture tank were recovered.
In step 1), the culture medium is injected into the culture tank by using the liquid inlet passage. Specifically, the amount of feed liquid is set, and the set weight M of the culture tank is obtained based on the amount of feed liquid in0 Starting a liquid inlet pump, injecting the culture medium in the liquid storage bag into the culture tank through a liquid inlet pipeline, and determining the quantity of the culture medium to be injected into the culture tank by measuring the weight of the culture tank in real time by utilizing a weight sensor; measured in real time weight M of culture tank sent by weighing sensor t And the set weight M of the culture tank in0 Comparing, obtaining a weight difference M according to formula (III) in ：
M in ＝M in0 -M t (III)
According to M in Control ofAnd the liquid inlet passage is opened and closed.
Further, M in Greater than 0, open the liquid inlet passage, M 1 And when the pressure is equal to or less than 0, the liquid inlet passage is disconnected.
In step 2), oxygen and carbon dioxide are respectively injected into the incubator through the oxygen passage and the carbon dioxide passage, and the gas in the incubator is injected into the culture tank through the mixed gas suction passage. Measuring the gas concentration value of carbon dioxide in the incubator by using a carbon dioxide gas concentration sensor, and measuring the oxygen gas concentration value in the incubator by using an oxygen gas concentration sensor; will real-time oxygen gas concentration value 
And a real-time carbon dioxide gas concentration value->
Respectively with the preset oxygen gas concentration value->
And preset carbon dioxide concentration value->
By comparison, the difference in the desired concentration, i.e.the concentration difference +.>
And->
According to
The on-off time of the oxygen passage is regulated;
according to
Adjusting the on-off time of the carbon dioxide passage;
when (when)
Is->
When the gas in the incubator meets the set threshold range, the mixed gas suction passage is opened, and the gas in the incubator is sucked into the incubator;
when (when)
Is->
When at least one of the gas mixture suction passages does not satisfy the set threshold range, the gas mixture suction passage is closed.
Further, according to
The value of (2) is regulated in a stepwise manner by the opening and closing time of the oxygen passage and/or, as a function of +.>
And (3) adjusting the on-off time of the carbon dioxide passage switch in a grading regulation mode.
In one embodiment, a fan may be used to agitate the gas flow to speed up mixing and to more uniformly mix the gases.
In step 3), cells are injected by the liquid inlet passage and filtered by the circulating passageA metabolite. The circulating passage is provided with a filter and a circulating pump. After the stirrer is started, a circulation passage is opened according to the culture requirement, when the circulation passage is in an open state, under the drive of a circulation pump, the mixture of the cell culture medium and cells in the culture tank is discharged from a first circulation port on the culture tank into a filter of the circulation passage, the pore diameter of the filter can be 0.2-1 micron, water can be permeated and components of metabolic wastes generated by cell culture in the culture medium can not permeate, and therefore, part of the culture medium and metabolic wastes form waste liquid to be filtered and temporarily stored in a waste liquid cavity on the filter; and the rest part of the culture medium and the cells enter the culture tank again through a second circulation port on the culture tank. The filter is provided with a first outlet and a second outlet, and the rest culture medium and cells are conveyed into the culture tank again through the first outlet. When the waste liquid is required to be discharged, the waste liquid passage is opened, and the set waste liquid discharge amount is set to obtain the set weight M of the culture tank out0 The waste liquid is discharged into the waste liquid pipe through the second outlet, and then is discharged into the waste liquid barrel. Measured in real time weight M of culture tank sent by weighing sensor t And the preset weight M of the culture tank out0 Comparing, obtaining the weight difference M according to the formula (IV) out ：
M out ＝M t -M out0 (IV)
According to M out And controlling the on-off of the waste liquid passage.
Further, M out Greater than 0, open the waste liquid passage, M out And when the liquid waste channel is equal to or smaller than 0, the liquid waste channel is disconnected.
When liquid feeding is needed, the liquid feeding amount is set, and the set weight M of the culture tank is obtained according to the liquid feeding amount in0 Starting a liquid inlet pump, injecting the culture medium in the liquid storage bag into the culture tank through a liquid inlet pipeline, and determining the quantity of the culture medium to be injected into the culture tank by measuring the weight of the culture tank in real time by utilizing a weight sensor; measured in real time weight M of culture tank sent by weighing sensor t And the set weight M of the culture tank in0 Comparing, obtaining a weight difference M according to formula (III) in ：
M in ＝M in0 -M t (III)
According to M in And controlling the on-off of the liquid inlet passage.
Further, M in Greater than 0, open the liquid inlet passage, M 1 And when the pressure is equal to or less than 0, the liquid inlet passage is disconnected.
Further, in the cultivation process, the circulation passage can be opened or closed according to the needs of the user.
In the step 4), after the cell culture is completed, the culture medium is replaced by normal saline, and the specific operation is as follows: after a fixed amount of waste liquid was discharged, an equal amount of physiological saline was added, and then the above operation was repeated until the medium was completely replaced. If the initial liquid amount in the culture tank is 400 ml, 200 ml of waste liquid is discharged first, then 200 ml of physiological saline is added, the concentration of the culture medium is reduced to 50%, and after ten times of repeated operation, the concentration of the culture medium is reduced to 1/2 10 The replacement is considered to be completed. And then enters a concentration link, continuously discharges waste liquid, and reduces the liquid volume in the culture tank.
In the concentration process, the liquid volume in the culture tank is controlled by the following control method: setting the weight of the target culture tank as M, and measuring the weight M of the culture tank sent by a weighing sensor in real time t Comparing with the target culture tank weight M, obtaining a weight difference M according to the formula (V) 1 ：
M 1 ＝M t -M (Ⅴ)
According to M 1 And controlling the on-off of the waste liquid passage.
Further, M 1 Greater than 0, open the waste liquid passage, M 1 And when the liquid waste channel is equal to or smaller than 0, the liquid waste channel is disconnected.
In step 5), the final cells are recovered by using the cell recovery path. Specifically, under the drive of the recovery pump, the mixed solution containing cells in the culture tank enters the recovery bag through the recovery port and the recovery pipeline.
Further, in the step 5), the method further comprises the step of operating the circulating pump reversely to recover the finished cells in the circulating path.
In one embodiment, the step 2) includes pre-treating the filter;
in one embodiment, the liquid inlet passage further comprises a liquid level gauge. The liquid level change of the liquid inlet pipeline is monitored by a liquid level meter.
In one embodiment, the culture system is provided with an alarm module. The method comprises the steps of presetting alarm critical values of liquid level, oxygen gas concentration, carbon dioxide gas concentration, culture tank weight and temperature in a system, wherein the alarm critical values comprise an alarm critical upper limit and an alarm critical lower limit, and when each detection information in the system is higher than the alarm critical upper limit or lower than the alarm critical lower limit, an alarm is given.
In one embodiment, an ultraviolet sterilizing lamp is arranged in the incubator, and the incubator is sterilized by the ultraviolet sterilizing lamp.
The parameters of the liquid inlet passage, the circulating passage and the waste liquid passage in the liquid flow assembly, such as liquid inlet amount and waste liquid discharge amount, can be set on site and corresponding operation can be immediately executed by a user according to the culture condition in the culture process; and the system can be set in advance, and the system is used for operation in a timing starting mode.
In summary, the system of the invention realizes a constant temperature culture environment, adopts a perfusion mode to perform cell culture, can discharge waste liquid in the culture process, prevent the accumulation of harmful metabolites, is beneficial to achieving higher cell culture density, can reduce subsequent processing steps, can recover finished cells without operations such as centrifugation, simplifies the operations, can improve the culture efficiency, is easy to industrialize, and realizes a totally-enclosed integrated process from cell activation, infection and amplification to finished product recovery. The radial shearing force is reduced as much as possible by axially stirring the cells and the culture solution in the tank body, so that the cells can be effectively protected, and the cell yield is improved. The system disclosed by the invention is used for separately feeding air, so that the content of each gas component in the culture process is stable, the influence of the change of external gas on the cell culture in the cell culture process can be avoided, the content of each gas component is unchanged, meanwhile, the manual operation is reduced, the cost is reduced, the risk of misoperation in the culture process is reduced, and the culture efficiency is improved. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Claims (8)
1. A totally enclosed cell culture system comprising at least the following:
an incubator (1);
a culture tank (2) for culturing cells, which is provided in the incubator; the culture tank (2) is provided with a stirrer (2.5), the stirrer is arranged in the culture tank, and the stirrer axially stirs cells and culture solution in the culture tank; the stirrer (2.5) comprises: a central shaft (2.51); and at least two blades (2.52) rotationally symmetrical with the axis of the central shaft and connected with the central shaft, wherein each blade comprises a blade body, the blade body spirally extends, the axial length of the blade body accounts for 20 to 35 percent of the length of the central shaft, the rotation angle of the blade body is 15 to 50 degrees, the maximum radial length is 20 to 54mm, and the radial length from the bottom to the top is gradually reduced;
An air flow assembly (3) communicated with the incubator (1) for adjusting the concentration of oxygen and carbon dioxide in the incubator (1);
a liquid flow component (4) communicated with the culture tank (2) and used for regulating the liquid flow in the culture tank (2), filtering the metabolites produced by cell culture and recovering finished cells; and the flow assembly (4) comprises: a liquid inlet passage (4.1), a circulation passage (4.2), a waste liquid passage (4.3) and a recovery passage (4.4); the liquid inlet passage (4.1) and the circulating passage (4.2) are respectively communicated with the culture tank (2), the liquid inlet passage (4.1) is used for feeding liquid into the culture tank (2), and the circulating passage (4.2) is used for filtering metabolites produced by cells; the waste liquid passage (4.3) is communicated with the circulating passage (4.2) for discharging the metabolites, and the recovery passage (4.4) is communicated with the culture tank (2) for recovering finished cells; the flow control parts of the liquid inlet passage (4.1), the circulating passage (4.2), the waste liquid passage (4.3) and the recovery passage (4.4) are controlled by the central controller (6); the circulating passage (4.2) comprises a circulating pipeline (4.2.1), a circulating pump (4.2.2) and a filter (4.2.3), the circulating pipeline (4.2.1) is communicated with the culture tank (2), the filter (4.2.3) is arranged on the circulating pipeline (4.2.1) and is communicated with the circulating pipeline (4.2.1), the circulating pipeline (4.2.1) is driven by the circulating pump (4.2.2), and the circulating pump (4.2.2) is controlled by the central controller (6);
A temperature regulating assembly (5) for regulating the temperature in the incubator;
and a central controller (6) connected with and controlling the air flow assembly, the liquid flow assembly and the temperature regulating assembly.
2. The totally enclosed cell culture system of claim 1, wherein the airflow assembly comprises: a separate air passage (3.1), a carbon dioxide passage (3.2) and an oxygen passage (3.3) respectively communicating with the incubator (1) for delivering gas into the incubator to form a mixed gas;
a mixed gas suction passage (3.4) which communicates the incubator (1) with the culture tank (2) and is used for inputting the mixed gas in the incubator (1) into the culture tank (2);
an exhaust gas discharge passage (3.5) communicating with the culture tank (2) for discharging exhaust gas generated during cell culture;
a gas concentration sensing module (3.6) including an oxygen gas concentration sensor (3.6.1) and a carbon dioxide gas concentration sensor (3.6.2); the gas concentration sensing module is used for measuring the real-time oxygen gas concentration value and the real-time carbon dioxide gas concentration value in the incubator respectively and providing detection information for the central controller (6).
3. The totally enclosed cell culture system of claim 2, wherein the gas flow assembly further comprises one, two, or three of the following features:
1) the carbon dioxide passage (3.2) comprises a carbon dioxide storage device (3.2.1), a carbon dioxide pressure reducing valve (3.2.2), a carbon dioxide passage switch (3.2.3) and a carbon dioxide pipeline (3.2.4); the carbon dioxide storage device (3.2.1) is connected with the carbon dioxide pipeline (3.2.4), the carbon dioxide pipeline (3.2.4) is driven by a carbon dioxide pressure reducing valve, and the carbon dioxide pipeline (3.2.4) is communicated with the incubator (1); a carbon dioxide passage switch (3.2.3) is arranged on the carbon dioxide passage, and the carbon dioxide passage switch (3.2.3) is controlled by the central controller (6);
2) The oxygen passage (3.3) comprises an oxygen storage device (3.3.1), an oxygen pressure reducing valve (3.3.2), an oxygen passage switch (3.3.3) and an oxygen pipeline (3.3.4); the oxygen storage device (3.3.1) is connected with the oxygen pipeline (3.3.4), the oxygen pipeline (3.3.4) is driven by an oxygen pressure reducing valve (3.3.2), and the oxygen pipeline (3.3.4) is communicated with the incubator (1); an oxygen passage switch (3.3.3) is arranged on the oxygen passage, and the oxygen passage switch (3.3.3) is controlled by the central controller (6);
3) The mixed gas suction passage (3.4) comprises a mixed gas suction pump (3.4.1) and a mixed gas suction pipeline (3.4.2); the mixed gas suction pipeline (3.4.2) is driven by a mixed gas suction pump (3.4.1), and the mixed gas suction pipeline (3.4.2) is used for injecting mixed gas from the incubator (1) into the incubator (2); the mixed gas suction pump (3.4.1) is controlled by the central controller (6).
4. The totally enclosed cell culture system of claim 2, further comprising one or more of the following features:
1) A gas discharge passage (3.7) is arranged in the incubator and is used for discharging gas in the incubator and keeping the pressure in the incubator stable;
2) The carbon dioxide passage (3.2) and the oxygen passage (3.3) are provided with a carbon dioxide passage incubator inlet (3.2.5) and an oxygen passage incubator inlet (3.3.5) on the incubator, and the carbon dioxide passage incubator inlet (3.2.5) and the oxygen passage incubator inlet (3.3.5) are both arranged at the upper part in the incubator;
3) The mixed gas passage is provided with a mixed gas passage incubator outlet (3.4.3), and the mixed gas passage incubator outlet (3.4.3), an oxygen gas concentration sensor (3.6.1) and a carbon dioxide gas concentration sensor (3.6.2) are arranged at the lower part in the incubator;
4) A fan (3.8) is arranged in the incubator and used for stirring air flow, accelerating mixing, enabling the air to be mixed more uniformly and accelerating heat exchange inside the incubator.
5. A totally enclosed cell culture system according to claim 2, wherein the central controller (6) comprises the following parts: the gas concentration comparison unit is used for comparing the real-time carbon dioxide gas concentration value C sent by the gas concentration sensing module tCO2 And a real-time oxygen gas concentration value C tO2 And a preset carbon dioxide concentration value C 0CO2 And a preset oxygen gas concentration value C 0O2 Respectively comparing, and obtaining the difference of the required concentration according to formulas (I) and (II), namely concentration difference C CO2 And C O2 ：
C CO2 = C 0CO2 - C tCO2 （I）
C O2 =C 0O2 -C tO2 （II）
A gas concentration switch control unit for controlling the opening and closing of the carbon dioxide passage, the oxygen passage and the mixed gas suction passage:
according to C O2 The on-off time of the oxygen passage is regulated;
according to C CO2 Adjusting the on-off time of the carbon dioxide passage;
when C O2 C (C) CO2 When the gas in the incubator meets the set threshold range, the mixed gas suction passage is opened, and the gas in the incubator is sucked into the incubator;
when C O2 C (C) CO2 When at least one of the gas mixture suction passages does not satisfy the set threshold range, the gas mixture suction passage is closed.
6. The totally enclosed cell culture system of claim 5, wherein the flow assembly further comprises one or more of the following features:
1) the liquid inlet passage (4.1) comprises a liquid storage bag (4.1.1), a liquid inlet pipeline (4.1.2) and a liquid inlet pump (4.1.3); the liquid storage bag (4.1.1) is communicated with the liquid inlet pipeline (4.1.2); the liquid inlet pipeline (4.1.2) is driven by the liquid inlet pump (4.1.3), and the liquid inlet pipeline (4.1.2) is communicated with the culture tank (2); the liquid inlet pump (4.1.3) is controlled by the central controller (6);
2) The waste liquid channel (4.3) comprises a waste liquid pump (4.3.1), a waste liquid barrel (4.3.2) and a waste liquid pipeline (4.3.3), the waste liquid pipeline (4.3.3) is communicated with the circulating channel (4.2), the waste liquid barrel (4.3.2) is connected with the waste liquid pipeline (4.3.3), the waste liquid pipeline (4.3.3) is driven by the waste liquid pump (4.3.1), and the waste liquid pump (4.3.1) is controlled by the central controller (6);
3) The recovery passageway (4.4) is including retrieving pipeline (4.4.1), retrieve pump (4.4.2) and recovery bag (4.4.3), retrieve pipeline (4.4.1) with cultivate jar (2) intercommunication, retrieve pipeline (4.4.1) receive retrieve pump (4.4.2) drive, retrieve bag (4.4.3) with retrieve pipeline (4.4.1) intercommunication, retrieve pump (4.4.2) receive central controller (6) control.
7. The totally enclosed cell culture system of claim 1, further comprising one or more of the following features:
1) The liquid flow assembly (4) comprises a weighing sensor (4.5), wherein the weighing sensor (4.5) is used for measuring the weight of the culture tank in real time, is positioned at the bottom of the culture tank and provides detection information for the central controller (6);
2) The temperature regulating assembly (5) comprises a heating device (5.1) and a temperature sensor (5.2); the heating device (5.1) and the temperature sensor (5.2) are arranged in the incubator (1) and are respectively connected with the central controller (6), and the heating device (5.1) is used for heating the inner cavity of the incubator; the temperature sensor (5.2) is used for measuring a real-time temperature value in the incubator and providing detection information for the central controller (6); the heating device (5.1) is controlled by the central controller (6).
8. Use of the totally enclosed cell culture system according to any one of claims 1-7 for totally enclosed cell culture.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010167455.8A CN111334427B (en) | 2019-03-12 | 2019-03-12 | Totally-enclosed cell culture system |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010167455.8A CN111334427B (en) | 2019-03-12 | 2019-03-12 | Totally-enclosed cell culture system |
| CN201910184785.5A CN109825436A (en) | 2019-03-12 | 2019-03-12 | Totally-enclosed cell culture system |
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| CN201910184785.5A Division CN109825436A (en) | 2019-03-12 | 2019-03-12 | Totally-enclosed cell culture system |
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| WO2020181709A1 (en) * | 2019-03-12 | 2020-09-17 | 华道(上海)生物医药有限公司 | Fully enclosed cell incubation system |
| CN110218643B (en) * | 2019-06-19 | 2024-02-02 | 华道(上海)生物医药有限公司 | Magnetic stirring system with weighing function |
| CN113005094A (en) * | 2021-03-16 | 2021-06-22 | 中国人民解放军海军军医大学第一附属医院 | Universal chimeric antigen receptor modified T cell and preparation method and application thereof |
| CN112662631B (en) * | 2021-03-16 | 2021-06-29 | 合源生物科技(天津)有限公司 | CAR-T cell perfusion culture method |
| CN114317270B (en) * | 2022-03-12 | 2022-06-17 | 广州赛太特生物医学科技有限公司 | Cell culture device for biological gene research |
| CN115888981B (en) * | 2023-01-10 | 2023-07-21 | 上海润度生物科技有限公司 | Automatic ventilation device for separating waste gas of incubator |
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| CN111218403B (en) | 2021-01-29 |
| CN111218403A (en) | 2020-06-02 |
| CN109825436A (en) | 2019-05-31 |
| CN111334427A (en) | 2020-06-26 |
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