CN209989411U - Totally-enclosed cell culture system - Google Patents
Totally-enclosed cell culture system Download PDFInfo
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- CN209989411U CN209989411U CN201920312979.4U CN201920312979U CN209989411U CN 209989411 U CN209989411 U CN 209989411U CN 201920312979 U CN201920312979 U CN 201920312979U CN 209989411 U CN209989411 U CN 209989411U
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Abstract
The utility model provides a totally closed cell culture system includes following part at least: the system comprises an incubator, a culture tank, an airflow component, a liquid flow component, a temperature regulation component and a central controller. The system realize the constant temperature culture environment, adopt the mode of perfusion to carry out cell culture, realize from cell activation, infection, expand the totally closed integration process of finished product recovery. The cells and the culture solution are axially stirred in the tank body, so that the radial shearing force is reduced as much as possible, the cells can be effectively protected, and the cell yield is improved. The system separately admits air, guarantees that each gaseous component content is stable among the culture process, can guarantee that the change of external gas can not directly cause the influence to cell culture among the cell culture process, guarantees that gaseous each component content is unchangeable, accomplishes the control to cultivateing temperature, liquid measure and gas concentration at the culture in-process, keeps the cultivation environment stable, reduces manual operation simultaneously, reduce cost, reduces the risk of cultivateing in-process misoperation, raises the efficiency.
Description
Technical Field
The utility model relates to a cell culture technical field especially relates to a totally closed cell culture system.
Background
In recent years, CAR-T cell immunotherapy has been considered as one of the most promising therapies to combat cancer. It has many advantages over other therapies, such as CAR-T cells can have multiple targeting sites, improving the accuracy of tumor treatment, and the course of action is not limited by mhc (major histocompatibility complex); the CAR-T cell has wider tumor killing range and longer effect; strong technical property, strong reproducibility and the like. In 2018, the FDA approved two CD19CAR-T cell drugs (kymeriah and yescatta, respectively) that had good efficacy in the treatment of hematological malignancies. However, there are still many limitations to CAR-T cell immunotherapy, such as the production of CAR-T cells. In the CAR-T treatment process, T cells which are technically modified need to be cultured in vitro, after the number of the cells which meet the treatment requirement is reached (generally, a patient needs hundreds of millions or even billions of CAR-T cells), the cells are infused back into the body of the patient to kill cancer cells in a targeted manner, however, the CAR-T cells are limited by the current technical means, the in vitro culture time of the CAR-T cells is relatively long, and the clinical treatment period is prolonged.
Cell culture (cell culture) refers to a method for simulating in vivo environment (sterility, proper temperature, pH value, certain nutritional conditions and the like) in vitro to enable the cells to survive, grow and reproduce and maintain main structures and functions. The cell culture technology is an important and common technology in cell biology research methods, and a large number of cells can be obtained by culturing cells through the cell culture technology, and signal transduction, anabolism, growth and proliferation of the cells and the like of the cells can be researched through the cell culture technology.
Most of the existing cell culture is manually operated culture, when a large amount of cells need to be cultured facing industrialization, a large amount of labor cost and time cost need to be spent, and meanwhile, the risk of errors is greatly increased along with the increase of the burden of an operator; in addition, artificial culture cannot accurately control the environment for cell growth, which is not favorable for cell growth.
SUMMERY OF THE UTILITY MODEL
In view of the above-mentioned shortcomings of the prior art, it is an object of the present invention to provide a totally enclosed cell culture system.
To achieve the above and other related objects, the present invention provides a totally enclosed cell culture system, comprising at least the following components:
an incubator;
the culture tank is arranged in the incubator; the culture tank is provided with a stirrer, and the stirrer is arranged in the culture tank; for performing cell culture;
an airflow assembly in communication with the incubator; for regulating the oxygen and carbon dioxide concentrations in the incubator;
a liquid flow assembly in communication with the culture tank; used for regulating the liquid flow in the culture tank, filtering metabolites produced by cell culture and recovering finished cells;
a temperature regulating component; the temperature control device is arranged in the incubator and is used for adjusting the temperature in the incubator;
and the central controller is connected with the air flow assembly, the liquid flow assembly and the temperature regulating assembly.
As mentioned above, the utility model discloses a totally closed cell culture system has following beneficial effect:
the system realize the constant temperature culture environment, adopt the perfusion mode to carry out cell culture, realize from cell activation, infection, expand the totally closed integration process of finished product recovery. The system adopt the perfusion mode, rather than the mode of filling, can discharge the waste liquid at the culture in-process, prevent the accumulation of harmful metabolite, be favorable to reaching higher cell culture density, can reduce subsequent processing step, need not operations such as centrifugation and can carry out finished product cell and retrieve, the simplified operation can improve cultivation efficiency, easily industrialization realizes from cell activation, infection, the totally closed integration process of expanding finished product recovery. The cells and the culture solution are axially stirred in the tank body, so that the radial shearing force is reduced as much as possible, the cells can be effectively protected, and the cell yield is improved. The system separately admits air, guarantees that each gaseous component content is stable among the culture process, can guarantee that the gaseous change of external world can not directly cause the influence to cell culture in the cell culture process, guarantees that gaseous each component content is unchangeable, accomplishes the control to cultivateing temperature, liquid measure and gas concentration at the culture in-process, keeps the cultivation environment stable, reduces manual operation simultaneously, and reduce cost reduces the risk of operation error among the culture process, improves cultivation efficiency.
Drawings
FIG. 1 shows a signal transmission diagram of the totally enclosed cell culture system of the present invention;
FIG. 2 is a front structural view of the totally enclosed cell culture system of the present invention;
FIG. 3 is a back view of the totally enclosed cell culture system of the present invention;
FIG. 4 is a diagram showing the distribution of the components on the surface of the incubator of the totally enclosed cell culture system of the present invention;
FIG. 5 is a schematic view showing the communication relationship between the culture tank and the liquid flow module of the totally enclosed cell culture system of the present invention.
FIG. 6 shows the internal structure of the culture tank of the totally enclosed cell culture system of the present invention.
FIG. 7 is a schematic top view of a stirrer of a culture tank of the totally enclosed cell culture system according to the present invention.
Description of the element reference numerals
1 incubator
2 culturing pot
2.1 liquid inlet
2.2 first circulation opening
2.3 second circulation port
2.4 recovery port
2.5 Agitator
2.51 middle axle
2.52 blade
2.53 closed accommodation chamber
2.6 culture cover
2.61 cover body
2.62 exhaust section
2.63 air intake
2.7 inner recess
3 airflow assembly
3.1 air passages
3.1.1 air Filter
3.1.2 air line
3.1.3 Vent
3.2 carbon dioxide pathway
3.2.1 carbon dioxide storage device
3.2.2 carbon dioxide pressure reducing valve
3.2.3 carbon dioxide Access switch
3.2.4 carbon dioxide line
3.2.5 carbon dioxide passage incubator inlet
3.3 oxygen pathway
3.3.1 oxygen storage device
3.3.2 oxygen pressure reducing valve
3.3.3 oxygen passage switch
3.3.4 oxygen line
3.3.5 oxygen pathway incubator inlet
3.4 Mixed gas suction passage
3.4.1 Mixed gas suction Pump
3.4.2 mixed gas suction line
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 discharge passage
3.8 Fan
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 pathway
4.2.1 circulation line
4.2.2 circulating Pump
4.2.3 Filter
4.3 waste liquid pathway
4.3.1 waste liquid Pump
4.3.2 waste liquid bucket
4.3.3 waste liquid line
4.4 Recycling lanes
4.4.1 recovery line
4.4.2 recovery Pump
4.4.3 recovery bags
4.5 weighing sensor
5 temperature regulating assembly
5.1 heating device
5.2 temperature sensor
6 central controller
7 sterilizing lamp
8 stirring driver
Detailed Description
The following description is provided for illustrative purposes, and other advantages and features of the present invention will become apparent to those skilled in the art from the following detailed description.
Please refer to fig. 1-7. It should be understood that the structure, ratio, size and the like shown in the drawings attached to the present specification are only used for matching with the content disclosed in the specification, so as to be known and read by those skilled in the art, and are not used for limiting the limit conditions that the present invention can be implemented, so that the present invention has no technical essential meaning, and any structure modification, ratio relationship change or size adjustment should still fall within the scope that the technical content disclosed in the present invention can cover without affecting the function that the present invention can produce and the purpose that the present invention can achieve. Meanwhile, the terms such as "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for convenience of description, and are not intended to limit the scope of the present invention, and changes or adjustments of the relative relationship thereof may be made without substantial technical changes, and the present invention is also regarded as the scope of the present invention.
It should be understood that the central controller of the present invention may be located at any position of the outer wall of the incubator, or located on the console outside the incubator, as long as it can be connected to other components of the totally enclosed cell culture system, and therefore, the position of the central controller is not shown in fig. 2-4.
As shown in fig. 1-4, the totally enclosed cell culture system provided by the present invention at least comprises the following parts:
an incubator 1; the incubator is used for providing a stable culture environment, including a stable gas environment and a temperature environment.
The culture tank 2 is arranged in the incubator; the culture tank 2 is provided with a stirrer which is arranged in the culture tank. For cell culture.
The airflow component 3 is communicated with the incubator 1; for regulating the oxygen and carbon dioxide concentrations in the incubator 1;
a liquid flow assembly 4 in communication with the culture tank 2; used for regulating the liquid flow in the culture tank 2, filtering metabolites produced by cell culture and recovering finished cells.
A temperature adjusting component 5; is arranged in the incubator 1 and is used for adjusting the temperature in the incubator.
A central controller 6; connecting the gas flow assembly, the liquid flow assembly and the temperature regulating assembly.
The culture system adopts a perfusion mode instead of a perfusion mode, waste liquid can be discharged in the culture process, accumulation of harmful metabolites is prevented, higher cell culture density can be achieved, subsequent treatment steps can be reduced, finished product cell recovery can be carried out without operations such as centrifugation, operation is simplified, cell recovery is facilitated, culture efficiency can be improved, and industrialization is easy.
The system adopts a stirring mode to culture cells, axially stirs the cells and the culture solution in the tank body in a culture system, and reduces radial shear force as much as possible so as to protect the cells and improve the cell yield.
Further, the airflow assembly 3 includes:
a separate air 3.1, carbon dioxide 3.2 and oxygen 3.3 passageway, respectively, communicating with the incubator 1 for delivering gases into the incubator to form a mixed gas.
And a mixed gas suction passage 3.4 communicating the incubator 1 and the culture tank 2, for introducing 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 the exhaust gas generated during the cell culture.
The gas concentration sensing module 3.6 comprises an oxygen gas concentration sensor 3.6.1 and a carbon dioxide gas concentration sensor 3.6.2; is connected with the central controller 6 and is respectively used for measuring the real-time oxygen gas concentration value and the real-time carbon dioxide gas concentration value in the incubator.
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 channel 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 pressure reducing valve 3.2.2 is arranged on the carbon dioxide pipeline 3.2.4, and the carbon dioxide pipeline 3.2.4 is communicated with the incubator 1; and 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 connected with 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 pipeline 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 pressure reducing valve 3.3.2 is arranged on the oxygen pipeline 3.3.4, 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 connected with the central controller 6.
In one embodiment, the oxygen access switch is selected from one or more of an oxygen storage device switch, an oxygen line switch, and an oxygen pressure relief valve switch. May be a solenoid valve.
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 line 3.4.2 is connected to the mixed gas suction pump 3.4.1, and the mixed gas suction pump 3.4.1 is connected to the central controller 6.
In one embodiment, a gas filter is provided in the mixed gas suction line 3.4.2. For filtering the gas entering the culture tank.
Further, the exhaust gas discharge passage 3.5 includes an exhaust gas discharge line. And a check valve is arranged on the waste gas discharge pipeline to prevent outside air from entering the tank through the waste gas discharge pipeline.
The exhaust gas discharge passage directly discharges exhaust gas to the outside of the system without communicating with the incubator 4.
Furthermore, a gas discharge passage 3.7 is arranged in the incubator and used for discharging gas in the incubator and keeping the gas pressure in the incubator stable.
Further, the gas exhaust passage 3.7 includes a gas exhaust line for exhausting the gas in the tank to maintain the gas pressure in the tank stable.
In one embodiment, the gas discharge passage 3.7 and the air passage 3.1 are the same passage.
In one embodiment, the carbon dioxide and oxygen passageways 3.2, 3.3 are provided with carbon dioxide passageway incubator inlet 3.2.5 and oxygen passageway incubator inlet 3.3.5 on the incubator, and the carbon dioxide passageway incubator inlet 3.2.5 and oxygen passageway incubator inlet 3.3.5 are both provided in the upper portion of the incubator. The cold air easily sinks the end, and the hot air rises, can let gas mixture reach whole environmental gas concentration unanimous more fast at the top.
In one embodiment, the mixed gas pathway is provided with a mixed gas pathway incubator outlet 3.4.3 on the incubator, the mixed gas pathway incubator outlet 3.4.3, the oxygen gas concentration sensor 3.6.1 and the carbon dioxide gas concentration sensor 3.6.2 being provided at a lower portion within the incubator. The concentration of the mixed gas can be reflected more accurately, 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, 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, to speed up mixing, to make the air mix more evenly, and to speed up heat exchange inside the incubator.
In one embodiment, the fan 3.8 is provided in the upper part of the incubator.
The liquid flow component 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 circulation 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 circulation passage 4.2 is used for filtering metabolites generated by cells; the waste liquid passage 4.3 is communicated with the circulation passage 4.2 for discharging the metabolite, and the recovery passage 4.4 is communicated with the culture tank 2 for recovering the finished product cells; the liquid inlet passage 4.1, the circulating passage 4.2, the waste liquid passage 4.3 and the recycling passage 4.4 are connected with the central controller 6.
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 connected with 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 connected with the central controller 6; the liquid in the liquid storage bag can be replaced according to the needs, and for example, the liquid can be culture medium or normal saline. The medium may be a liquid medium.
Furthermore, 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 cells is completed, the manifold is in a closed state, and a manifold cover may be provided to close the manifold, for example.
In one embodiment, the liquid inlet passage 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 and is used for detecting the liquid level change of the liquid inlet pipeline.
Circulation route 4.2 includes circulation pipeline 4.2.1, circulating pump 4.2.2 and filter 4.2.3, circulation pipeline 4.2.1 with cultivate jar 2 intercommunication, filter 4.2.3 locate on the circulation pipeline 4.2.1 and with circulation pipeline 4.2.1 intercommunication, circulation pipeline 4.2.1 with circulating pump 4.2.2 is connected, circulating pump 4.2.2 with central controller 6 is connected.
In one embodiment, the filter 4.2.3 may be a hollow fiber column.
In one embodiment, the waste liquid path 4.3 includes a waste liquid pump 4.3.1, a waste liquid barrel 4.3.2 and a waste liquid line 4.3.3, the waste liquid line 4.3.1 is connected to the circulation path 4.2, the waste liquid barrel 4.3.2 is connected to the waste liquid line 4.3.1, the waste liquid line 4.3.1 is connected to the waste liquid pump 4.3.1, and the waste liquid pump 4.3.1 is connected to the central controller 6.
In one embodiment, the recovery pathway 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 connected to 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 connected to the central controller 6.
In one embodiment, the culture system is provided with a recovery bag placing tray for placing the recovery bag. And the recycling bag placing plate is provided with anti-skid ribs.
Further, as shown in FIG. 5, the side wall of the culture tank 2 is provided with four ports:
the liquid inlet 2.1 is used for being communicated with a liquid inlet passage 4.1;
a first circulation port 2.2 and a second circulation port 2.3 for communicating with the circulation path 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 opened during the 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 by the circulation pump, the pore size of the filter 4.2.3 can be 0.2-1 μm, and the water and the components of the metabolic waste generated by the cell culture in the culture medium can permeate through the filter, while the cells cannot permeate through the filter, so that part of the waste solution formed by the culture medium and the metabolic waste is filtered, and the rest of the culture medium and the cells can enter the culture tank 2 again from the second circulation port 2.3.
During the culture, the circulation path may be opened or closed as required.
Furthermore, a waste liquid cavity is arranged on the filter 4.2.3 and used for temporarily storing waste liquid. The filter 4.2.3 is provided with a first outlet and a second outlet, the first outlet being used for re-feeding the remaining part of the culture medium and the cells into the culture tank. The second outlet is used for communicating the waste liquid cavity with the waste liquid pipeline 4.3.1 and discharging waste liquid.
The first circulation port 2.2 and the recovery port 2.4 are both arranged at the bottom of the side wall of the culture tank so as to sufficiently suck out cells and 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 used to determine tank weight in real time, located at the bottom of the tank and connected to the central controller 6.
The temperature adjusting component 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 the real-time temperature value in the incubator. The temperature adjusting component can enable the culture tank in the culture box to be in a constant temperature environment, and the temperature of gas entering the culture tank is guaranteed to be constant.
The heating device 5.1 may be a heating plate. Attached to the inner wall of the incubator.
The heating plate and the temperature sensor are both commercially available products.
As shown in fig. 6 and 7, the stirrer 2.5 comprises: the middle shaft 2.51; and at least two blades 2.52 which are rotationally symmetrical about the axis of the central shaft and are connected with the central shaft, each blade comprises a blade body, the blade bodies extend spirally, the axial length of each blade body accounts for 20-35% of the length of the central shaft, the rotation angle of each blade body is 15-50 degrees, the maximum radial length is 20-54 mm, and the radial length from the bottom to the top is gradually reduced. With the blade having the above shape, it is possible to reduce the shearing force for the T cells while stirring the T cells and the culture system uniformly.
Further, the radial length of the vane body decreases in a linear relationship with the axial height from the bottom to the top thereof.
Furthermore, the spiral shape is formed by cutting off a conical surface on the basis of a positive spiral surface and then keeping a part inside the conical surface, and the half cone angle of the conical surface is 20-45 degrees. The technical effect of facilitating the axial up-and-down rolling of cells and culture solution is achieved, and meanwhile, the space of radial vortex formed by the blades and the side wall of the tank body is reduced, so that the 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 an inner recess 2.7 in the centre thereof for fixing the culture tank.
And a stirring driver 8 is arranged in the culture system and used for driving the stirrer. The stirring drive is connected to the central control unit 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 the magnets driving the stirrer in rotation. In this case, the stirring driver 8 is a magnetic driver that generates a magnetic force action on the magnet to drive the stirrer to rotate. Adopt the magnetic stirring principle to drive the agitator with non-contact's form, compare in the mode that adopts the pivot of motor direct drive agitator, can guarantee the cleanness of the inside cell culture environment of culture tank and maintain convenient. The reason for this is that, the rotating shaft of the stirrer is directly driven by the motor, and the motor is usually required to be disposed outside the culture tank, so the rotating shaft of the stirrer must extend out of the culture tank, and therefore, a tight seal is required to be ensured between the rotating shaft of the stirrer and the culture tank in the culture environment for a long time, which increases the complexity of the system and makes it difficult to maintain, and the abrasion of the sealing member itself may cause pollution to the culture system.
The blade of agitator adopts helicoid and specific axial dimension, radial dimension's design, reaches axial stirring cell and culture solution in the jar body in cultivateing the system, and the radial shearing force of minimize to protect the cell, improve the effect of cell productivity.
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, and an air inlet part 2.63 and an air outlet part 2.62 which are arranged on the lid body and used for conveying and discharging air to the inner space of the tank body, wherein the length of the air inlet part 2.63 in the direction vertical to the bottom surface of the lid body is larger than that of the air outlet part 2.62; when cell culture, the inlet unit is used for conveying gas to the cell culture tank, and when cell culture is carried out, the inlet unit adopts a non-contact conveying mode to convey gas, namely the structure of the inlet unit is not in contact with the liquid level of a culture solution, so that the arrangement has the advantages that the inlet pipe extends into the liquid level to generate bubbles in a culture system to damage T cells, the non-contact conveying can prevent the bubbles from being generated, 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 beneficial effect is that compared with the arrangement that the length of the air inlet part is equal to or shorter than that of the air outlet part, the oxygen and carbon dioxide concentration in the culture system can be adjusted more quickly. The gas components of the inlet gas are adjusted along with different stages of the culture, the proportion of the carbon dioxide in the inlet gas is properly increased in the initial stage, and a certain amount of carbon dioxide is generated by the respiration of cells along with the culture, so that the proportion of the carbon dioxide in the inlet gas can be reduced.
In one embodiment, an ultraviolet sterilizing lamp is provided in the incubator 4 for sterilizing the incubator.
In one embodiment, the culture system is provided with an alarm module and is connected to the central controller 6.
Further, the culture tank is made of a non-air-permeable material.
The culture tank and/or its accessories are in gas or liquid exchange with the outside only through various passages.
The incubator also comprises an incubator cover which separates the incubator from the external environment, so that a relatively independent environment is formed in the incubator.
The totally enclosed structure means that in the whole process from activation, infection, amplification to finished product recovery of cell culture, the whole culture environment (including 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 from the outside environment, so that the environment in the incubator is in an adjustable range.
The central controller can be a single chip microcomputer which can be an 8-bit minimum system. The central controller may also be selected from different brands and models, or a higher number of controllers or processors. The central controller may be used to install the associated control programs. After installing the relevant control program, the central controller can receive the signals of the liquid flow assembly, the air flow assembly and the temperature adjusting assembly and the instruction of a user, and adjust the parameters of parts in the assemblies according to the requirement so as to ensure that the system runs stably.
The utility model provides a totally closed cell culture system's hardware architecture. The central controller of the totally-enclosed cell culture system of the utility model can be provided with control programs with different settings according to the needs.
After installing the relevant control program, the central controller 6 can receive the signal of the gas concentration sensing module and control the opening and closing of the oxygen passage switch, the carbon dioxide passage switch and the mixed gas suction pump according to the requirement, thereby controlling the opening and closing of the oxygen passage, the carbon dioxide passage and the mixed gas suction passage and controlling the air inflow; the device can receive signals of the weighing sensor, and controls the opening and closing of the liquid inlet pump, the waste liquid pump, the circulating pump and the recovery pump according to requirements, so as to control the opening and closing of the liquid inlet passage, the waste liquid passage, the circulating passage and the recovery passage and control the flowing condition of liquid; the signal of the temperature sensor can be received, the heating device is adjusted according to the requirement, and the temperature in the incubator is controlled; the opening and closing of the stirring driver can be controlled.
In one embodiment, the central controller 6 may be programmed to:
a gas concentration comparison unit for comparing the real-time oxygen gas concentration value sent by the gas concentration sensing module
And real-time carbon dioxide gas concentration value
With a predetermined oxygen gas concentration value
And a preset carbon dioxide concentration value
Respectively comparing to obtain 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
Adjusting the on-off time of the oxygen passage;
according to
Adjusting the on-off time of the carbon dioxide passage;
when in useAnd
when the gas mixture is in the range of the set threshold value, the mixed gas suction passage is opened to suck the gas in the incubator into the culture tank.
When in use
And
and closing the mixed gas suction passage when at least one of the gas mixture suction passages does not satisfy the set threshold range.
Oxygen gas concentration valueCarbon dioxide concentration value
And the threshold range can be set according to the requirements of the cells to be cultured. In a preferred manner, in the case of the preferred mode,
and
the threshold range may be selected from-0.1% to 0.1%.
When in use
And
when the gas volume in the culture tank and the flow rate of the mixed gas suction pump are determined together, the mixed gas suction passage is opened by timing control, the gas in the culture tank is sucked into the culture tank, and the timing time is determined according to the gas volume in the culture tank and the flow rate of the mixed gas suction pump. The timing control is thatAnd
when the predetermined threshold range is satisfied, the mixed gas suction passage is not opened immediately, but is controlled to be opened and closed according to the time set by the system.
Can be based on
The on-off time of the carbon dioxide access is adjusted by adopting a graded regulation and control mode. For example according to
The value of (a) is graded,
the smaller, the twoThe shorter the carbon dioxide passage is open between the secondary detection compartments.
Can be based on
The on-off time of the oxygen passage switch is adjusted by adopting a graded regulation and control mode. For example according to
The value of (a) is graded,the smaller the oxygen passage duration between two detections.
The carbon dioxide passage or the oxygen passage has constant gas flow rate during ventilation, and the ventilation quantity can be adjusted by controlling the on-off time of the passage switch. The longer the opening time, the greater the ventilation. The method is simple and accurate in ventilation control and controllable in accessory cost.
Furthermore, in order to ensure that the gas is uniformly mixed, the gas concentration value can be measured by the gas concentration sensing module after the oxygen or carbon dioxide passage is cut off and the gas is sufficiently mixed. Namely, after the passage is disconnected and mixed for a period of time after the gas is injected, the gas concentration value is measured by the gas concentration sensing module. Generally, the shorter the length of aeration, the shorter the required off-mix time. The gas is oxygen or carbon dioxide. The passage means an oxygen passage or a carbon dioxide passage.
Taking the culture box with the size of 373mm × 330mm × 250mm as an example:
in one embodiment of the present invention, the substrate is,
the value of (a) is divided into seven levels,
when the ventilation state is kept, the gas flow rate is constant; when in use
When the concentration is more than or equal to 2%, controlling a carbon dioxide access switch to be turned on for 1.5 seconds and then turned off, then waiting for 12 seconds to uniformly mix the gas, reading the concentration value of a carbon dioxide gas concentration sensor, and continuously comparing the concentration value with a set value; when in useWhen the carbon dioxide is detected to be in the open state, the carbon dioxide access switch is controlled to be closed after being opened for 1 second, then the concentration value of the carbon dioxide gas concentration sensor is read after waiting for 9 seconds, and the concentration value is continuously compared with a set value; when in use
When the carbon dioxide is detected to be in the open state, the carbon dioxide access switch is controlled to be closed after being opened for 0.8 second, then the concentration value of the carbon dioxide gas concentration sensor is read after waiting for 3 seconds, and the concentration value is continuously compared with a set value; when in use
When the carbon dioxide is detected to be in the open state, the carbon dioxide access switch is controlled to be closed after being opened for 0.6 second, the concentration value of the carbon dioxide gas concentration sensor is directly read, and the comparison with the set value is continued; when in use
When the carbon dioxide is detected to be in the open state, the carbon dioxide access switch is controlled to be closed after being opened for 0.5 second, the concentration value of the carbon dioxide gas concentration sensor is directly read, and the comparison with the set value is continued; when in use
When the carbon dioxide is detected to be in the open state, the carbon dioxide access switch is controlled to be closed after being opened for 0.3 second, the concentration value of the carbon dioxide gas concentration sensor is directly read, and the comparison with the set value is continued; when in useAt this time, the carbon dioxide passage switch is kept in the off state.
A program may be provided in the central controller 6 to convert the volume of the liquid into a weight according to the density of the liquid, and then control the amount of the liquid to be introduced and the amount of the waste liquid to be discharged according to the load cell. When no liquid is added at the beginning of the culture, peeling off the culture tank, wherein the weight of the culture tank is the weight of the content in the culture tank during the culture process.
The following programs may be provided in the central controller 6:
a weight comparison unit for measuring the weight M of the culture tank in real time sent by the weighing sensor during liquid feedingtAnd the user-instructed weight M of the culture tankin0Comparing to obtain weight difference M according to formula (III)in：
Min＝Min0-Mt(III)
A liquid inlet passage on-off control unit for controlling the liquid inlet passage according to MinAnd controlling the on-off of the liquid inlet passage.
Further, said is according to MinControlling the on-off of the liquid inlet passage as follows:
Mingreater than 0, opening the liquid inlet passage, MinAnd when the liquid inlet passage is equal to or less than 0, the liquid inlet passage is cut off.
The central controller also comprises a waste liquid channel on-off control unitAnd (5) Yuan. During liquid drainage, the weight comparison unit measures the weight M of the culture tank sent by the weighing sensor in real timetAnd the user-instructed weight M of the culture tankout0Comparing to obtain the weight difference M according to the formula (IV)out：
Mout＝Mt-Mout0(Ⅳ)
Waste liquid passage on-off control unit according to MoutAnd controlling the on-off of the waste liquid channel.
MoutGreater than 0, opening the waste passage, MoutAnd when the liquid inlet passage is equal to or less than 0, the liquid inlet passage is cut off.
The central controller 6 may be programmed to perform the following actions:
preset temperature value T0The temperature value may be a temperature value suitable for cell culture; the temperature sensor provides the real-time temperature value T in the incubator for the central controllertAnd T0Comparison when T istLess than T0When the heating device is started, the central controller controls the heating device to start; when T istGreater than or equal to T0And when the temperature is higher than the set temperature, the central controller controls the heating device to stop running.
The central controller 6 may be programmed to perform the following actions:
alarm critical values of the liquid flow assembly, the air flow assembly and the temperature adjusting assembly can be preset, and when the central controller receives that the detection information of the liquid flow assembly, the air flow assembly and the temperature adjusting assembly exceeds the limit, the central controller controls the alarm module to give an alarm.
The utility model discloses also can adopt the mode among the prior art to set up central controller 6, make central controller 6 can realize controlling airflow component, liquid stream subassembly, the function of the subassembly and the stirring driver that adjust the temperature can.
The utility model provides an adopt totally closed cell culture system's application method does:
1) liquid feeding: setting liquid inlet quantity, and injecting a culture medium into the 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, respectively comparing the gas concentration values with the set values, and injecting the gas in the incubator into the incubator when the oxygen and the carbon dioxide in the incubator reach the standard; setting the temperature of the incubator, and preheating the incubator;
3) and (3) continuous culture: injecting cells, injecting factors, starting a stirrer, injecting a culture medium into the culture tank during continuous culture, filtering metabolites and discharging waste liquid;
4) replacement and concentration: after the cell culture is finished, replacing the culture medium with normal saline, concentrating after the replacement is finished, continuously discharging waste liquid, and reducing the liquid volume in the culture tank;
5) and (3) finished product recovery: the agitator was stopped and the finished cells in the culture tank were recovered.
To sum up, the system realize the constant temperature culture environment, adopt the perfusion mode to carry out cell culture, realize from cell activation, infection, expand to the totally closed integration process of finished product recovery. The system separately admit air, each gaseous component content is stable in the assurance culture process, can ensure that the gaseous change of external world can not directly cause the influence to cell culture in the cell culture process, guarantee that gaseous each component content is unchangeable, reduce manual operation simultaneously, reduce cost reduces the risk of cultivateing in-process misoperation, improvement cultivation efficiency. Therefore, the utility model effectively overcomes various defects in the prior art and has high industrial utilization value.
The above embodiments are merely illustrative of the principles and effects of the present invention, and are not to be construed as limiting the invention. Modifications and variations can be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which may be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A totally enclosed cell culture system comprising at least the following:
an incubator (1);
the culture tank (2) is arranged in the incubator; the culture tank (2) is provided with a stirrer (2.5), and the stirrer is arranged in the culture tank;
an airflow assembly (3) in communication with the incubator (1);
a liquid flow assembly (4) in communication with the culture tank (2);
the temperature adjusting component (5) is arranged in the incubator;
and the central controller (6) is connected with the air flow assembly, the liquid flow assembly and the temperature regulating assembly.
2. The fully enclosed cell culture system of claim 1, wherein the airflow assembly comprises:
a separate air passage (3.1), carbon dioxide passage (3.2) and oxygen passage (3.3) communicating with the incubator (1), respectively;
a mixed gas suction passage (3.4) for communicating the incubator (1) and the culture tank (2);
an exhaust gas discharge passage (3.5) communicating with the culture tank (2);
the gas concentration sensing module (3.6) comprises an oxygen gas concentration sensor (3.6.1) and a carbon dioxide gas concentration sensor (3.6.2); is connected with the central controller (6).
3. The fully enclosed cell culture system of claim 2, wherein the airflow 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 pressure reducing valve (3.2.2) is arranged on the carbon dioxide pipeline (3.2.4), 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 connected with 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 pressure reducing valve (3.3.2) is arranged on the oxygen pipeline (3.3.4), 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 connected with 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 connected with the mixed gas suction pump (3.4.1), and the mixed gas suction pump (3.4.1) is connected with the central controller (6).
4. The fully closed cell culture system according to claim 2, further comprising one or more of the following features:
1) a gas discharge passage (3.7) is arranged in the incubator;
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) on the incubator, and 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) are arranged at the lower part in the incubator.
5. A totally enclosed cell culture system as claimed in claim 1, wherein a fan (3.8) is provided in the incubator.
6. A totally enclosed cell culture system as claimed in claim 1, wherein 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 waste liquid passage (4.3) is communicated with the circulating passage (4.2), and the recovery passage (4.4) is communicated with the culture tank (2); the liquid inlet passage (4.1), the circulating passage (4.2), the waste liquid passage (4.3) and the recycling passage (4.4) are respectively connected with the central controller (6).
7. The fully closed cell culture system according to claim 6, 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 connected with 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 connected with the central controller (6);
2) the circulation passage (4.2) comprises a circulation pipeline (4.2.1), a circulation pump (4.2.2) and a filter (4.2.3), the circulation pipeline (4.2.1) is communicated with the culture tank (2), the filter (4.2.3) is arranged on the circulation pipeline (4.2.1) and is communicated with the circulation pipeline (4.2.1), the circulation pipeline (4.2.1) is connected with the circulation pump (4.2.2), and the circulation pump (4.2.2) is connected with the central controller (6);
3) the waste liquid passage (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 (4.3.1) is communicated with the circulating passage (4.2), the waste liquid barrel (4.3.2) is connected with the waste liquid pipeline (4.3.1), the waste liquid pipeline (4.3.1) is connected with the waste liquid pump (4.3.1), and the waste liquid pump (4.3.1) is connected with the central controller (6);
4) the recycling passage (4.4) comprises a recycling pipeline (4.4.1), a recycling pump (4.4.2) and a recycling bag (4.4.3), the recycling pipeline (4.4.1) is communicated with the culture tank (2), the recycling pipeline (4.4.1) is connected with the recycling pump (4.4.2), the recycling bag (4.4.3) is communicated with the recycling pipeline (4.4.1), and the recycling pump (4.4.2) is connected with the central controller (6).
8. A totally enclosed cell culture system according to claim 1, wherein the liquid flow module (4) comprises a load cell (4.5), the load cell (4.5) being located at the bottom of the culture tank and being connected to the central controller (6).
9. A totally enclosed cell culture system according to claim 1, wherein the tempering 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).
10. The fully closed cell culture system according to claim 1, wherein the agitator comprises: a middle shaft; and at least two blades which are rotationally symmetrical about the axis of the central shaft and are connected with the central shaft, each blade comprises a blade body, the blade bodies extend spirally, the axial length of each blade body accounts for 20-35% of the length of the central shaft, the rotation angle of each blade body is 15-50 degrees, the maximum radial length is 20-54 mm, and the radial length from the bottom to the top is gradually reduced.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201920312979.4U CN209989411U (en) | 2019-03-12 | 2019-03-12 | Totally-enclosed cell culture system |
| US17/216,699 US20220348851A1 (en) | 2019-03-12 | 2019-07-31 | Fully enclosed cell culture system |
| PCT/CN2019/098474 WO2020181709A1 (en) | 2019-03-12 | 2019-07-31 | Fully enclosed cell incubation system |
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| CN201920312979.4U CN209989411U (en) | 2019-03-12 | 2019-03-12 | Totally-enclosed cell culture system |
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| CN209989411U true CN209989411U (en) | 2020-01-24 |
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