CN209778895U - totally-enclosed cell culture box - Google Patents
totally-enclosed cell culture box Download PDFInfo
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- CN209778895U CN209778895U CN201920312927.7U CN201920312927U CN209778895U CN 209778895 U CN209778895 U CN 209778895U CN 201920312927 U CN201920312927 U CN 201920312927U CN 209778895 U CN209778895 U CN 209778895U
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Abstract
The utility model provides a totally closed cell culture case includes following part at least: a box body; the culture tank is arranged in the box body; the culture tank comprises a stirrer, a tank body and a cover body, and the stirrer is arranged in the tank body; the box internal environment adjusting component is used for adjusting the gas environment and the temperature environment in the box body; the tank interior environment adjusting component is used for adjusting the liquid environment and the gas environment in the culture tank; a central controller. The system realize constant temperature culture environment. 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, improves cultivation efficiency.
Description
Technical Field
The utility model relates to a cell culture technical field especially relates to a totally closed cell culture case.
Background
in recent years, CAR-T cell immunotherapy has been considered as one of the most promising therapies to combat cancer. It has many incomparable advantages over other therapies, such as CAR-T cells can have multiple targeting sites, improve the accuracy of tumor treatment, and the course of action is not restricted 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 incubator.
To achieve the above and other related objects, a first aspect of the present invention provides a totally enclosed cell incubator, comprising at least:
a box body;
A culture tank; for cell culture. The culture tank is arranged in the box body; the culture tank comprises a cover body assembly, a tank body and a stirrer, the stirrer is arranged in the tank body, and the cover body is positioned at the upper part of the tank body and can be covered with the tank body;
an in-box environment adjusting component for adjusting the gas environment and the temperature environment in the box body;
the tank internal environment adjusting component is used for adjusting the liquid environment and the gas environment in the culture tank;
a central controller; and the tank internal environment adjusting component are connected.
As mentioned above, the utility model discloses a totally closed cell culture case has following beneficial effect:
The incubator provides a stable cell culture environment, adopts the perfusion mode to carry out cell culture, realizes the totally closed integration process of all steps of cell activation, infection, amplification to 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 incubator of the present invention;
FIG. 2 is a front structural view of the totally enclosed cell incubator of the present invention;
FIG. 3 is a back structure view of the totally enclosed cell incubator of the present invention;
FIG. 4 is a diagram showing the distribution of components on the surface of a totally enclosed cell incubator body;
FIG. 5 shows the internal structure of the culture tank of the totally enclosed cell culture chamber of the present invention.
FIG. 6 is a top view of the agitator of the culture tank of the totally enclosed cell culture chamber according to the present invention.
Description of the element reference numerals
1 case body
2 culturing pot
2.1 Agitator
2.1.1 medial axis
2.1.2 blade body
2.1.3 closed accommodation Chamber
2.2 tank body
2.2.1 inner recess
2.3 cover body
2.3.1 cover body
2.3.2 air intake
2.3.2.1 air inlet pipe
2.3.2.2 air inlet
2.3.3 exhaust section
2.3.3.2 air outlet
3 in-box environment regulating 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 Access switch
3.2.2 carbon dioxide line
3.2.3 carbon dioxide pathway tank inlet
3.3 oxygen pathway
3.3.1 oxygen passage switch
3.3.2 oxygen line
3.3.3 oxygen passage case Inlet
3.4 gas concentration sensing module
3.4.1 oxygen gas concentration sensor
3.4.2 carbon dioxide gas concentration sensor
3.5 heating device
3.6 temperature sensor
3.7 gas discharge passage
3.8 Fan
3.9 sterilizing lamp
4 in-tank environment adjusting assembly
4.1 Mixed gas suction passage
4.1.1 Mixed gas suction Pump
4.1.2 mixed gas suction line
4.1.3 Mixed gas passage Box Outlet
4.2 exhaust gas discharge passage
4.3 liquid inlet passage interface
4.4 Filter
4.5 recovery path interface
4.6 weighing sensor
4.7 first circulation port
4.8 second circulation port
5 Central controller
6 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. 1-6. 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 housing, or may be located on the console outside the housing, as long as it can be connected to other components in the totally enclosed cell culture chamber, 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 incubator provided by the present invention at least comprises the following parts:
A box body 1;
The culture tank 2 is used for carrying out cell culture, and the culture tank 2 is arranged in the box body; the culture tank 2 comprises a stirrer 2.1, a tank body 2.2 and a cover body 2.3, wherein the stirrer 2.1 is arranged in the tank body 2.2, and the cover body 2.3 is positioned at the upper part of the tank body and can be covered with the tank body;
An in-box environment adjusting unit 3 for adjusting the gas environment and the temperature environment in the box body;
An in-tank environment adjusting unit 4 for adjusting the liquid environment and the gas environment in the culture tank;
A central controller 5; and the tank internal environment adjusting component are connected.
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.
further, the in-tank environment adjusting unit 3 includes:
The separated air passage 3.1, carbon dioxide passage 3.2 and oxygen passage 3.3 are respectively communicated with the box body 1. For delivering gas into the tank to form a mixed gas.
A gas concentration sensing module 3.4 including an oxygen gas concentration sensor 3.4.1 and a carbon dioxide gas concentration sensor 3.4.2; arranged in the box body and respectively connected with a central controller (5). Respectively used for measuring the real-time oxygen gas concentration value and the real-time carbon dioxide gas concentration value in the box body.
and the heating device 3.5 is arranged in the box body 1, and the heating device 3.5 is connected with the central controller 5. Used for heating the inner cavity of the box body.
And the temperature sensor 3.6 is arranged in the box body 1 and is connected with the central controller 5. And is used for measuring the real-time temperature value in the box body.
Heating device 3.5 and temperature sensor 3.6 can make the culture tank that is located the box be in the constant temperature environment, and guarantee that the gaseous temperature that gets into in the culture tank is invariable.
Further, the air passage 3.1 comprises an air line 3.1.2; the air line 3.1.2 communicates with the tank 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 introduced into the cabinet 1.
The carbon dioxide passage 3.2 comprises a carbon dioxide passage switch 3.2.1 and a carbon dioxide pipeline 3.2.2; the carbon dioxide pipeline 3.2.1 is communicated with the box body 1; and a carbon dioxide passage switch 3.2.1 is arranged on the carbon dioxide pipeline 3.2.2, and the carbon dioxide passage switch 3.2.1 is connected with the central controller 5.
In one embodiment, the carbon dioxide passage switch may be a solenoid valve.
The oxygen passage 3.3 comprises an oxygen passage switch 3.3.1 and an oxygen pipeline 3.3.2; the oxygen pipeline 3.3.1 is communicated with the box body 1; an oxygen passage switch 3.3.1 is arranged on the oxygen pipeline 3.3.2, and the oxygen passage switch 3.3.1 is connected with the central controller 5.
In one embodiment, the oxygen passage switch may be a solenoid valve.
the carbon dioxide passage 3.2 and the oxygen passage 3.3 are provided with a carbon dioxide passage box inlet 3.2.3 and an oxygen passage box inlet 3.3.3 on the box body, and the carbon dioxide passage box inlet 3.2.3 and the oxygen passage box inlet 3.3.3 are both arranged at the upper part in the box body. 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.
The heating device 3.5 may be a heating plate. Attached to the inner wall of the box body.
the heating plate and the temperature sensor are both commercially available products.
In one embodiment, the housing is provided with a gas vent passage 3.7 for venting the gas in the housing to maintain a stable gas pressure in the housing.
In one embodiment, the gas discharge passage 3.7 and the air passage 3.1 are the same passage.
The air passage 3.1 and/or the gas discharge passage 3.7 is provided with a vent 3.1.3 on the tank, the vent 3.1.3 being remote from the carbon dioxide passage tank inlet 3.2.5, the oxygen passage tank inlet 3.3.5, the mixed gas passage tank outlet 3.4.3, the oxygen gas concentration sensor 3.6.1 and the carbon dioxide gas concentration sensor 3.6.2.
In one embodiment, a fan 3.8 is provided in the box. The gas mixing device is used for stirring gas flow, quickening mixing, enabling gas to be mixed more uniformly and quickening heat exchange inside the box body.
In one embodiment, the fan 3.8 is provided in the upper part of the housing.
In one embodiment, a sterilization lamp 3.9 is provided in the housing 1 for sterilizing the housing.
The in-tank environment conditioning assembly 4 includes:
A mixed gas suction passage 4.1 communicating the tank 1 and the culture tank 2, and connected to the central controller 5. For feeding the mixed gas in the tank 1 into the culture tank 2.
a gas concentration sensing module 3.4 including an oxygen gas concentration sensor 3.4.1 and a carbon dioxide gas concentration sensor 3.4.2; arranged in the box body and respectively connected with the central controller 5. Respectively used for measuring the concentration value of oxygen gas and the concentration value of carbon dioxide gas in the box body.
an exhaust gas discharge passage 4.2 communicating with the culture tank 2. Used for discharging the waste gas generated in the cell culture process to the outside of the box body.
and the liquid inlet passage interface 4.3 is used for communicating the culture tank 2 with the liquid inlet passage. The liquid inlet passage is used for feeding liquid into the culture tank 2.
A filter 4.4 for communicating with the culture tank 2. The filter is provided with a receiving port for receiving the culture materials in the culture tank, a waste liquid port for connecting the waste liquid pipe and a feedback port for returning the filtered culture materials to the culture tank. Filtering the metabolite produced by the cells and removing waste liquid.
In one embodiment, the filter 4.4 may be a hollow fiber column.
A recovery passage port 4.5 provided in the culture tank 2. Is used for communicating the culture tank 2 with a recovery passage and is used for recovering finished cells.
The mixed gas suction passage 4.1 comprises a mixed gas suction pump 4.1.1 and a mixed gas suction pipeline 4.1.2; the mixed gas suction pipeline 4.1.2 is connected with a mixed gas suction pump 4.1.1, and the mixed gas suction pump 4.1.1 is connected with the central controller 5. The mixed gas suction line 4.1.2 is used for injecting the mixed gas from the tank 1 into the culture tank 2.
in one embodiment, a gas filter is provided on the mixed gas intake line 4.1.2. For filtering the gas entering the culture tank.
Further, the exhaust gas discharge passage 4.2 comprises 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 tank body without communicating with the tank body 1.
In one embodiment, the mixed gas passageway has a mixed gas passageway housing outlet 4.1.3 in the housing, and the mixed gas passageway housing outlet 4.1.3, oxygen gas concentration sensor 3.4.1 and carbon dioxide gas concentration sensor 3.4.2 are provided in a lower portion of the housing. 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.
Further, as shown in fig. 2 and 6, the side wall of the culture tank 2 is provided with four ports:
And the liquid inlet passage interface 4.3 is used for communicating the culture tank 2 with the liquid inlet passage. The liquid inlet passage is used for feeding liquid into the culture tank 2;
A recovery passage interface 4.5 for communicating the culture tank 2 with the recovery passage. For recovery of the product cells;
A first circulation port 4.7 and a second circulation port 4.8 for connecting the filter 4.4 to the culture tank 2 and filtering the metabolic products produced by the cells.
As shown in FIGS. 2 and 6, 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 4.7 into the filter 4.4, the filter 4.4 has a semi-permeable membrane with a pore size of 0.2-1 μm, and can permeate water and the components of the metabolic waste generated by the cell culture in the culture medium, but cannot permeate the cells, 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 are introduced into the culture tank 2 again from the second circulation port 4.8.
the first circulation port 4.7 and the collection passage port 4.5 are provided at the bottom of the side wall of the culture tank to sufficiently suck out the cells and the liquid in the culture tank 2.
In one embodiment, the opening and closing of the mixed gas suction passage is regulated by regulating the opening and closing of the mixed gas suction pump.
In one embodiment, the in-tank environmental conditioning assembly 4 includes a load cell 4.6, the load cell 4.6 being used to determine the real-time tank weight, being located below the tank and connected to the central controller 5.
as shown in fig. 5 and 6, the stirrer 2.1 comprises: the middle shaft is 2.1.1; and at least two blades which are rotationally symmetrical around the axis of the central shaft and are connected with the central shaft, each blade comprises a blade body 2.1.2, the blade bodies 2.1.2 extend spirally, the axial length of the blade bodies 2.1.2 accounts for 20 to 35 percent of the length of the central shaft, the rotating angle of the blade bodies 2.1.2 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. With the blade having the above shape, it is possible to reduce the shearing force to the cultured cells while uniformly stirring the cultured cells and the culture system.
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 the cells and the culture solution can be 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, the cultured cells are protected, and the yield of the cultured cells is improved.
The bottom surface of the culture tank comprises an inner concave part 2.2.1 positioned at the center of the bottom surface and used for fixing the culture tank.
And a stirring driver 6 is arranged in the incubator and used for driving the stirrer. The stirring driver is connected with the central controller 5.
in one embodiment, the blade further comprises a closed housing 2.1.3 at the maximum radial length of the blade body for housing the magnets driving the stirrer in rotation. In this case, the stirring driver 6 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.
further, as shown in fig. 5, the lid body 2.3 of the cell culture tank 2 includes a lid main body 2.3.1, and an air inlet portion 2.3.2 and an air outlet portion 2.3.3 provided in the lid main body for supplying and discharging air to and from the internal space of the tank, wherein the length of the air inlet portion 2.3.2 in the direction perpendicular to the bottom surface of the lid main body is larger than that of the air outlet portion 2.3.3; when the cell culture is carried out, the gas inlet part is used for conveying gas into the cell culture tank, and when the cell culture is carried out, the gas inlet part conveys the gas in a non-contact conveying mode, namely the structure of the gas inlet part is not in contact with the liquid level of a culture solution, so that the gas inlet part and the gas inlet pipe in the prior art stretch into the liquid level, bubbles are generated in a culture system to damage cells in culture, the non-contact conveying can prevent the bubbles from being generated, and the yield of the cultured 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.
Further, the air inlet portion includes an air inlet pipe 2.3.2.1, the air outlet portion includes an air outlet pipe 2.3.3.1, and the length of the air inlet pipe 2.3.2.1 in the direction perpendicular to the bottom surface of the cover body is greater than that of the air outlet pipe 2.3.3.1.
In one embodiment, the inlet duct 2.3.2.1 and the outlet duct 2.3.3.1 each extend perpendicularly from the bottom surface of the lid body;
In an embodiment, the air inlet portion 2.3.2 and the air outlet portion 2.3.3 further include an air inlet 2.3.2.2 and an air outlet 2.3.3.2 protruding from the cover body, respectively, for introducing an external air source into the air inlet pipe and guiding out air in the air outlet pipe.
In one embodiment, the incubator is provided with an alarm assembly and is connected to the central controller 5.
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 totally-enclosed cell culture box provided by the utility model can be used for totally-enclosed cell culture.
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 in-tank environment adjusting assembly and the environment adjusting assembly in the incubator and the instruction of a user, and adjust the parameters of parts in the assemblies as required so as to ensure that each assembly of the incubator runs stably.
the utility model provides a totally closed cell culture case's hardware architecture. The central controller of the totally-enclosed cell culture box of the utility model can be provided with control programs with different settings according to the needs.
After the relevant control programs are installed, the central controller can receive signals 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 requirements so as to control the opening and closing of the oxygen passage, the carbon dioxide passage and the mixed gas suction passage and control the air inflow; the weighing sensor can receive signals of the weighing sensor, and when the weighing sensor is connected with an external liquid inlet passage, a waste liquid passage, a circulation passage and a recovery passage, a control program can be set to respectively control the external liquid inlet passage, the waste liquid passage, the circulation passage and the recovery passage so as to control the liquid flowing condition; the signal of the temperature sensor can be received, the heating device can be adjusted according to the requirement, and the temperature in the box body can be controlled; the opening and closing of the stirring driver can be controlled.
In one embodiment, the central controller may be programmed to:
A gas concentration comparison unit for comparing the real-time oxygen gas concentration value sent by the gas concentration sensing moduleAnd real-time carbon dioxide gas concentration valueWith a predetermined oxygen gas concentration valueAnd a preset carbon dioxide concentration valueRespectively comparing to obtain the difference of the required concentration according to the formulas (I) and (II), namely the concentration differenceAnd CCO 2:
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 toadjusting the on-off time of the oxygen passage;
According toAdjusting the on-off time of the carbon dioxide passage;
When in useAndWhen the gas in the tank body is in the preset threshold range, the mixed gas suction passage is opened, and the gas in the tank body is sucked into the culture tank.
when in useAndAnd 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 valueConcentration of carbon dioxideValue ofAnd 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,AndThe threshold range may be selected from-0.1% to 0.1%.
when in useAndWhen 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 tank body 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 thatAndwhen 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 onthe on-off time of the carbon dioxide access is adjusted by adopting a graded regulation and control mode. For example according toThe value of (a) is graded,The smaller, the twoThe shorter the carbon dioxide passage is open between the secondary detection compartments.
Can be based onthe on-off time of the oxygen passage switch is adjusted by adopting a graded regulation and control mode. For example according tothe value of (a) is graded,The smaller the oxygen passage duration between two detections.
Number of stages of classification andThe grading level can be flexibly designed. The number of hierarchical levels may generally be 1-10 levels. For example, the stages may be classified into 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
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 useWhen 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 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 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 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 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 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 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 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 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.
The values of (a) are also divided into seven levels, When the ventilation state is kept, the gas flow rate is constant; when in usewhen the concentration value is more than or equal to 2%, controlling an oxygen passage switch to be opened for 1.5 seconds and then closed, waiting for 12 seconds, reading the concentration value of the oxygen gas concentration sensor, and continuously comparing the concentration value with a set value; when in useWhen the oxygen concentration sensor is used, the oxygen passage switch is controlled to be opened for 1 second and then closed, then the concentration value of the oxygen gas concentration sensor is read after the oxygen passage switch is waited for 9 seconds, and the concentration value is continuously compared with a set value; when in use When the oxygen concentration sensor is used, the oxygen passage switch is controlled to be opened for 0.8 second and then closed, then the concentration value of the oxygen gas concentration sensor is read after waiting for 3 seconds, and the comparison with the set value is continued; when in usethen the oxygen passage switch is controlled to be opened for 0.6 secondThen closing, directly reading the concentration value of the oxygen gas concentration sensor, and continuously comparing the concentration value with a set value; when in usewhen the oxygen concentration sensor is used, the oxygen passage switch is controlled to be opened for 0.5 second and then closed, the concentration value of the oxygen gas concentration sensor is directly read, and the comparison with the set value is continued; when in useWhen the oxygen concentration sensor is used, the oxygen passage switch is controlled to be opened for 0.3 second and then closed, the concentration value of the oxygen gas concentration sensor is directly read, and the comparison with the set value is continued; when in useAt this time, the oxygen passage switch is kept in the off state.
the central controller may be programmed to convert the volume of the liquid into weight according to the density of the liquid, and then control the amount of the liquid to be fed and the amount of the waste liquid to be discharged according to the weighing sensor. 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.
Alarm critical values of the in-tank environment adjusting assembly and the in-tank environment adjusting assembly are preset, and when the central controller receives detection information exceeding the limit of the in-tank environment adjusting assembly and the in-tank environment adjusting assembly, the alarm module is controlled to give an alarm.
The method for totally-enclosed cell culture by utilizing the totally-enclosed cell culture box provided by the utility model adopts the following steps:
1) The culture tank is respectively connected with the liquid inlet passage and the recovery passage;
2) Liquid inlet process: setting liquid inlet quantity, and injecting a culture medium into the culture tank;
3) 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;
4) 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;
5) 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;
6) And (3) finished product recovery: the agitator was stopped and the finished cells in the culture tank were recovered.
To sum up, the incubator 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 incubator 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 (12)
1. A totally-enclosed cell culture box is characterized by at least comprising the following parts:
A box body (1);
The culture tank (2) is arranged in the box body; the culture tank (2) comprises a stirrer (2.1), a tank body (2.2) and a cover body (2.3), wherein the stirrer (2.1) is arranged in the tank body (2.2), and the cover body (2.3) is positioned at the upper part of the tank body and can be covered with the tank body;
An in-box environment adjusting unit (3) for adjusting the gas environment and the temperature environment in the box body;
an in-tank environment adjusting unit (4) for adjusting the liquid environment and the gas environment in the culture tank;
And the central controller (5) is connected with the in-tank environment regulating assembly and the in-tank environment regulating assembly.
2. A totally enclosed cell culture chamber as claimed in claim 1, wherein the in-chamber environmental conditioning assembly (3) comprises:
A separated air passage (3.1), a carbon dioxide passage (3.2) and an oxygen passage (3.3) which are respectively communicated with the box body (1);
A gas concentration sensing module (3.4) comprising an oxygen gas concentration sensor (3.4.1) and a carbon dioxide gas concentration sensor (3.4.2); arranged in the box body and respectively connected with a central controller (5);
The heating device (3.5), the heating device (3.5) is arranged in the box body (1); the heating device (3.5) is connected with the central controller (5);
the temperature sensor (3.6), temperature sensor (3.6) locate in box (1) with central controller (5) are connected.
3. The hermetically sealed cell incubator of claim 2, wherein the in-chamber environmental conditioning assembly further comprises one or more of the following features:
1) the carbon dioxide passage (3.2) comprises a carbon dioxide passage switch (3.2.1) and a carbon dioxide pipeline (3.2.2); the carbon dioxide pipeline (3.2.1) is communicated with the box body (1); a carbon dioxide passage switch (3.2.1) is arranged on the carbon dioxide pipeline (3.2.2), and the carbon dioxide passage switch (3.2.1) is connected with the central controller (5);
2) The oxygen passage (3.3) comprises an oxygen passage switch (3.3.1) and an oxygen pipeline (3.3.2); the oxygen pipeline (3.3.1) is communicated with the box body (1); an oxygen passage switch (3.3.1) is arranged on the oxygen pipeline (3.3.2), and the oxygen passage switch (3.3.1) is connected with the central controller (5);
3) a gas discharge passage (3.7) is arranged in the box body;
4) the carbon dioxide passage (3.2) and the oxygen passage (3.3) are provided with a carbon dioxide passage box inlet (3.2.3) and an oxygen passage box inlet (3.3.3) on the box body, and the carbon dioxide passage box inlet (3.2.3) and the oxygen passage box inlet (3.3.3) are arranged at the upper part in the box body;
5) the heating device (3.5) is a heating plate and is attached to the inner wall of the box body;
6) a fan (3.8) is arranged in the incubator (1);
7) A sterilizing lamp (3.9) is arranged in the incubator.
4. The totally enclosed cell culture chamber according to claim 1, wherein the in-tank environmental conditioning assembly (4) comprises:
A mixed gas suction passage (4.1) which communicates the box body (1) and the culture tank (2) and is connected with the central controller (5);
An exhaust gas discharge passage (4.2) communicating with the culture tank (2);
a liquid inlet passage interface (4.3) arranged on the culture tank (2);
the filter (4.4) is arranged in the box body (1), and is provided with a receiving port for receiving culture substances in the culture tank, a waste liquid port for connecting a waste liquid pipe and a feedback port for returning the filtered culture substances to the culture tank;
A recovery passage interface (4.5) provided on the culture tank (2).
5. the totally enclosed cell incubator as claimed in claim 4, wherein the mixed gas suction path (4.1) comprises a mixed gas suction pump (4.1.1) and a mixed gas suction line (4.1.2); the mixed gas suction pipeline (4.1.2) is connected with a mixed gas suction pump (4.1.1), and the mixed gas suction pump (4.1.1) is connected with the central controller (5).
6. a totally enclosed cell culture chamber as claimed in claim 1, wherein the in-tank environmental conditioning assembly (4) comprises a load cell (4.6) located below the culture tank and connected to the central controller (5).
7. a totally enclosed cell culture chamber as claimed in claim 1, characterized in that the agitator (2.1) comprises: a medial axis (2.1.1); and at least two blades which are rotationally symmetrical around the axis of the central shaft and are connected with the central shaft, each blade comprises a blade body (2.12), the blade bodies (2.12) extend spirally, the axial length of each blade body (2.12) accounts for 20 to 35 percent of the length of the central shaft, the rotating angle of each blade body (2.12) 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.
8. A totally enclosed cell culture chamber as claimed in claim 7, wherein the blade further comprises a closed housing chamber (2.1.3) at the maximum radial length of the blade body for housing a magnet driving rotation of the agitator.
9. the totally enclosed cell culture chamber according to claim 7, wherein the bottom surface of the tank body comprises an inner recess (2.2.1) at the center thereof, and the blade is provided with a notch matching the inner recess, the axial distance between the notch and the inner recess being at least 2 mm.
10. The totally enclosed cell culture chamber according to claim 1, wherein the cover body comprises a cover body (2.3.1), an air inlet part (2.3.2) and an air outlet part (2.3.3), the air inlet part (2.3.2) and the air outlet part (2.3.3) are respectively arranged on the cover body (2.3.1), and the length of the air inlet part (2.3.2) in the direction perpendicular to the bottom surface of the cover body is larger than the length of the air outlet part (2.3.3) in the corresponding direction.
11. The hermetically sealed cell incubator as claimed in claim 10, wherein the air inlet part includes an air inlet pipe (2.3.2.1), and the air outlet part includes an air outlet pipe (2.3.3.1), and a length of the air inlet pipe (2.3.2.1) in a direction perpendicular to the bottom surface of the cover body is greater than a length of the air outlet pipe (2.3.3.1) in the corresponding direction.
12. the fully enclosed cell culture chamber according to claim 11, further comprising one or more of the following features:
1) The air inlet pipe (2.3.2.1) and the exhaust pipe (2.3.3.1) respectively extend vertically from the bottom surface of the cover body;
2) The air inlet part (2.3.2) and the air outlet part (2.3.3) respectively comprise a bulge arranged at the air inlet (2.3.2.2) and the air outlet (2.3.3.2) of the cover body.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110982697A (en) * | 2020-03-03 | 2020-04-10 | 广州中安基因科技有限公司 | Cell culture device for biotechnology research and development |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110982697A (en) * | 2020-03-03 | 2020-04-10 | 广州中安基因科技有限公司 | Cell culture device for biotechnology research and development |
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