CN111218403A

CN111218403A - Totally enclosed cell culture method

Info

Publication number: CN111218403A
Application number: CN202010168036.6A
Authority: CN
Inventors: 余学军; 徐鹏; 方勇军; 李力; 梁辉
Original assignee: Huadao Shanghai Biological Medicine Co ltd
Current assignee: Huadao Shanghai Biological Medicine Co ltd
Priority date: 2019-03-12
Filing date: 2020-03-11
Publication date: 2020-06-02
Anticipated expiration: 2040-03-11
Also published as: CN111218403B; CN109825436A; CN111334427A; CN111334427B

Abstract

The invention provides a totally-enclosed cell culture method by adopting a totally-enclosed cell culture system, which comprises the following steps: 1) a liquid inlet process; 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) continuously culturing; 4) replacement and concentration; 5) and (5) recovering a finished product. At present, no automatic equipment specially used for CAR-T cell culture is seen at home, and compared with artificial laboratory culture, the equipment culture can greatly save labor cost and obviously improve culture efficiency.

Description

Totally enclosed cell culture method

Technical Field

The invention relates to the technical field of cell culture, in particular to a totally-enclosed cell culture method.

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.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to provide a totally enclosed cell culture process.

In order to achieve the above objects and other related objects, the present invention provides a method for performing a totally enclosed cell culture using a totally enclosed cell culture system, comprising the steps of:

1) liquid inlet process: 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.

As described above, the totally enclosed cell culture method of the present invention has the following beneficial effects:

the method realizes a constant-temperature culture environment, adopts a perfusion mode to culture the cells, and realizes a totally-enclosed integrated process from cell activation, infection and amplification to finished product recovery. The system provided by the invention adopts a perfusion mode instead of a perfusion mode, can discharge waste liquid in the culture process, prevents accumulation of harmful metabolites, is beneficial to achieving higher cell culture density, can reduce subsequent treatment steps, can recover finished product cells without operations such as centrifugation and the like, simplifies the operation, can improve the culture efficiency, is easy to industrialize, and realizes a totally-enclosed integrated process from cell activation, infection and amplification to finished product recovery. The 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.

At present, no automatic equipment specially used for CAR-T cell culture exists in China, compared with manual laboratory culture, the equipment can greatly save labor cost, remarkably improve culture efficiency and realize industrial production, 15 equipment can be managed by 1 person at the same time, the per-capita productivity ratio is 15:1 compared with manual culture, and the production process meets the requirements of a drug traceability system and action plan requirements issued by the national drug supervision and management bureau in 2019 and about accelerating intelligent drug supervision. Compared with the similar foreign equipment (mainly 2 devices, namely a FlexFactory cell culture system of GE company in America, namely a German and American day-whirly CliniMACS Prodigy cell culture system, namely the GE and American day-whirly CliniMACS Prodigy systems, the device import cost is huge, the price of each set of device is more than 250 ten thousand yuan of RMB on average), the price of the device is one-fifteenth to one-twentieth of the foreign and similar equipment, and the cost advantage is obvious.

Drawings

FIG. 1 shows a signal transmission diagram of a totally enclosed cell culture system according to the invention;

FIG. 2 is a front 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 parts on the surface of the incubator of the totally enclosed cell culture system of the present invention;

FIG. 5 is a schematic diagram showing the communication between the culture tank and the liquid flow assembly 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 top view showing a structure of a stirrer of a culture tank of the totally enclosed cell culture system according to the present invention.

FIG. 8 shows the CAR-T cell subpopulation analysis.

FIG. 9 shows the results of quantitative measurements of CAR-T functional cell (infection efficiency) expression.

FIG. 10 shows the CAR-T cytokine assay results.

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 of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.

Please refer to fig. 1-10. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.

It should be understood that the central controller according to the present invention may be located at any position on the outer wall of the incubator, or on a console located outside the incubator, etc., as long as it is capable of connecting with other components of the totally enclosed cell culture system, and therefore, the position of the central controller is not shown in fig. 2-4.

The invention provides a method for totally-enclosed cell culture by adopting a totally-enclosed cell culture system, which comprises the following steps:

As shown in FIGS. 1 to 4, the totally enclosed cell culture system of the present invention comprises at least the following components:

an incubator 1 for providing a stable cultivation environment including a stable gas environment and a temperature environment.

A culture tank 2 for cell culture, which is provided in the incubator; the culture tank 2 is provided with a stirrer, and the stirrer is arranged in the culture tank;

a gas flow assembly 3, communicating with the incubator 1, for regulating the oxygen and carbon dioxide concentrations in the incubator 1;

the liquid flow component 4 is communicated with the culture tank 2 and is used for adjusting the liquid flow in the culture tank 2, filtering metabolites generated by cell culture and recovering finished cells;

the temperature adjusting component 5 is used for adjusting the temperature in the incubator;

and the central controller 6 is connected with and controls the air 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.

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; the concentration value of the real-time oxygen gas and the concentration value of the real-time carbon dioxide gas in the incubator are respectively measured, and the gas concentration sensing module provides detection information for the central controller 6.

Further, the air passage 3.1 comprises an air line 3.1.2; the air line 3.1.2 communicates with the incubator 1.

In one embodiment, the air 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 pipeline 3.2.4 is driven by a carbon dioxide pressure reducing valve, and the carbon dioxide pipeline 3.2.4 is communicated with the incubator 1; 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 controlled by the central controller 6.

In one embodiment, the carbon dioxide passage switch is selected from one or more of a carbon dioxide storage device switch, a carbon dioxide 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 pipeline 3.3.4 is driven by an oxygen pressure reducing valve 3.3.2, and the oxygen pipeline 3.3.4 is communicated with the incubator 1; an oxygen passage switch 3.3.3 is arranged on the oxygen passage, and the oxygen passage switch 3.3.3 is controlled by the central controller 6.

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 driven by a mixed gas suction pump 3.4.1, and the mixed gas suction line 3.4.2 is used for injecting mixed gas from the incubator 1 into the culture tank 2; the mixed gas suction pump 3.4.1 is controlled by a central controller 6.

In one embodiment, 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.

Further, the central controller 6 includes the following parts:

a gas concentration comparison unit for comparing the real-time oxygen gas concentration value C sent by the gas concentration sensing module_tO2And real-time carbon dioxide gas concentration value C_tCO2And a predetermined oxygen gas concentration value C_0O2And a preset carbon dioxide concentration value C_0CO2Respectively comparing the two solutions, and obtaining the difference value of the required concentration according to the formulas (I) and (II), namely the concentration difference C_O2And C_CO2：

C_O2＝C_0O2-C_tO2(I)

C_CO2＝C_0CO2-C_tCO2(II)

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 C_O2Adjusting the on-off time of the oxygen passage;

according to C_CO2Adjusting the on-off time of the carbon dioxide passage;

when C is present_O2And C_CO2When 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 C is present_O2And C_CO2And 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 value C_0O2Carbon dioxide concentration value C_0CO2And the threshold range can be set according to the requirements of the cells to be cultured. In a preferred mode, C_O2And C_CO2The threshold range may be selected from-0.1% to 0.1%.

In one embodiment, when C_O2And C_CO2When 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 means that when C_O2And C_CO2When 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.

Further, the opening and closing of the oxygen passage are controlled by controlling the opening and closing of the oxygen passage switch; the opening and closing of the carbon dioxide passage is controlled by controlling the opening and closing of the carbon dioxide passage switch, and the opening and closing of the mixed gas suction passage is controlled by controlling the opening and closing of the mixed gas suction pump.

In one embodiment, a programmed algorithm may be used to control the opening and closing of the oxygen access switch, the carbon dioxide access switch, and the mixed gas inhalation access. According to the current gas concentration measurement value, different on-off time of a gas passage switch is controlled by a program, so that the actual gas concentration value in the incubator is close to or equal to a set value.

In one embodiment, the opening and closing of the mixed gas suction passage is regulated by regulating the opening and closing of the mixed gas suction pump.

In one possible embodiment, according to C_CO2The on-off time of the carbon dioxide access is adjusted by adopting a graded regulation and control mode. For example according to C_CO2Value classification of C_CO2The smaller the length of time that the carbon dioxide passage between the two detection intervals is open.

In one possible embodiment, according to C_O2The on-off time of the oxygen passage switch is adjusted by adopting a graded regulation and control mode. For example according to C_O2Value classification of C_O2The smaller the oxygen passage duration between two detections.

C_CO2Number of classification stages and C_O2The 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, C_CO2Has a value of seven grades, C_CO2≥2％、1％≤C_CO2＜2％、0.8％≤C_CO2＜1％、0.5％≤C_CO2＜0.8％、0.3％≤C_CO2＜0.5％、0.1％＜C_CO2＜0.3％、C_CO2Less than or equal to 0.1 percent; when the ventilation state is kept, the gas flow rate is constant; when C is present_CO2When 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 the content is less than or equal to 1 percent, C_CO2If the concentration value is less than 2%, controlling a carbon dioxide access switch to be turned on for 1 second and then turned off, then waiting for 9 seconds, reading the concentration value of the carbon dioxide gas concentration sensor, and continuously comparing the concentration value with a set value; when the content of C is more than or equal to 0.8 percent_CO2If the concentration value is less than 1%, controlling a carbon dioxide access switch to be turned on for 0.8 second and then turned off, waiting for 3 seconds, reading the concentration value of the carbon dioxide gas concentration sensor, and continuously comparing the concentration value with a set value; when the content of C is more than or equal to 0.5 percent_CO2When the concentration value is less than 0.8%, controlling a carbon dioxide access switch to be turned on for 0.6 second and then turned off, directly reading the concentration value of the carbon dioxide gas concentration sensor, and continuously comparing the concentration value with a set value; when the content of C is more than or equal to 0.3 percent_CO2When the concentration value is less than 0.5%, controlling a carbon dioxide access switch to be turned on for 0.5 second and then turned off, directly reading the concentration value of the carbon dioxide gas concentration sensor, and continuously comparing the concentration value with a set value; when 0.1% < C_CO2When the concentration value is less than 0.3%, controlling a carbon dioxide access switch to be turned on for 0.3 second and then turned off, directly reading the concentration value of the carbon dioxide gas concentration sensor, and continuously comparing the concentration value with a set value; when C is present_CO2When the carbon dioxide passage is less than or equal to 0.1 percent, the carbon dioxide passage switch keeps a closed state.

C_O2The value of (A) is also classified into seven grades, C_O2≥2％、1％≤C_O2＜2％、0.8％≤C_O2＜1％、0.5％≤C_O2＜0.8％、0.3％≤C_O2＜0.5％、0.1％＜C_O2＜0.3％、C_O2Less than or equal to 0.1 percent; when the ventilation state is kept, the gas flow rate is constant; when C is present_O2When the oxygen content is more than or equal to 2 percent, the oxygen passage switch is controlled to be opened for 1.5 seconds and then closed, and then the time waits for 12 secondsThen, reading the concentration value of the oxygen gas concentration sensor, and continuously comparing the concentration value with a set value; when the content is less than or equal to 1 percent, C_O2If the concentration value is less than 2%, controlling an oxygen passage switch to be turned on for 1 second and then turned off, waiting for 9 seconds, reading the concentration value of the oxygen gas concentration sensor, and continuously comparing the concentration value with a set value; when the content of C is more than or equal to 0.8 percent_O2When the concentration value is less than 1%, controlling an oxygen passage switch to be turned on for 0.8 second and then turned off, waiting for 3 seconds, reading the concentration value of the oxygen gas concentration sensor, and continuously comparing the concentration value with a set value; when the content of C is more than or equal to 0.5 percent_O2When the concentration value is less than 0.8%, controlling an oxygen passage switch to be turned on for 0.6 second and then turned off, directly reading the concentration value of the oxygen gas concentration sensor, and continuously comparing the concentration value with a set value; when the content of C is more than or equal to 0.3 percent_O2When the concentration value is less than 0.5%, controlling an oxygen passage switch to be turned on for 0.5 second and then turned off, directly reading the concentration value of the oxygen gas concentration sensor, and continuously comparing the concentration value with a set value; when 0.1% < C_O2When the concentration value is less than 0.3%, controlling an oxygen passage switch to be turned on for 0.3 second and then turned off, directly reading the concentration value of the oxygen gas concentration sensor, and continuously comparing the concentration value with a set value; when C is present_O2When the oxygen content is less than or equal to 0.1 percent, the oxygen passage switch keeps the closed state.

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 flow control components of the liquid inlet passage 4.1, the circulating passage 4.2, the waste liquid passage 4.3 and the recovery passage 4.4 are controlled by the central controller 6.

The flow control component can be a flow pump or a flow control switch.

The liquid inlet passage 4.1 comprises a liquid storage bag 4.1.1, a liquid inlet pipeline 4.1.2 and a liquid inlet pump 4.1.3, and the liquid storage bag 4.1.1 is communicated with the liquid inlet pipeline 4.1.2; the liquid inlet pipeline 4.1.2 is driven by the liquid inlet pump 4.1.3, and the liquid inlet pipeline 4.1.2 is communicated with the culture tank 2; the liquid inlet pump 4.1.3 is controlled by a central controller 6; the liquid in the liquid storage bag can be replaced according to the 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. And sending the liquid level information in the liquid inlet pipeline to the central controller 6 in real time.

The circulation path 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 driven by the circulation pump 4.2.2, and the circulation pump 4.2.2 is controlled by the central controller 6.

In one embodiment, the filter 4.2.3 may be a hollow fiber column.

In one embodiment, the waste liquid path 4.3 includes a waste liquid pump 4.3.1, a waste liquid tank 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 tank 4.3.2 is connected to the waste liquid line 4.3.1, the waste liquid line 4.3.1 is driven by the waste liquid pump 4.3.1, and the waste liquid pump 4.3.1 is controlled by the central controller 6.

In one embodiment, the recovery pathway 4.4 includes a recovery conduit 4.4.1, a recovery pump 4.4.2 and a recovery bag 4.4.3, the recovery conduit 4.4.1 is in communication with the culture tank 2, the recovery conduit 4.4.1 is driven by the recovery pump 4.4.2, the recovery bag 4.4.3 is in communication with the recovery conduit 4.4.1, and the recovery pump 4.4.2 is controlled by the central controller 6.

In one embodiment, the culture system is provided with a recovery bag placing tray for placing 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 under the driving of 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, therefore, 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 enter the culture tank 2 again from the second circulation port 2.3.

During the culture process, the circulation path can be opened or closed according to the needs of users.

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, being located at the bottom of the tank and providing sensed information to a central controller 6.

The central controller contains a liquid control module: the central controller can receive a user instruction and controls the on-off of the liquid inlet passage, the circulation passage, the waste liquid passage and the recovery passage according to the user instruction.

In the process of culturing, the user can carry out the instruction, sets up feed liquor volume, waste liquid discharge volume as required.

In one embodiment, the central controller 6 may convert the volume of the liquid into weight according to the density of the liquid, and then control the liquid feeding amount and the waste liquid discharging amount according to a 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.

In one embodiment, the central controller 6 comprises:

a weight comparison unit for measuring the weight M of the culture tank in real time sent by the weighing sensor during liquid feeding_tAnd the user-instructed weight M of the culture tank_in0Comparing to obtain weight difference M according to formula (III)_in：

M_in＝M₀-M_t(III)

A liquid inlet passage on-off control unit for controlling the liquid inlet passage according to M_inAnd controlling the on-off of the liquid inlet passage.

Further, said is according to M_inControlling the on-off of the liquid inlet passage as follows:

M_ingreater than 0, opening the liquid inlet passage, M_inAnd 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 unit. During liquid drainage, the weight comparison unit measures the weight M of the culture tank sent by the weighing sensor in real time_tAnd the user-instructed weight M of the culture tank_out0Comparing to obtain the weight difference M according to the formula (IV)_out：

M_out＝M_t-M_out0(Ⅳ)

Waste liquid passage on-off control unit according to M_outAnd controlling the on-off of the waste liquid channel.

M_outGreater than 0, opening the waste passage, M_outAnd when the liquid inlet passage is equal to or less than 0, the liquid inlet passage is cut off.

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 and providing detection information for the central controller 6; the heating means 5.1 are controlled by the central control 6. 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.

In one embodiment, the 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 controller_tAnd T₀Comparison when T is_tLess than T₀When the heating device is started, the central controller controls the heating device to start; when T is_tGreater 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 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 controlled by the central controller 6.

In one embodiment, the blade further comprises a closed housing cavity 2.53 at the maximum radial length of the blade body for housing 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 blades of the stirrer adopt the design of a spiral surface and specific axial size and radial size, and axially stir cells and culture solution in the tank body in a culture system, so that the radial shear force is reduced as much as possible, the cells are protected, and the cell yield is improved.

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 driven by the central controller 6. And when the central controller receives that the detection information of the liquid flow component, the air flow component and the temperature regulating component exceeds the limit, the central controller controls the alarm module to give an alarm.

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.

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 door 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.

In the step 1), a culture medium is injected into the culture tank by using a liquid inlet passage. Specifically, the liquid inlet amount is set, and the set weight M of the culture tank is obtained according to the liquid inlet amount_in0Starting a liquid inlet pump to inject the culture medium in the liquid storage bag into the culture tank through a liquid inlet pipeline, and determining the amount of the culture medium to be injected into the culture tank by measuring the weight of the culture tank in real time by using a weight sensor; real-time determination of the weight M of the culture tank transmitted by the weighing cell_tAnd the set weight M of the culture tank_in0Comparing to obtain weight difference M according to formula (III)_in：

M_in＝M_in0-M_t(III)

According to M_inAnd controlling the on-off of the liquid inlet passage.

Further, M_inGreater than 0, opening the liquid inlet passage, M₁And when the liquid inlet passage is equal to or less than 0, the liquid inlet passage is cut off.

In step 2), oxygen and carbon dioxide are injected into the incubator through the oxygen passage and the carbon dioxide passage, respectively, and the gas in the incubator is injected into the culture tank through the mixed gas suction passage. Measuring the gas concentration value of carbon dioxide in the incubator by using a carbon dioxide gas concentration sensor, and measuring the gas concentration value of carbon dioxide in the incubator by using an oxygen gas concentration sensorMeasuring the concentration value of oxygen gas in the incubator; real-time oxygen gas concentration value C_tO2And real-time carbon dioxide gas concentration value C_tCO2Respectively corresponding to a predetermined oxygen gas concentration value C_0O2And a preset carbon dioxide concentration value C_0CO2Comparing to obtain the difference of the required concentration according to formulas (I) and (II), namely the concentration difference C_O2And C_CO2：

C_O2＝C_0O2-C_tO2(I)

C_CO2＝C_0CO2-C_tCO2(II)

According to C_O2Adjusting the on-off time of the oxygen passage;

according to C_CO2Adjusting the on-off time of the carbon dioxide passage;

when C is present_O2And C_CO2When the gas in the incubator meets the set threshold range, opening a mixed gas suction passage, and sucking the gas in the incubator into the incubator;

Further, according to C_O2The on-off time of the oxygen passage is adjusted in a graded regulation mode, and/or according to C_CO2The on-off time of the carbon dioxide access switch is adjusted by adopting a graded regulation and control mode.

In one embodiment, a fan may be used to agitate the gas stream to speed up the mixing and to make the gas mixing more uniform.

In the step 3), the liquid inlet passage is used for injecting cells, and the circulating passage is used for filtering metabolites. And a filter and a circulating pump are arranged on the circulating passage. Starting the stirrer, starting culture, opening a circulation path according to culture needs, discharging a mixture of cell culture medium and cells in the culture tank from a first circulation port on the culture tank into a filter of the circulation path under the driving of a circulation pump when the circulation path is in an open state, wherein the pore diameter of the filter can be 0.2-1 micron, and the filter can permeate water and components of metabolic waste generated by cell culture in the culture mediumThe cells cannot penetrate through the cells, so that part of the culture medium and metabolic waste form waste liquid which is filtered and temporarily stored in a waste liquid cavity on the filter; and the rest part of the culture medium and the cells enter the culture tank again from a second circulation port on the culture tank. The filter is provided with a first outlet and a second outlet, and the rest part of the culture medium and the cells are conveyed into the culture tank again through the first outlet. When the waste liquid needs to be discharged, the waste liquid passage is opened, and the waste liquid discharge amount is set to obtain the set weight M of the culture tank_out0And the waste liquid is discharged into the waste liquid pipe through the second outlet and then into the waste liquid barrel. Real-time determination of the weight M of the culture tank transmitted by the weighing cell_tAnd predetermined weight M of culture tank_out0Comparing to obtain weight difference M according to formula (IV)_out：

M_out＝M_t-M_out0(IV)

According to M_outAnd controlling the on-off of the waste liquid channel.

Further, M_outGreater than 0, opening the waste passage, M_outAnd when 0 or less, the waste liquid passage is cut off.

When liquid needs to be fed, the liquid feeding amount is set, and the set weight M of the culture tank is obtained according to the liquid feeding amount_in0Starting a liquid inlet pump to inject the culture medium in the liquid storage bag into the culture tank through a liquid inlet pipeline, and determining the amount of the culture medium to be injected into the culture tank by measuring the weight of the culture tank in real time by using a weight sensor; real-time determination of the weight M of the culture tank transmitted by the weighing cell_tAnd the set weight M of the culture tank_in0Comparing to obtain weight difference M according to formula (III)_in：

M_in＝M_in0-M_t(III)

According to M_inAnd controlling the on-off of the liquid inlet passage.

Further, during the culture process, the circulation path can be opened or closed according to the needs of the user.

In the step 4), after the cell culture is finished, replacing the culture medium with physiological saline, and specifically, the method comprises the following steps: after a certain amount of waste liquid was discharged, an equal amount of physiological saline was supplied, and the above operation was repeated until the medium was completely replaced. If the initial liquid amount in the culture tank is 400 ml, 200ml of waste liquid is discharged, 200ml of normal saline is added, the concentration of the culture medium is reduced to 50% of the original concentration, and after the operation is repeated for ten times, the concentration of the culture medium is reduced to the original concentration (1/2)¹⁰The substitution is considered to be completed. Then enter the concentration link, continuously discharge the waste liquid, reduce the liquid volume in the culture tank.

In the concentration process, the volume of the liquid in the culture tank is controlled, and the control method comprises the following steps: setting the target culture tank weight as M, and measuring the culture tank weight M sent by the weighing sensor in real time_tComparing the weight M with the target culture tank weight M to obtain the weight difference M according to the formula (V)₁：

M₁＝M_t-M (Ⅴ)

According to M₁And controlling the on-off of the waste liquid channel.

Further, M₁Greater than 0, opening the waste passage, M₁And when 0 or less, the waste liquid passage is cut off.

And 5) recovering the finished product cells by using the cell recovery passage. Specifically, under the drive of the recovery pump, the mixed liquid containing the cells in the culture tank enters the recovery bag through the recovery pipeline through the recovery port.

Further, in the step 5), the method also comprises reversely operating the circulating pump to recover the finished cells in the circulating path.

In one embodiment, said step 2) comprises pre-treating said filter;

in one embodiment, the liquid inlet passage further comprises a level gauge. And monitoring the liquid level change of the liquid inlet pipeline by using a liquid level meter.

In one embodiment, the culture system is provided with an alarm module. Alarm critical values of liquid level, oxygen gas concentration, carbon dioxide gas concentration, weight and temperature of the culture tank in the system are preset, the alarm critical values comprise an alarm critical upper limit and an alarm critical lower limit, and when all detection information in the system is higher than the alarm critical upper limit or lower than the alarm critical lower limit, an alarm is given.

In one embodiment, an ultraviolet sterilizing lamp is arranged in the incubator, and the incubator is sterilized by the ultraviolet sterilizing lamp.

The opening and closing of a liquid inlet passage, a circulation passage and a waste liquid passage in the liquid flow component, and the parameters of liquid inlet amount and waste liquid discharge amount can be set on site by a user according to the culture condition and immediately execute corresponding operations in the culture process; or the system can be set in advance and operated by adopting a timing starting mode.

The culture methods are useful for the culture of CAR-T cells.

It is to be understood that the scope of the invention is not to be limited to the specific embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts.

Example 1

In one embodiment of the present invention, CAR-T cells were cultured (hereinafter referred to as CCS culture) using the totally closed cell culture method according to the process flow and parameters shown in Table 1, and compared with the conventional manual CAR-T cell culture method (control). Specifically, the method comprises the following steps:

1.1 culture method and results:

CCS culture CAR-T cell process: on the first day, 1E +09 PBMC cells are taken for separation and sorting, and CD3/CD28 is added for activation; the next day, infection was performed when the total number of cells was not less than 1E7, infection schedule: infection density is 2E5-1E6/ml, MOI is 1-10; performing cell amplification from the third day to the tenth day, stirring at the rotating speed of 20-100r/min, and performing fluid infusion in a perfusion mode: the rotating speed of a circulating pump is 20-150r/min, and the perfusion time is 40-60 min; cells were harvested on day eleven: concentrating the cell suspension to 100-200ml, performing perfusion replacement, replenishing the frozen stock solution at a low rotation speed of 20-150r/min, and finally filling and freezing, wherein the culture result is shown in table 2.

TABLE 1 Primary Process flow for culturing CAR-T cells

DAY2-DAY10, temperature 37 ℃, CO₂The content was 5%.

Traditional hand-cultured CAR-T cell process: on the first day, 1E +09 PBMC cells are taken for separation and sorting, and CD3/CD28 is added for activation; the next day, infection was performed when the total number of cells was not less than 1E7, infection schedule: infection density is 2E5-1E6/ml, MOI is 1-10; performing cell expansion from the third day to the tenth day, and performing fed-batch culture based on density; cells were harvested on day eleven: the cells were collected by low speed centrifugation, resuspended and filled with the frozen stock solution, and finally frozen, and the culture results are shown in table 2.

TABLE 2 CCS and conventional hand-prepared CAR-T cell expansion results

And (4) conclusion: from the results in Table 2, it can be seen that the expansion fold of CAR-T cells prepared by CCS is significantly higher than that of conventional manual, and the culture medium usage is significantly less than that of conventional manual due to Perfusion (Perfusion) feeding method.

1.2 CAR-T cell subpopulation analysis

The CAR-T cells obtained at 1.1 were analyzed for cell subsets, detected using Life flow cytometer, Flowjo analysis software. As shown in table 3 and fig. 8, the CCS-generated CAR-T cells were clustered closer to 1 for CD4 and CD 8: 1. CCS and the traditionally manually prepared CAR-T cell subset are normal in grouping, and the detection is qualified.

TABLE 3 CAR-T cell subpopulation detailed data

1.3 CAR-T functional cell (infection efficiency) expression quantification

Performing flow detection by using recombinant human CD19 protein with His label and anti-His PE, wherein the protein sequence is shown as SEQ ID NO: 1, specifically:

MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRM GTWSTR。

flow assay results are shown in table 4 and fig. 9, CCS and traditional hand-prepared CAR-T cells contain functional cells that meet assay standards.

TABLE 4 CAR-T functional cell (infection efficiency) expression quantification detailed data

Sample name	The infection efficiency%	Whether it is qualified or not
			CAR-T (CCS production)	49.4	Is that
CAR-T (traditional hand-made)	52.0	Is that
			PBMC	0.0	Is that

1.4 CAR-T cell specific killing Activity assay

Using cells K562-CD19 expressing CD19 and K562 not expressing CD19 as Target cells (Target), and CAR-T (CD19) and PBMC as effector cells (E), respectively, after testing the transfection efficiency, the cells were transfected according to CAR +: Target ═ 0.25:1, 0.5:1, 1: effect-target ratios of 1, 2:1, 4:1 Effector cells were incubated with target cells at 37 ℃ for 16h and then assayed for LDH (lactate dehydrogenase) release in the supernatant by ELISA (Promega, cytoxicity Assay, 0000383099), with the results shown in Table 5. The increase of the effective target ratio of the CAR-T cells prepared by the two modes to the target cells (K562-CD19) is positively correlated with the killing ratio, but the non-target cells are not correlated, representing that the two CAR-T cell preparations have specific killing activity, and indicating that the killing performance of the CAR-T group is the positive cells transfected by the CART virus. In combination, the two CAR-T cell preparations were tested.

TABLE 5CAR-T cell-specific killing Activity assay details

Note: the negative part of the table is marked as 0

1.5CAR-T cytokine Release assay

The CAR-T (CD19) prepared in the two ways was incubated with K562-CD19 cells expressing CD19 and K562 cells negative in CD19 expression, respectively, for 16hr, and then cell culture supernatants were collected and the cytokine contents were measured using CBA kit manufactured by BD, as shown in table 6 and fig. 10. Therefore, cytokine release of the CAR-T cells prepared in the two modes and the target cells (K562-CD19) is obviously improved, but the non-target cells are irrelevant, and through T test, the release of the supernatant cytokines of the CAR-T cells prepared in the two modes and the target cells (K562-CD19) in coculture is not significantly different, P is more than 0.05, and the CAR-T cell products prepared in the two modes have specific killing activity and are not significantly different. Untransfected T cells (UTD, PBMC) did not significantly increase target cytokines, indicating that elevated factor release from the CAR-T group is a function of CAR-T virus transfected positive cells.

And combining the above two modes, the prepared CAR-T cell product is qualified in detection. The methods of the invention, however, enable the production of CAR-T cells in large quantities.

TABLE 6 CAR-T cell-specific killing Activity assay details

In conclusion, the system provided by the invention realizes a constant-temperature culture environment, adopts a perfusion mode to culture cells, can discharge waste liquid in the culture process, prevents the accumulation of harmful metabolites, is beneficial to achieving higher cell culture density, can reduce subsequent treatment steps, can recover finished product cells without operations such as centrifugation and the like, simplifies the operation, can improve the culture efficiency, is easy to industrialize, and realizes a totally-closed integrated process from cell activation, infection and amplification to 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 disclosed by the invention separately feeds air, ensures the stable content of each gas component in the culture process, can ensure that the change of external gas in the cell culture process does not directly influence the cell culture, ensures the constant content of each gas component, simultaneously reduces manual operation, reduces the cost, reduces the risk of misoperation in the culture process, improves the culture efficiency, can realize industrial production and has obvious cost advantage. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can 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.

Sequence listing

<110> Huadao (Shanghai) biopharmaceutical Co., Ltd

<120> totally enclosed cell culture method

<150>2019101847855

<151>2019-03-12

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<170>SIPOSequenceListing 1.0

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Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met

1 5 10 15

Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp

20 25 30

Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln

35 40 45

Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu

50 55 60

Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile

65 70 7580

Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu

85 90 95

Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr

100 105 110

Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp

115 120 125

Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro

130 135 140

Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala

145 150 155 160

Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro

165 170 175

Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro

180 185 190

Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser

195 200 205

Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser

210 215 220

Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp

225 230 235 240

Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala

245 250 255

Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu

260 265 270

Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly

275 280 285

Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu

290 295 300

Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg

305 310 315 320

Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val

325 330 335

Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu

340 345 350

Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala

355 360 365

Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp

370 375 380

Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly

385 390 395 400

Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu

405 410 415

Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu

420 425 430

Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly

435 440 445

Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu

450 455 460

Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser

465 470 475 480

Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Gly

485 490 495

Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln

500 505 510

Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala

515 520 525

Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp

530 535 540

Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg

545 550 555

Claims

1. A method for totally-enclosed cell culture by adopting a totally-enclosed cell culture system comprises the following steps:

2. The method of claim 1, wherein the fully closed cell culture system comprises at least the following components:

an incubator (1);

a culture tank (2) for performing cell culture and provided in the incubator; the culture tank (2) is provided with a stirrer (2.5), and the stirrer is arranged in the culture tank;

a gas flow assembly (3) in communication with the incubator (1) for regulating oxygen and carbon dioxide concentrations in the incubator (1);

the liquid flow component (4) is communicated with the culture tank (2) and is used for adjusting the liquid flow in the culture tank (2), filtering metabolites produced by cell culture and recovering finished cells;

a temperature adjustment assembly (5) for adjusting the temperature within the incubator;

and the central controller (6) is connected with and controls the air flow assembly, the liquid flow assembly and the temperature regulating assembly.

3. The method of claim 2, wherein the gas flow assembly 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 incubator (1) and used for conveying gas into the incubator to form mixed gas;

a mixed gas suction passage (3.4) which communicates the incubator (1) and the culture tank (2) and is used 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 exhaust gas generated during 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); the concentration value of the real-time oxygen gas and the concentration value of the real-time carbon dioxide gas in the incubator are respectively measured, and the gas concentration sensing module provides detection information for the central controller (6).

4. A method of conducting a closed cell culture using a closed cell culture system according to claim 3, wherein the airflow component further comprises one, two or three of the following features:

1) the carbon dioxide passage (3.2) comprises a carbon dioxide storage device (3.2.1), a carbon dioxide pressure reducing valve (3.2.2), a carbon dioxide passage switch (3.2.3) and a carbon dioxide pipeline (3.2.4); the carbon dioxide storage device (3.2.1) is connected with the carbon dioxide pipeline (3.2.4), the carbon dioxide pipeline (3.2.4) is driven by a carbon dioxide pressure reducing valve, and the carbon dioxide pipeline (3.2.4) is communicated with the incubator (1); a carbon dioxide passage switch (3.2.3) is arranged on the carbon dioxide passage, and the carbon dioxide passage switch (3.2.3) is controlled by the central controller (6);

2) the oxygen passage (3.3) comprises an oxygen storage device (3.3.1), an oxygen pressure reducing valve (3.3.2), an oxygen passage switch (3.3.3) and an oxygen pipeline (3.3.4); the oxygen storage device (3.3.1) is connected with the oxygen pipeline (3.3.4), the oxygen pipeline (3.3.4) is driven by an oxygen pressure reducing valve (3.3.2), and the oxygen pipeline (3.3.4) is communicated with the incubator (1); an oxygen passage switch (3.3.3) is arranged on the oxygen passage, and the oxygen passage switch (3.3.3) is controlled by the central controller (6);

3) the mixed gas suction passage (3.4) comprises a mixed gas suction pump (3.4.1) and a mixed gas suction pipeline (3.4.2); the mixed gas suction line (3.4.2) is driven by a mixed gas suction pump (3.4.1), and the mixed gas suction line (3.4.2) is used for injecting mixed gas into the culture tank (2) from the culture box (1); the mixed gas suction pump (3.4.1) is controlled by the central controller (6).

5. A method of conducting a blind cell culture using a blind cell culture system according to claim 3, further comprising one or more of the following features:

1) the incubator is internally provided with a gas discharge passage (3.7) for discharging gas in the incubator and keeping the gas pressure in the incubator stable;

2) the carbon dioxide passage (3.2) and the oxygen passage (3.3) are provided with a carbon dioxide passage incubator inlet (3.2.5) and an oxygen passage incubator inlet (3.3.5) on the incubator, and the carbon dioxide passage incubator inlet (3.2.5) and the oxygen passage incubator inlet (3.3.5) are both arranged at the upper part in the incubator;

3) the mixed gas passage is provided with a mixed gas passage incubator outlet (3.4.3) 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;

4) the incubator is internally provided with a fan (3.8) for stirring air flow, accelerating mixing, enabling the air to be mixed more uniformly and accelerating heat exchange inside the incubator.

6. A method for closed cell culture using a closed cell culture system according to claim 3, wherein the central controller (6) comprises the following parts:

a gas concentration comparison unit for comparing the real-time carbon dioxide gas concentration value sent by the gas concentration sensing module

And real-time oxygen gas concentration value

And a preset carbon dioxide concentration value

And a predetermined oxygen gas 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 carbon dioxide passage, the oxygen 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 use

And

when the gas in the incubator meets the set threshold range, opening a mixed gas suction passage, and sucking the gas in the incubator into the incubator;

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.

7. A method of conducting a closed cell culture using a closed cell culture system according to claim 2, wherein the flow module (4) comprises: a liquid inlet passage (4.1), a circulation passage (4.2), a waste liquid passage (4.3) and a recovery passage (4.4); the liquid inlet passage (4.1) and the circulating passage (4.2) are respectively communicated with the culture tank (2), the liquid inlet passage (4.1) is used for feeding liquid into the culture tank (2), and the circulating passage (4.2) is used for filtering metabolites generated by cells; the waste liquid passage (4.3) is communicated with the circulation passage (4.2) and is used for discharging the metabolite, and the recovery passage (4.4) is communicated with the culture tank (2) and is used for recovering finished cells; the flow control components of the liquid inlet passage (4.1), the circulating passage (4.2), the waste liquid passage (4.3) and the recycling passage (4.4) are controlled by the central controller (6).

8. The method of conducting a totally enclosed cell culture using a totally enclosed cell culture system as claimed in claim 7, wherein the flow assembly further comprises one or more of the following features:

1) the liquid inlet passage (4.1) comprises a liquid storage bag (4.1.1), a liquid inlet pipeline (4.1.2) and a liquid inlet pump (4.1.3); the liquid storage bag (4.1.1) is communicated with the liquid inlet pipeline (4.1.2); the liquid inlet pipeline (4.1.2) is driven by the liquid inlet pump (4.1.3), and the liquid inlet pipeline (4.1.2) is communicated with the culture tank (2); the liquid inlet pump (4.1.3) is controlled by the central controller (6);

2) the 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 driven by the circulation pump (4.2.2), and the circulation pump (4.2.2) is controlled by 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 driven by the waste liquid pump (4.3.1), and the waste liquid pump (4.3.1) is controlled by the central controller (6);

4) the recovery passage (4.4) comprises a recovery pipeline (4.4.1), a recovery pump (4.4.2) and a recovery bag (4.4.3), the recovery pipeline (4.4.1) is communicated with the culture tank (2), the recovery pipeline (4.4.1) is driven by the recovery pump (4.4.2), the recovery bag (4.4.3) is communicated with the recovery pipeline (4.4.1), and the recovery pump (4.4.2) is controlled by the central controller (6).

9. A method for conducting a closed cell culture using a closed cell culture system according to claim 2, wherein the liquid flow module (4) comprises a load cell (4.5), the load cell (4.5) being adapted to measure the tank weight in real time, being located at the bottom of the tank and providing the measurement information to the central controller (6).

10. A method for closed cell culture using a closed cell culture system according to claim 2, wherein the temperature conditioning assembly (5) comprises a heating device (5.1) and a temperature sensor (5.2); the heating device (5.1) and the temperature sensor (5.2) are arranged in the incubator (1) and are respectively connected with the central controller (6), and the heating device (5.1) is used for heating the inner cavity of the incubator; the temperature sensor (5.2) is used for measuring the real-time temperature value in the incubator and providing detection information for the central controller (6); the heating device (5.1) is controlled by the central controller (6).

11. A method of closed cell culture using a closed cell culture system according to claim 2, wherein the agitator (2.5) comprises: a medial axis (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.

12. The method of conducting whole cell culture using a whole cell culture system according to claim 2, wherein oxygen and carbon dioxide are injected into the culture tank through an oxygen passage and a carbon dioxide passage of the whole cell culture system, respectively, and the gas in the culture tank is injected into the culture tank through a mixed gas suction passage; in the step 2), the real-time oxygen gas concentration value C is measured_tO2And real time twoCarbon oxide gas concentration value C_tCO2Respectively corresponding to a predetermined oxygen gas concentration value C_0O2And a preset carbon dioxide concentration value C_0CO2Comparing to obtain the difference of the required concentration according to formulas (I) and (II), namely the concentration difference C_O2And C_CO2：

C_O2＝C_0O2-C_tO2(I)

C_CO2＝C_0CO2-C_tCO2(II)

According to C_O2Adjusting the on-off time of the oxygen passage;

according to C_CO2Adjusting the on-off time of the carbon dioxide passage;

13. The method of claim 12, wherein the whole cell culture is performed according to C_O2The on-off time of the oxygen passage is adjusted in a graded regulation mode, and/or according to C_CO2The on-off time of the carbon dioxide access switch is adjusted by adopting a graded regulation and control mode.

14. The method of claim 7, wherein the method comprises filtering the metabolite using a circulation path of the totally enclosed cell culture system, the circulation path having a filter and a circulation pump; said step 2) comprises pre-treating said filter; in the step 5), the method also comprises reversely operating the circulating pump to recover the finished cells in the circulating path.