CN111154645A - Stirrer, cell culture tank and culture method - Google Patents

Abstract

The present invention relates to a cell culture tank agitator, a cell culture tank, and a cell culture method. Wherein the agitator comprises: a middle shaft; and at least two blades which are rotationally symmetrical about the axis of the central shaft and are connected with the central shaft, each blade comprises a blade body, the blade bodies extend spirally, the axial length of each blade body accounts for 20-35% of the length of the central shaft, the rotation angle of each blade body is 15-50 degrees, the maximum radial length is 20-54 mm, and the radial length from the bottom to the top is gradually reduced. The stirrer, the cell culture tank and the cell culture method have the advantages of small stirring shearing force, high cell yield and the like.

Description

Stirrer, cell culture tank and culture method

Technical Field

The invention relates to a stirrer, a cell culture tank and a culture method.

Background

The cellular immunotherapy therapy is characterized by that the autoimmune cells of human body are collected, cultured in vitro to make their quantity be several times greater, and the target killing function is enhanced, then the above-mentioned material can be returned into human body to kill pathogen and cancer cell in blood and tissue.

For the current widespread cancer treatment, the killing of tumor cells during the treatment process lacks selectivity, and the tumor cells are killed simultaneously, and a large number of normal cells are killed. Therefore, targeted therapy has been the subject of intense research for cancer therapy.

And a method of cellular immunity, for example, CAR-T (Chimeric Antigen Receptor T-cell immunotherapy) is a therapeutic method of obtaining T lymphocytes carrying specific receptors recognizing tumor antigens by genetic modification. The chimeric antigen receptor can specifically bind to the antigen on the surface of tumor cells, thereby preventing CAR-T cells from damaging normal cells of a human body and realizing targeted therapy. 8/10/2017, Nowa announced that FDA officially approved its breakthrough CAR-T therapy Kymriah (tisagenlecucel), marketed under the name CTL 019. Is used for treating patients with refractory relapsed B-cell Acute Lymphoblastic Leukemia (ALL) under 25 years old and pricing $ 47.5 ten thousand. On 18.10.2017, another CAR-T therapy Yescatta (axicabtagene ciloleucel) was approved by the FDA and was marketed under the name KTE-C19. For the treatment of diffuse large B-cell lymphoma (DLBCL), primary mediastinal B-cell lymphoma (PMBCL) and Transformed Follicular Lymphoma (TFL), priced at $ 37.3 ten thousand. The step of obtaining CAR-T cells is typically gene transduction of cultured T cells (CAR transduction), followed by expansion of the transduced cells to yield a plurality of CAR-T cells; CAR-T cells are difficult to culture efficiently on a large scale and are one of the reasons why cellular immunotherapy is costly. Animal cells are sensitive to the external environment due to the absence of cell walls, and such indicators as carbon dioxide concentration, contamination of contaminants in culture systems, and the like all have a significant effect on the large-scale culture yield of animal cells, particularly T cells.

At the laboratory scale, a method for cell expansion is currently and generally adopted, in which a culture container is a reaction container common to laboratories, such as a cell culture flask, and a solution needs to be changed after a period of culture, and a technician needs to centrifuge a mixture of cells and a culture solution and resuspend the culture solution of a centrifuged product during the solution changing process, so as to continue the culture, and finally, during harvesting, the centrifugation operation still needs to be performed, and the product can be obtained after washing with physiological saline. The culture yield of the cells has a great relationship with the experimental skill of an experimental operator, and the time for centrifugally collecting the cells in the liquid changing process, the force for blowing and sucking the cells in the heavy suspension process, and the time and the force for gently shaking after the heavy suspension all influence the experimental result. Therefore, the culture process is random, which results in poor consistency of cultured cells and difficulty in large-scale culture.

In the prior art, in the industrial level mixing, for example, in the fermentation device described in the chinese patent application publication No. CN107760524A, the present inventors have found that if the stirring paddle commonly used in the industry, such as a hydrofoil paddle and a turbine paddle, is used to stir the cells and the culture solution, the strong shear stress generated during the stirring process breaks the T cells, and the cell yield is low.

In the prior art, most of the solutions for reducing the shear force in the cell culture system are provided with a plurality of stirring elements on a stirrer shaft. For example, the agitator system disclosed in chinese patent application publication No. CN102639221A, which has at least three agitator elements arranged in the axial direction, adopts a structure of inclined blades + standard disk agitators to reduce the shear force in the cell culture system. The inventors found that the use of the stirrer not only requires a large number of parts and thus makes assembly and maintenance complicated, but also causes a large shear stress on cells during T cell culture and affects cell productivity regardless of whether the blades are disposed obliquely or vertically.

In the prior art, there is also a cell culture tank without stirring function, for example, a cell amplification device described in chinese patent application publication No. CN107267389A, which discharges a culture solution and cells by moving a piston, but the inventors found that, in order to prevent the cells from settling, the operation of centrifuging and discharging the culture solution must be frequently performed because there is no stirring inside the amplification device, which consumes a lot of time and materials, and that, at the same time, the cells are subjected to a great pressure in some edge regions by finally collecting the amplified cells and pushing the cells by the piston, which causes the cells to be damaged or to be adhered to the edge of the culture tank, thereby reducing the yield of the cells.

The main reason why the productivity in industrial-scale large-scale culture is lower than that in laboratory-scale culture is that the culture solution and T cells are mixed by using common industrial homogeneous mixing equipment, such as a propeller-type stirrer, and a vortex is generated during the stirring, and the shear stress of the vortex breaks the T cells, so that the productivity of the cells is low.

In view of the foregoing, there is a need in the art for an agitator for a cell culture tank, and culture method, to facilitate scale-up culture of CAR-T cells.

Disclosure of Invention

It is an object of the present invention to provide an agitator for a cell culture tank.

It is an object of the present invention to provide a cell culture tank.

It is an object of the present invention to provide a cell culture method.

According to one aspect of the invention, the stirrer of the cell culture tank comprises a central shaft 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, 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.

In an embodiment of the agitator, the radial length of the blade body tapers in a linear relationship with the axial height from the bottom to the top thereof.

In the embodiment of the stirrer, 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.

In an embodiment of the agitator, the maximum radial length of the blade body is selected in the following range: 20-36mm or 25-45mm or 30-54 mm.

A cell culture tank according to another aspect of the present invention comprises a tank body and an agitator provided in an inner space of the tank body; the stirrer comprises a middle shaft; and at least two blades which are rotationally symmetrical about the axis of the central shaft and are connected with the central shaft, each blade comprises a blade body, the blade bodies extend spirally, the axial length of each blade body from the bottom of the central shaft accounts for 20-35% of the height of the tank body, the maximum radial length accounts for 50-90% of the radial length of the tank body, the rotation angle of the spiral is 15-50 degrees, and the radial length from the bottom to the top is gradually reduced.

In an embodiment of the cell culture tank, the blade further comprises a closed accommodation chamber located at the maximum radial length of the blade body for accommodating a magnet for driving the stirrer to rotate.

In an embodiment of the cell culture tank, the bottom surface of the tank body comprises an inner recess at the center thereof for positioning connection with an external tray; the blade is provided with concave portion assorted breach, the breach with the axial distance of concave portion is 2mm at least.

In the embodiment of the cell culture tank, one end of the middle shaft, which is far away from the blade, is matched with a support hole in a hollow area of a cylindrical fixture block; and the middle shaft is also provided with a step surface extending in the radial direction at the lower end of the supporting hole.

A culture method according to still another aspect of the present invention comprises:

a) in a cell culture tank, mixing the transduced immune cells with a culture solution, continuously stirring in the culture process, and amplifying and culturing the immune cells;

wherein the cell culture tank comprises a tank body and a stirrer arranged in the inner space of the tank body;

the stirrer comprises a central shaft 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, the blade bodies extend spirally, the axial length of each blade body is 20-35% of the height of the tank body from the bottom of the central shaft, the maximum radial length of each blade body is 50-90% of the radial length of the tank body, the rotation angle of each spiral is 15-50 degrees, and the radial length from the bottom to the top is gradually reduced.

In an embodiment of the cultivation process, the rotation speed of the stirrer is 20 to 60 revolutions per minute.

The improved effect of the invention at least comprises that the blades of the stirrer adopt a spiral surface and the design of specific axial dimension and radial dimension, and the invention achieves the purposes of axially stirring cells and culture solution in the tank body in a culture system and reducing radial shearing force as much as possible so as to protect the cells and improve the cell yield.

Drawings

The above and other features, properties and advantages of the present invention will become more apparent from the following description of the embodiments with reference to the accompanying drawings, in which:

FIG. 1 is a schematic configuration diagram of an embodiment of a cell culture system.

FIG. 2 is a schematic structural view of an embodiment of the cell culture tank.

FIG. 3 is a schematic cross-sectional structure of an embodiment of a cell culture tank.

FIG. 4 is a schematic view showing the external structure of an embodiment of the tank body of the cell culture tank.

FIG. 5 is a schematic structural view of an embodiment of a lid assembly for a cell culture tank.

Figure 6 is a schematic cross-sectional view of the can lid assembly of figure 5.

Fig. 7 is a schematic structural diagram of an embodiment of a cylindrical cartridge.

FIG. 8 is a schematic diagram of the structure of an embodiment of the agitator of the cell culture tank.

Fig. 9 is a schematic view of another angular configuration of the agitator of fig. 8.

FIG. 10 is a top view of the agitator of FIG. 8

FIG. 11 is a schematic diagram of the agitator agitation process of the cell culture tank.

FIG. 12 is a schematic structural view of an embodiment of the cell culture tank.

Detailed Description

The following discloses many different embodiments or examples for implementing the subject technology described. Specific examples of components and arrangements are described below to simplify the present disclosure, but these are merely examples and do not limit the scope of the invention. For example, if a first feature is formed over or on a second feature described later in the specification, this may include embodiments in which the first and second features are formed in direct contact, and may also include embodiments in which additional features are formed between the first and second features, such that the first and second features may not be in direct contact. Additionally, reference numerals and/or letters may be repeated among the various examples throughout this disclosure. This repetition is for the purpose of simplicity and clarity and does not in itself dictate a relationship between the various embodiments and/or configurations discussed. Further, when a first element is described as being coupled or coupled to a second element, the description includes embodiments in which the first and second elements are directly coupled or coupled to each other, as well as embodiments in which one or more additional intervening elements are added to indirectly couple or couple the first and second elements to each other.

Further, it is to be understood that the positional or orientational relationships indicated by the directional terms such as "front, rear, upper, lower, left, right", "transverse, vertical, horizontal" and "top, bottom" and the like are generally based on the positional or orientational relationships shown in the drawings and are presented only for convenience in describing and simplifying the invention, and in the absence of a contrary explanation, these directional terms are not intended to indicate and imply that the referenced device or element must have a particular orientation or be constructed and operated in a particular orientation and therefore should not be construed as limiting the scope of the invention; the terms "inside" and "outside" refer to the inner and outer parts relative to the outline of each part itself, and the terms "first" and "second" are used to define the parts, and are used only for the convenience of distinguishing the corresponding parts, and the terms do not have any special meaning unless otherwise stated, and therefore, the scope of the present invention should not be construed as being limited.

Also, this application uses specific language to describe embodiments of the application. Reference throughout this specification to "one embodiment," "an embodiment," and/or "some embodiments" means that a particular feature, structure, or characteristic described in connection with at least one embodiment of the present application is included in at least one embodiment of the present application. Therefore, it is emphasized and should be appreciated that two or more references to "an embodiment" or "one embodiment" or "some embodiments" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, some features, structures, or characteristics of one or more embodiments of the present application may be combined as appropriate.

As shown in fig. 1 to 3, the T cell culture system includes a cell culture tank 1, a circulation system 2, and pipes 41, 42, 43, 44, 45 connected to the cell culture tank and to the outside. The cell culture tank 1 includes a tank 11, a lid assembly 12 provided at an upper end of the tank 11, and a stirrer 13 provided in an inner space 11 of the tank for stirring the T cells and the culture solution. Four ports are arranged on the side wall of the tank body 11, a first inlet 110 is used for inputting liquid or initial transduced T cells into the internal space of the tank body 11, in the cell amplification culture stage, the liquid input into the first inlet 110 is culture solution, and after the amplification culture is finished, the liquid input into the first inlet 110 is normal saline for washing the T cells; the first outlet 111 is used for outputting cell culture products to the outside, and in the embodiment, the output is a mixture of washed T cells and physiological saline; the second inlet 112 and the second outlet 113 are connected to the circulation system 2, during the cell culture process, under the driving of a peristaltic pump (not shown) of the circulation system 2, the mixture of the cell culture fluid and the cells is discharged from the second outlet 113 into the filtering membrane module 21 of the circulation system 2, the filtering membrane module 21 may be a hollow filtering membrane module, the filtering membrane module 21 has a semi-permeable membrane, the pore size of which may be 0.2 μm, and the membrane can permeate water and the components of metabolic waste generated by the cell culture in the culture fluid, but the cells themselves cannot permeate, so that a part of the culture fluid and the metabolic waste is filtered, and the rest of the culture fluid and the cells enter the inner space of the tank 11 again through the second inlet 112. The first outlet 111 and the second outlet 113 are disposed at the bottom of the sidewall of the tank body to sufficiently suck out the cells and the liquid inside the tank body 11. Specifically, as shown in fig. 1 and 2, the circumferential positions of the first inlet 110 and the first outlet 111 are the same, and the first inlet 110 is axially higher than the first outlet 111 by at least 30mm to facilitate the operation of inserting the pipeline, but it can be understood by those skilled in the art that the height of the first inlet cannot be too high, otherwise the culture solution falls to the culture system too fast when being introduced, and directly impacts the liquid surface from top to bottom, possibly damaging T cells, and the distance that the first inlet 110 is axially higher than the first outlet 111 may be 30-50 mm. The positions of the second inlet 112 and the second outlet 113 are axially symmetrical to the positions of the first inlet 110 and the first outlet 111 about the axis of the can body 11, so as to facilitate the die drawing process during processing.

As shown in fig. 2, 3, 11 and 12, the structure for driving the stirrer 13 inside the tank 11 to rotate may be a magnetic stirring structure as shown in fig. 12. The tray 3 of the magnetic stirring structure includes a tray body 31 for supporting the pot 11 at the bottom thereof and a magnetic stirring actuator 32, and as shown in fig. 3, a magnet 14 is provided in a magnet housing chamber 131 of the stirrer 13. With continued reference to fig. 11, the magnetic force of the magnet 14 contained in the stirrer 13 and the driver 32, driven by the magnetic driver 31, drives the stirrer 13 located inside the tank 11 to rotate, so as to stir the cells and the culture solution, and make the cells, the culture solution and the oxygen fully contact. The principle of driving the stirrer 13 to rotate by magnetic force is the working principle of a conventional magnetic stirrer in the chemical and biological field, that is, after the power is turned on, the motor of the driver 32 drives the magnet of the driver to rotate, and further drives the magnet 14 to drive the stirrer 13 to rotate, which is a content easily understood by those skilled in the art, and therefore, the detailed description is omitted. The bottom of the can 11 may be provided with an inner concave portion 115 to match the can 11 with a corresponding outer convex portion 311 of the external tray 31 to fix the can 11, so that the can 11 can be assembled on the tray 3 more conveniently and quickly. Compared with the prior art that the rotating shaft of the stirrer is directly driven by the motor, the non-contact type driving stirrer 13 adopting the magnetic stirring principle in the above embodiment has the main advantages of ensuring the cleanness and convenient maintenance of the cell culture environment inside the tank body 11. The reason for this is that the motor is used to directly drive the rotating shaft of the stirrer, and the motor is usually required to be disposed outside the tank 11, so the rotating shaft of the stirrer must extend out of the tank 11, and therefore a tight seal in the culture environment for a long time is required to be ensured between the rotating shaft of the stirrer and the tank 11, 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. In addition, the side wall of the tank 11 may be at least partially transparent, so that an operator can observe the culture conditions in the tank 11 conveniently, such as the liquid level, the mixing uniformity of the culture solution and the cells, and preferably, as shown in fig. 2 and 4, the side wall of the tank 11 may be provided with corresponding liquid level scales, so as to facilitate quantitative observation and recording of liquid level changes by the operator. The liquid level scale includes a maximum allowable liquid level 117, i.e., the highest position of the culture system to which cells and culture medium are added must not exceed the maximum allowable liquid level, and the position thereof can be used as a reference for the depth of the air inlet part of the culture tank 1 extending into the tank 11 and the height of the blade of the agitator 13. The diameter of the tank 11 of the embodiment of fig. 2 and 4 is 80mm, and the maximum allowable liquid level 117 is on the scale of 300 mL. For different culture scale requirements, the diameter of the tank 11 can be correspondingly increased, and the height is unchanged, so the height of the maximum allowable liquid level is also unchanged, for example, the diameter of the tank 11 can be enlarged to 100mm and 120mm, the maximum allowable liquid level is 450mL and 650mL, but the height of the tank 11 is unchanged, so the position of the maximum allowable liquid level 117 in the tank 11 is unchanged.

Referring to fig. 3 and 4, an open end 118 of the tank 11, that is, an upper portion of the tank 11 in the embodiment of the drawings, is provided with a threaded connection portion 119 extending from the open end 118 to the other end of the tank, and a threaded connection portion 120 corresponding to an inner wall of a cover body of the cover assembly 12, as shown in fig. 5 and 6, the threaded connection portion 120 of the cover body is an internal thread, and the threaded connection portion 119 of the tank 11 is an external thread, which is easily understood by those skilled in the art, the threaded connection portion 120 may also be an external thread, and the threaded connection portion 119 may also be an internal thread, so that the tank 11 and the cover assembly 12 are connected by using a threaded connection structure, which is convenient for an operator to install. As shown in fig. 2 to 4, the end of the threaded connection 119 of the can 11 is provided with a stop protrusion 1110 protruding radially from the side wall of the can 11, which acts as a stop during the tightening of the cap assembly 12, i.e. prompts the operator to stop the tightening. Of course, a groove (not shown) may be correspondingly formed on the sidewall of the cover body, and the groove is engaged with the limiting protrusion 1110 after being screwed down, so as to prevent the cover assembly 12 from being loosened or even opened due to misoperation and the like, and ensure the sterile environment inside the tank 11 during the cultivation process.

As shown in fig. 1, 3 and 5 to 7, the lid assembly 12 of the cell culture tank 1 includes a lid body 121, and an air inlet 122 and an air outlet 123 provided in the lid body 121 for supplying and discharging air to and from the internal space of the tank 11, wherein the air inlet 122 is longer than the air outlet 123 in a direction perpendicular to the bottom surface of the lid body 121 as shown in fig. 3 and 5; during cell culture, the gas inlet part 122 is used for conveying gas into the tank body 11 of the cell culture tank 1, and during cell culture, the gas inlet part 122 conveys the gas in a non-contact conveying mode, namely the structure of the gas inlet part 122 is not in contact with the liquid level of a culture solution, so that the gas inlet pipe in the prior art stretches into the liquid level, bubbles are generated in a culture system to damage T cells, the non-contact conveying can prevent the generation of the bubbles, and the yield of the T cells is improved. Meanwhile, the length of the air inlet part 122 in the direction perpendicular to the bottom surface of the cap body 121 is greater than that of the air outlet part 123, which has an advantageous effect in that the oxygen and carbon dioxide concentrations in the culture system can be adjusted more rapidly than in a case where the length of the air inlet and the length of the air outlet are equal or shorter. 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. Specifically, as shown in fig. 3 and 5 to 6, an example of a specific structure of the air intake part 122 and the air exhaust part 123 may be such that the air intake part 122 includes an air intake pipe, and the air exhaust part 123 includes an air exhaust pipe, and the length of the air intake pipe in a direction perpendicular to the bottom surface of the cover body 121 is greater than that of the air exhaust pipe. Although other configurations may be employed, such as a configuration in which the exhaust portion 123 is provided with only an exhaust port, without an exhaust pipe. The gas inlet 122 and the gas outlet 123 are both provided with gas discharge pipes, so that the gas exchange between the culture system and the outside is quicker, and the gas components in the culture gas can be regulated more quickly. Further, the specific structure of the intake pipe and the exhaust pipe may be as shown in fig. 3 and 5 to 6, which extend vertically from the bottom surface of the cover body 121. Although other arrangements exist, such as extending radially from the sidewall of the cap body 121, then bending, and then extending in a direction perpendicular to the bottom surface of the cap body 121. The structure that the bottom surface of the direct cover body 121 extends vertically as shown in the figure is adopted, so that the processing and the installation are convenient, and the air inlet pipe and the air outlet pipe are convenient to clean. In addition, referring to fig. 1, fig. 3 and fig. 5 to fig. 6, the air inlet portion 122 and the air outlet portion 123 further include an air inlet 124 and an air outlet 125, respectively, protruding from the cover body 121, for introducing an external air source into the air inlet pipe and for discharging air from the air outlet pipe. Although other structures for introduction and removal exist, such as openings directly formed in the cover body 121 itself for direct contact with the intake pipe and the exhaust pipe. The arrangement of the air inlet 124 and the air outlet 125 which are arranged in a protruding way is adopted, so that the insertion and the disassembly of the pipeline, the air inlet pipe and the air exhaust pipe can be facilitated, and the sealing of the cell culture tank 1 is also ensured. The specific structure of the air inlet 124 and the air outlet 125 provided with the protrusions is not limited to the pagoda type interface in the figure, and may be a common interface structure such as a screw interface and a quick-connect interface.

With continued reference to FIGS. 3, 5-7, the connection structure between the agitator 13 and the cover assembly 12 in the cell culture tank 1 may be detachable, so that the cleaning and maintenance operations can be facilitated for the operator. When cleaning, the operator first removes cover assembly 12, and then removes agitator 13 from the cover, so can fully wash cover assembly 12 and agitator 13, guarantee the clean degree of cultivation system. The specific structure of the detachable connection may be a cylindrical snap structure as shown in fig. 3, 5 to 7, and the cover assembly 12 further includes a hollow cylindrical snap body 126 having a bottom surface integrated with the bottom surface of the cover body 121, and the cylindrical snap body 126 includes a set of opposite side surfaces having the snap holes 127 and a set of opposite side surfaces having the wedge-shaped snap grooves 1261. Correspondingly, one end of the middle shaft 132 of the stirrer 13 is matched with the support hole 129 arranged in the hollow area 1200 of the cylindrical fixture block 128 to form a shaft hole, as shown in fig. 3, the middle shaft 132 is further provided with a step surface 136 extending radially at the lower end of the support hole 129, namely, at the end close to the blade, which has the beneficial effect that when the middle shaft rotates, the middle shaft may rub against the matched support hole 129 to generate debris, and the step surface 136 is arranged to prevent the debris from falling into the culture system from the support hole 129, so as to keep the culture environment clean. The cylindrical latch 128 further includes a set of opposing outer side walls having wedge-shaped latches 1200 for engaging with the wedge-shaped slots 1261 and a set of opposing outer side walls having snap hooks 1201 for engaging with the snap holes 127, and the agitator 13 is detachably connected to the cover assembly 12 through the snap structure of the cylindrical latch 128 and the cylindrical snap body 126.

In addition, the specific form of the cover assembly 12 and the tank 11 can be that the cover assembly 12 and the tank 11 are connected in an openable manner, such as a threaded detachable connection structure shown in fig. 2 to 6, or other common openable connection structures, such as a cover assembly and a tank connected by a movable hinge. With continued reference to fig. 2 and fig. 3, the vertical position of the air outlet 1221 of the air inlet portion 122 in the tank 11 is higher than the air inlet 1231 of the air outlet portion 123, and the air outlet 1231 of the air inlet portion 122 is higher than the maximum allowable liquid level 117 of the tank 11, so as to prevent the air inlet portion 122 from directly contacting the liquid level to generate bubbles to damage T cells. Preferably, the gas outlet of the gas inlet part is 30-50mm higher than the highest liquid level, so as to ensure that the distance between the gas outlet 1221 and the liquid level can quickly and sufficiently convey gas into the culture system while ensuring that T cells are not damaged, and the T cells can fully contact with fresh oxygen. It is understood that the cover assembly 12 may also be non-openable with respect to the tank 11, i.e. the cover assembly 12 is in the form of a closed top surface of the tank 11, and the relative positions of the air outlet 1221 of the air inlet 122 and the air inlet 1231 of the air outlet 123 of the air outlet 122 and the maximum allowable liquid level 117 of the tank 11 are the same as the above-mentioned embodiments in which the cover assembly 12 and the tank 11 are openable. The effect of preventing the generation of bubbles in the culture system and ensuring the sufficient and rapid exchange of gas substances in the culture system can be achieved, and of course, the structure that can not be opened, that is, the structure that the tank body 11 is integrated with the gas inlet part 122 and the gas outlet part 123, is adopted, so that the number of parts is reduced, the cost is reduced, but the cleaning of the tank body and the gas inlet part can be inconvenient.

Referring to fig. 3, 8 to 11, the stirrer 13 of the cell culture tank 1 has a structure including a central shaft 132 and two or more blades 133 which are rotationally symmetrical about the axis of the central shaft 132 and are connected to the central shaft 132, and the number of the blades may be two as shown in fig. 3. Each blade 133 comprises a blade body, as shown in fig. 9 and 10, the blade body extends in a spiral shape, the dimensional parameters of the blade body include that the axial length of the blade body accounts for 20% to 35% of the height of the tank 11, and the central axis 132 of the stirrer is basically arranged along the height of the tank 11, that is, the axial length of the blade body accounts for 20% to 35% of the length of the central axis 132; the spiral rotation angle of the blade body is 15-50 degrees, in the illustrated embodiment 18 degrees, the maximum radial length of the blade body is 50-90% of the radial length of the tank body 11, the corresponding diameters of the tank body 11 of the illustrated embodiment can be 80mm, 100mm and 120mm respectively, the maximum radial length of the corresponding blade body ranges from 20-36mm or 25-45mm or 30-54mm respectively, and the radial length of the blade body from the bottom to the top is gradually reduced. With the blade 133 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. This is because, as shown in fig. 11, the magnet 14 housed in the agitator 13 and the magnetic force of the actuator 32 act on each other by the magnetic actuator 31 to rotate the agitator 13 located inside the tank 11. Compared with the configuration of the plane type blade in the prior art, the spiral blade has smaller shearing force to fluid and gently supports cells and culture solution, and the axial and radial dimensions of the blade body are designed, so that the size of the blade relative to the tank body is larger, the space for forming tangential vortex in the radial space between the blade body and the tank body is reduced, and T cells are further protected; meanwhile, the radial length of the blade body from the bottom to the top is reduced, so that the 'lifted' cells and the culture solution can smoothly flow and circulate, and the cells and the culture solution can be quickly and fully contacted with the gas input into the culture system while being fully stirred and mixed in the axial direction of the stirring body. In particular, in the configuration in which the gas inlet 122 delivers gas into the tank 11 of the cell culture tank 1 in a non-contact manner as shown in the embodiment of FIG. 3, the upward and downward movement in the axial direction is more advantageous in that the cells and the culture medium are sufficiently contacted with the oxygen gas to be supplied. By adopting the culture agitator 13 with the above structure, in the process of T cell expansion culture, the mixture of transduced T cells and culture solution in the cell culture tank 1 can be continuously agitated in the culture process to expand and culture immune cells, preferably, the agitating speed of the agitator is about 20-60 rpm, and the slower agitating speed is combined with the blade 133 with the above shape, which is beneficial to further reducing the shearing force in the agitation system and improving the yield of T cells.

Further, as shown in fig. 8 to 10, a specific structure in which the radial length of the blade body is gradually reduced from the bottom to the top may be gradually reduced in a linear relationship with the axial height, for example, formed by a portion which remains inside a conical surface after being cut off on the basis of a positive helicoid, the half-cone angle of the conical surface being 20 to 45 degrees; or an inclined spiral surface with an included angle of 20-45 degrees between a straight generatrix and an axis or a conical spiral surface with a half cone angle of 20-45 degrees of a corresponding conical surface can be used as a basis, and the spiral shape of the blade is obtained after the conical surface is cut off. The beneficial effect that so obtains lies in when reaching the technical effect that does benefit to the cell of axial roll up and down and culture solution, reduces the space that blade 133 and jar 11 lateral wall formed radial vortex to reduce the shearing force that the stirring process produced, thereby protect T cell, improve its productivity. For embodiments in which the bottom of the can 11 is provided with an internal recess 115 to mate the can 11 with a corresponding external protrusion 311 of the external disk 31 to secure the can 11, the bottom of the blade 133 of the agitator 13 is also provided with a notch 134 corresponding to the internal recess 115 of the can 11, and it will be readily appreciated that a gap is also required between the notch 134 and the internal recess 115 to prevent the blade 133 of the agitator 13 from interfering with the internal recess 115 when rotating. The axial clearance between the notch 134 and the inner recess 115 is at least 2 mm. The notch 134 is adopted, which has the advantage of facilitating the installation of the stirrer 13. Specifically, during the assembly process, the tank 11 is quickly fitted and fixedly mounted with the corresponding male portion 311 of the tray body 31 through the arrangement of the female portion 115, and then the agitator 13 is mounted inside the tank 11 through the fitting of the blade notch 134 and the female portion 115.

Further, the specific structure for realizing the detachable connection of the stirrer 13 and the cover assembly 12 may include the cylindrical snap-fit connection structure of the cover assembly 12 and the stirrer 13 described above, and may also be other connection structures, for example, one end of the central shaft 132 is processed into an external thread or an internal thread structure to be connected with the cover assembly 12, and the like.

In summary, the beneficial effects of the cell culture system, the cell culture tank 1, the cover assembly 12 and the stirrer 13 according to the above embodiment at least include:

1. the blades of the stirrer adopt a spiral surface and a design of specific axial size and radial size, so that cells and culture solution are axially stirred in the tank body 11 in a culture system, and radial shear force is reduced as much as possible to protect the cells and improve the cell yield;

2. the design of cover body assembly 12 for cultivate the system and can carry out gas exchange with the external world fully fast, also easily with jar body 11, agitator 13 installation dismantlement simultaneously, conveniently wash the maintenance.

3. The inlet and outlet of the tank body 11 and the cooperation of the cell culture system enable the supplement of fresh culture solution and the discharge of waste liquid to be continuously performed in a circulating manner in the cell culture process, and the cell culture efficiency is improved.

Although the present invention has been described in the above embodiments, it is not intended to limit the present invention, and those skilled in the art can make variations and modifications without departing from the spirit and scope of the present invention, such as applying the T cell culture of the above embodiments to the culture of other types of immune cells, and even other types of mammalian cells. Therefore, any modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention are within the protection scope defined by the claims of the present invention, unless the technical essence of the present invention departs from the content of the present invention.

Claims (10)

1. An agitator for a cell culture tank, comprising:

a middle shaft; and

the axial line of the middle shaft is rotationally symmetrical and connected with at least two blades of the middle 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 middle 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.

2. The agitator of claim 1, wherein a radial length of the blade body tapers in a linear relationship with an axial height from a bottom to a top of the blade body.

3. The stirrer according to claim 1, wherein the helical shape is formed by cutting off a conical surface on the basis of a positive helical surface and then remaining inside the conical surface, and the conical surface has a half-cone angle of 20 to 45 degrees.

4. The agitator of claim 1, wherein the blade body maximum radial length is selected from the range of:

20-36mm or 25-45mm or 30-54 mm.

5. A cell culture tank, comprising:

a tank body;

the stirrer is arranged in the inner space of the tank body;

the stirrer comprises a middle shaft; and

the axial line of the middle shaft is rotationally symmetrical and is connected with at least two blades of the middle shaft, each blade comprises a blade body, the blade bodies extend spirally, the axial length of each blade body from the bottom of the middle shaft accounts for 20-35% of the height of the tank body, the maximum radial length accounts for 50-90% of the radial length of the tank body, the rotation angle of the spiral is 15-50 degrees, and the radial length from the bottom to the top is gradually reduced.

6. The cell culture tank of claim 5, wherein the blade further comprises a closed receiving chamber at a maximum radial length of the blade body for receiving a magnet for driving the agitator to rotate.

7. The cell culture canister of claim 6, wherein the bottom surface of the canister body includes an internal recess in the center thereof for positioning connection with an external tray; the blade is provided with concave portion assorted breach, the breach with the axial distance of concave portion is 2mm at least.

8. The cell culture tank of claim 5,

one end of the middle shaft, which is far away from the blade, is matched with a supporting hole in a hollow area of a cylindrical clamping block; and the middle shaft is also provided with a step surface extending in the radial direction at the lower end of the supporting hole.

9. A method of cell culture comprising:

a) in a cell culture tank, mixing the transduced immune cells with a culture solution, continuously stirring in the culture process, and amplifying and culturing the immune cells;

wherein the cell culture tank comprises a tank body and a stirrer arranged in the inner space of the tank body;

the stirrer comprises a central shaft 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, the blade bodies extend spirally, the axial length of each blade body is 20-35% of the height of the tank body from the bottom of the central shaft, the maximum radial length of each blade body is 50-90% of the radial length of the tank body, the rotation angle of each spiral is 15-50 degrees, and the radial length from the bottom to the top is gradually reduced.

10. The culture method according to claim 9, further comprising:

the rotating speed of the stirrer is 20-60 revolutions per minute.

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