Cell perfusion culture method and device
By using centrifugal force through a rotating tubular container to achieve continuous cell concentration and perfusion culture, the problems of membrane fouling and high shear in traditional perfusion systems are solved, enabling efficient, low-cost cell separation and stable production.
Patent Information
- Application Number
- CN202511348491.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-20
- Publication Date
- 2025-11-14
AI Technical Summary
Traditional perfusion culture systems suffer from problems such as membrane fouling, high shear force, low separation efficiency, and high maintenance costs, making it difficult to achieve efficient, low-cost, and sustainable cell concentration and separation.
A rotatable tubular container is used to push cells against the container wall using centrifugal force. The cells are fed through the first opening and discharged through the second opening, achieving continuous cell concentration. When the set concentration is reached, feeding and centrifugation are stopped, and the transport direction is switched to return the concentrated cells to the culture tank for perfusion culture.
It achieves efficient, low-shear, and low-cost cell concentration and separation, improves perfusion stability and capacity, and is suitable for large-scale cell production and industrial biopharmaceutical processes.
Smart Images
Figure CN120944799A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioengineering, and more particularly to a method and apparatus for cell perfusion culture. Background Technology
[0002] In the fields of biopharmaceuticals and cell culture, perfusion culture is an animal cell culture technique that significantly increases cell density and product yield by continuously replenishing fresh culture medium and retaining cells in a reactor. This technique utilizes cell retention devices (such as alternating tangential flow systems) to maintain a cell concentration of up to 10⁻⁶. 7 Up to 10 9 With a yield of 1000 cells / ml and a culture period of several months, the protein yield is 3-5 times higher than that of traditional methods.
[0003] Perfusion culture is a key technology for efficiently culturing cells to improve the yield of target products. Traditional perfusion systems typically use methods such as membrane filtration, directional flow filtration, or gravity sedimentation to separate cells from the culture medium, but these methods have drawbacks such as membrane fouling, high shear forces, low separation efficiency, or high maintenance costs. Summary of the Invention
[0004] The purpose of this invention is to provide a cell perfusion culture method and apparatus that can achieve efficient, low-shear, low-cost, and sustainable cell concentration and separation, thereby improving the stability and production capacity of perfusion.
[0005] In a first aspect, the present invention provides a cell perfusion culture method, comprising the following steps: A container is configured to hold liquid, the container having a first opening in the bottom central region and a second opening in the top central region; The cell culture medium containing cells in the cell culture tank is injected into the container through the first opening to supply the cells. The container is rotated to use centrifugal force to push the cells closer to the container wall; Liquid is continuously discharged from the second opening, enabling continuous cell concentration. The cell concentration is monitored in real time, and the feeding, rotation, and discharging operations are stopped when the cell concentration reaches the preset value. By switching the delivery direction of the first opening, the concentrated cells are returned to the cell culture tank to achieve perfusion culture.
[0006] The beneficial effects of the method of the present invention are as follows: A rotatable container capable of holding liquid is configured, the container having a first opening in the central region of the bottom and a second opening in the central region of the top; culture medium containing cells from a cell culture tank is injected into the container through the first opening for feeding; the container is controlled to rotate, using centrifugal force to push the cells close to the container wall; liquid is continuously discharged from the second opening, achieving continuous cell concentration; cell concentration is monitored in real time, and when the cell concentration reaches a set value, feeding, rotation (centrifugation), and discharge operations are stopped; the conveying direction of the first opening is switched, and the concentrated cells are returned to the cell culture tank to achieve perfusion culture. This method eliminates the need for membrane components, provides continuous and stable operation, has a simple structure, is easy to control, and achieves efficient, low-shear, low-cost, and sustainable cell concentration and separation, improving the stability and capacity of perfusion, and is suitable for large-scale cell production and industrial biopharmaceutical processes.
[0007] Optionally, continuously feeding the cell culture medium containing cells from the cell culture tank into the container through the first opening includes: According to the set perfusion interval and frequency, the cell culture medium containing cells in the cell culture tank is injected into the container through the first opening. The beneficial effect is that by controlling the perfusion interval and frequency to inject the cell culture medium containing cells from the cell culture tank into the container through the first opening, dynamic steady-state cell culture can be achieved, ensuring product quality while improving production efficiency.
[0008] Optionally, the delivery direction of the first opening is switched to return the concentrated cells to the cell culture vessel to achieve perfusion culture, including: By reversing the pump's rotation to switch the delivery direction of the first opening, the concentrated cells are returned to the cell culture tank, achieving perfusion culture. The beneficial effect is that reversing the pump's rotation to switch the delivery direction of the first opening improves production efficiency and the continuity of cell culture, avoiding instability in cell culture tank concentration.
[0009] Optionally, the rotation speed of the container ranges from 100 rpm to 3000 rpm; and / or the container can be connected to multiple modules to achieve multi-stage concentration. The advantages are that controlling the rotation speed of the container within the range of 100 rpm to 3000 rpm avoids cell damage due to sudden stress, adapting to different cell cultures; and setting the container to be connected to multiple modules for multi-stage concentration improves production efficiency.
[0010] Optionally, the cells in the culture medium are animal cells, insect cells, or microbial cells.
[0011] In a second aspect, the present invention provides a cell perfusion culture device applicable to any possible combination of the culture methods described in the first aspect above, comprising a drive mechanism, a container, a first power pump, a second power pump, a cell culture tank, and a controller; The drive mechanism is connected to the container and is used to drive the container to rotate; The container has a first opening located in the bottom center region and a second opening located in the top center region; One end of the first power pump is connected to the first opening through a pipe, and the other end is connected to the cell culture tank through a pipe. It is used to supply material to the container and to transport the concentrated cells back to the cell culture tank by rotating the pump in the opposite direction. The second power pump is connected to the second opening via a pipe and is used for draining liquid and discharging material; The controller is electrically connected to the first power pump, the second power pump, and the drive mechanism respectively, and is used to control the working status of the first power pump, the second power pump, and the drive mechanism.
[0012] The beneficial effects of the device of the present invention are as follows: by setting up a driving mechanism, a container, a first power pump, a second power pump, a cell culture tank, and a controller; the driving mechanism is connected to the container and is used to drive the container to rotate; the container has a first opening in the bottom central region and a second opening in the top central region; one end of the first power pump is connected to the first opening through a pipe, and the other end is connected to the cell culture tank through a pipe, for feeding the container and transporting the concentrated cells back to the cell culture tank by rotating the pump in the opposite direction; the second power pump is connected to the second opening through a pipe for discharging liquid; the controller is electrically connected to the first power pump, the second power pump, and the driving mechanism respectively, for controlling the working state of the first power pump, the second power pump, and the driving mechanism. It eliminates the need for membrane components, is continuous and stable, has a simple structure, is easy to control, and achieves efficient, low-shear, low-cost, and sustainable cell concentration and separation, improving the stability and capacity of perfusion, and is suitable for large-scale cell production and industrial biopharmaceutical processes.
[0013] Optionally, the container is a rotatable centrifuge tube; Optionally, the rotatable centrifuge tube is made of COP material and has a spiral groove on its inner wall; the spiral groove converts axial flow into spiral laminar flow, reduces the Reynolds number, and avoids cell damage caused by turbulence.
[0014] Optionally, the first opening and the second opening are connected to the container via a shaft seal. The advantage of this is that connecting the first and second openings to the container via a shaft seal ensures that the pipes connected by the two openings remain stationary when the centrifuge tube rotates.
[0015] Optionally, the container is conical, has an inner conical structure, or is cylindrical. The advantage of this is that designing the container as conical, having an inner conical structure, or being cylindrical can improve separation efficiency.
[0016] Optionally, the container is made of a temperature- and corrosion-resistant material. The advantage of this is that using a temperature- and corrosion-resistant material for the container can improve its service life.
[0017] Optionally, the cell culture vessel is a stirred bioreactor; and / or the container is a test tube, centrifuge tube, or culture vessel. Attached Figure Description
[0018] Figure 1 A flowchart of a cell perfusion culture method provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of a cell perfusion culture device provided in an embodiment of the present invention.
[0019] Explanation of reference numerals in the attached figures: 1. Container; 2. Second opening; 3. First opening; 4. Cell culture jar; 5. First power pump; 6. Controller; 7. Second power pump. Detailed Implementation
[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art. The terms "comprising" and similar expressions used herein mean that the element or object preceding the word covers the element or object listed following the word and its equivalents, but do not exclude other elements or objects.
[0021] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.
[0022] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.
[0023] In the description of this application, the term "and / or" is used to describe the logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and A and B exist simultaneously. Additionally, the character " / " in this document generally indicates that the preceding and following objects have an "or" logical relationship.
[0024] In this application, terms such as “first” and “second” are used only to distinguish one entity or operation from another, and do not necessarily require or imply any actual quantity, hierarchy or order relationship between these entities or operations.
[0025] Without further limitations, the use of terms such as “comprising,” “including,” “having,” or other similar open-ended expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.
[0026] As understood in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceeding" are understood to exclude the stated number; expressions such as "above," "below," and "within" are understood to include the stated number. Furthermore, in the description of the embodiments in this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times," unless otherwise explicitly specified.
[0027] In the description of the embodiments of this application, the space-related expressions used, such as "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "vertical," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential," indicate the orientation or positional relationship based on the orientation or positional relationship shown in the specific embodiments or drawings. They are only for the purpose of describing the specific embodiments of this application or for the reader's understanding, and do not indicate or imply that the device or component referred to must have a specific position, a specific orientation, or be constructed or operated in a specific orientation. Therefore, they should not be construed as limitations on the embodiments of this application.
[0028] Unless otherwise expressly specified or limited, the terms "installation," "connection," "linking," "fixing," and "setting," as used in the description of the embodiments of this application, should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral arrangement; it can be a direct connection or an indirect connection through an intermediate medium; it can be a relationship of two components combined together, an interaction relationship between two components, or a connection within two structures. Those skilled in the art to which this application pertains can understand the specific meaning of the above terms in the embodiments of this application according to the specific circumstances.
[0029] In this specification, references to "one embodiment" or "some embodiments" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in one or more embodiments of the invention. Therefore, the phrases "in one embodiment," "in some embodiments," "in other embodiments," and "in still other embodiments" appearing in different parts of this specification do not necessarily refer to the same embodiment, but rather mean "one or more, but not all, embodiments," unless otherwise specifically emphasized. In embodiments of the invention, "exemplarily" or "for example" are used to indicate that they are examples, illustrations, or descriptions. Any embodiment or design described as "exemplarily" or "for example" in embodiments of the invention should not be construed as preferred or advantageous over other embodiments or designs. Specifically, the use of "exemplarily" or "for example" is intended to present the relevant concepts in a concrete manner.
[0030] like Figure 1 As shown, the present invention provides a cell perfusion culture method, comprising the following steps: S101, a rotatable container capable of holding liquid is configured, the container having a first opening in the bottom central region and a second opening in the top central region. Exemplarily, the container is preferably a tubular container, such as a test tube, centrifuge tube, or culture vessel, which can rotate around a central axis to form a centrifugal force field. Under centrifugal force, cells are pushed to the region near the container wall, while cell debris and culture medium components remain in the region near the central axis of the container. The supernatant, free of intact cells, is then continuously discharged from the second opening in the top center of the container, thereby achieving continuous feeding and discharging for cell concentration with high efficiency.
[0031] In some embodiments, the rotational speed of the container ranges from 100 rpm to 3000 rpm, with the specific speed optimized according to the cells used. The centrifugal force can also be precisely adjusted by regulating the centrifugation speed, for example, through an acceleration control module (the acceleration time from 0 to 3000 rpm can be set within the range of 30-180 seconds), to prevent cell damage due to sudden force and to adapt to different cell cultures. In other embodiments, the container can be connected to multiple modules to achieve multi-stage concentration, thereby improving production efficiency.
[0032] S102, the culture medium containing cells in the cell culture tank is injected into the container through the first opening to supply the material.
[0033] In some embodiments, to achieve dynamic steady-state cell culture and improve production efficiency while ensuring product quality, continuously injecting the cell-containing culture medium from the cell culture tank into the container through a first opening for feeding includes: injecting the cell-containing culture medium from the cell culture tank into the container through the first opening according to a set perfusion interval and frequency. Preferably, the perfusion tubing uses medical-grade silicone tubing (inner diameter 4mm ± 0.1mm), equipped with a pulse damper to eliminate fluid fluctuations, and all components in contact with the fluid are USP Class VI biocompatibility certified.
[0034] In other embodiments, the cells in the culture medium are animal cells, insect cells, or microbial cells.
[0035] S103, control the rotation of the container, use centrifugal force to push the cells to the vicinity of the container wall, and leave cell debris and culture medium components in the central axis area of the container, which can form a clear boundary layer between cells and debris, and achieve high separation efficiency.
[0036] S104, the supernatant containing cell debris is continuously discharged from the second opening, achieving continuous cell concentration. Preferably, the second opening adopts a cone-shaped drainage design, which not only allows the supernatant to be discharged quickly, but also reduces the amount of debris residue.
[0037] S105 monitors cell concentration in real time. When the cell concentration reaches the set value, it stops centrifugation, feeding, and discharging operations, thus avoiding cell damage caused by over-concentration.
[0038] S106, switch the delivery direction of the first opening, and return the concentrated cells to the cell culture tank to achieve perfusion culture.
[0039] In some embodiments, in order to improve production efficiency and the continuity of cell culture, and to avoid unstable cell culture tank concentration, the delivery direction of the first opening is switched to return the concentrated cells to the cell culture tank to achieve perfusion culture. This includes: switching the delivery direction of the first opening by reversing the pump to return the concentrated cells to the cell culture tank to achieve perfusion culture.
[0040] The advantages of this invention are that, compared to traditional methods such as membrane filtration, directional flow filtration, or gravity sedimentation for separating cells from culture medium, which suffer from membrane fouling, high shear forces, low separation efficiency, or high maintenance costs, this invention utilizes the centrifugal force of a tubular container to push cells from the cell culture medium injected at the bottom center to a position near the tube wall. This allows for the continuous discharge of cell-free supernatant at the top center, achieving cell concentration. Once the concentration reaches a set value, centrifugation is stopped, and the concentrated cells are exported through the first opening and returned to the cell culture tank for perfusion culture. This achieves efficient, low-shear, low-cost, and sustainable cell concentration and separation, improving perfusion stability and production capacity. It is suitable for large-scale cell production and industrial biopharmaceutical processes.
[0041] To facilitate understanding, this embodiment further elaborates on the specific implementation process of the above method in conjunction with a specific application scenario, which includes the following steps: 1. Centrifugal concentration stage: A tubular container is used, which can rotate around a central axis to generate a centrifugal force field; Cell culture medium is continuously injected through the first opening located in the central area at the bottom of the container. Under centrifugation, cells are pushed to the area near the container wall, while cell debris and culture medium components remain in the area near the central axis of the container. The supernatant, which contains no intact cells, is continuously discharged from the second opening at the center of the top of the container; Cells are concentrated by achieving continuous feeding and continuous discharging, thereby improving concentration efficiency.
[0042] 2. Cell reinfusion stage: The cell concentration is monitored in real time, and centrifugation, feeding and discharging operations are stopped when the preset concentration is reached. Switch the delivery direction of the first opening at the bottom to export the concentrated cells; The concentrated cells are then reinfused into the original cell culture tank or other bioreactor; To achieve cell perfusion and maintain a stable cell concentration in the cell culture vessel.
[0043] Compared with the prior art, the present invention has the following advantages: 1. No filter membrane is required, avoiding membrane fouling and shear damage; 2. Enables continuous and automated cell concentration and separation, suitable for large-scale production; 3. Centrifugal force is controllable and gentle, with minimal impact on cell viability; 4. Simple structure, stable operation, and low maintenance cost.
[0044] like Figure 2 As shown, applied to the above-mentioned culture method, the present invention also provides a cell perfusion culture device, including a drive mechanism (not shown), a container 1, a first power pump 5, a second power pump 7, a cell culture tank 4, and a controller 6; the drive mechanism is connected to the container 1 and is used to drive the container 1 to rotate; the container 1 has a first opening 3 located in the bottom center region and a second opening 2 located in the top center region; one end of the first power pump 5 is connected to the first opening 3 through a pipe, and the other end is connected to the cell culture tank 4 through a pipe, for feeding materials to the container 1 and transporting concentrated cells back to the cell culture tank 4 by rotating the pump in the opposite direction; the second power pump 7 is connected to the second opening 2 through a pipe, for discharging liquid and material; the controller 6 is electrically connected to the first power pump 5, the second power pump 7, and the drive mechanism respectively, for controlling the working state of the first power pump 5, the second power pump 7, and the drive mechanism (such as automatically controlling the centrifugation speed, pump speed, pump direction switching, and feeding / discharging time). The culture apparatus provided by this invention utilizes the centrifugal force of container 1 to push cells from the cell culture medium injected from the bottom center to a position near the tube wall, thereby continuously discharging cell-free supernatant from the top center, achieving cell concentration. When the concentration reaches a set value, centrifugation is stopped, and the concentrated cells are exported through the first opening and returned to the cell culture tank to achieve perfusion culture. This enables efficient, low-shear, low-cost, and sustainable cell concentration and separation, improving the stability and production capacity of perfusion.
[0045] In some embodiments, in order to ensure that the two connected pipes remain stationary when the centrifuge tube rotates, the first opening 3 and the second opening 2 are connected to the container 1 by a shaft seal.
[0046] In other embodiments, to improve separation efficiency, the container 1 is conical, has an inner conical structure, or is cylindrical.
[0047] In some other embodiments, in order to improve the service life of the device, the container 1 is made of a temperature- and corrosion-resistant material.
[0048] In some embodiments, the cell culture tank 4 is a stirred bioreactor used for the master culture of cells.
[0049] The working process of the above-mentioned cell perfusion culture device is as follows: Step 1: Cell centrifugation and concentration 1. The cell culture medium is drawn from the cell culture tank 4 through the first power pump 5 and enters the first opening 3 at the center of the bottom of the centrifuge module; 2. Container 1 rotates at a set speed (e.g., 1000 rpm) driven by the drive mechanism, which can prevent cells from being damaged by sudden force and form a stable centrifugal force field; 3. Under the action of centrifugal force, cell particles migrate and deposit towards the tube wall, forming a high-concentration cell layer. At the same time, it can form a clear boundary between cells and debris, resulting in high separation efficiency. 4. Cell debris and culture medium components in the culture medium remain in the central axis region of container 1; 5. The supernatant is continuously discharged through the second opening 2 at the top of container 1, thereby separating the cells from the culture medium and achieving continuous feeding and discharging to concentrate the cells with high concentration efficiency. In some preferred embodiments, the second opening adopts a cone-shaped drainage design, which not only allows the supernatant to be discharged quickly, but also reduces the amount of debris residue.
[0050] 6. The controller 6 monitors the concentration level in real time. When the concentration reaches the set value (e.g., cell density > 5 × 10⁻⁶), it will trigger a trigger. 7 When the concentration reaches (cells / mL), the centrifugation, feeding, and discharging operations are automatically stopped, which can avoid cell damage caused by over-concentration.
[0051] Step 2: Cell reinfusion and perfusion 1. The system switches the pump function, changing the delivery direction of the first opening 3, which can improve production efficiency and the continuity of cell culture, and avoid unstable cell culture tank concentration; 2. Concentrated cells are discharged from the bottom of container 1 via the first power pump 5; 3. The concentrated cells are returned to the cell culture tank 4 for perfusion feeding; 4. The perfusion interval, frequency, and cell concentration can be automatically adjusted by the controller 6 to achieve dynamic steady-state culture.
[0052] Device structural details: Container 1 is arranged longitudinally and is preferably made of heat-resistant and corrosion-resistant materials; The inlet and outlet ports (first opening 3, second opening 2) can use standard aseptic connectors for easy and quick connection; Controller 6 can be embedded in a PLC system or configured with operating logic using the LabVIEW programming platform; The system supports integration with online sensors, such as cell densitometers, conductivity meters, pH and DO probes.
[0053] This embodiment illustrates a typical application process of the present invention, which can achieve efficient cell concentration and stable perfusion cell concentration without the use of a filter membrane, and is suitable for large-scale production of various cell lines such as CHO cells, HEK293, and Vero.
[0054] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.
Claims
1. A method for cell perfusion culture, characterized in that, Includes the following steps: A container is configured to hold liquid, the container having a first opening in the bottom central region and a second opening in the top central region; The cell culture medium containing cells in the cell culture tank is injected into the container through the first opening to supply the cells. The container is rotated to use centrifugal force to push the cells closer to the container wall; Liquid is continuously discharged from the second opening, enabling continuous cell concentration. The cell concentration is monitored in real time, and the feeding, rotating, and discharging operations are stopped when the cell concentration reaches the preset value. By switching the delivery direction of the first opening, the concentrated cells are returned to the cell culture tank to achieve perfusion culture.
2. The method according to claim 1, characterized in that, The process of continuously injecting cell-containing culture medium from a cell culture tank into the container through a first opening includes: According to the set perfusion interval and frequency, the culture medium containing cells in the cell culture tank is injected into the container through the first opening to supply the material.
3. The method according to claim 1, characterized in that, Switching the delivery direction of the first opening to return the concentrated cells to the cell culture vessel to achieve perfusion culture includes: By reversing the pump rotation to switch the delivery direction of the first opening, the concentrated cells are returned to the cell culture tank to achieve perfusion culture.
4. The method according to claim 1, characterized in that, The container has a rotational speed ranging from 100 rpm to 3000 rpm; and / or the container can be connected to multiple modules to achieve multi-stage concentration.
5. The method according to claim 1, characterized in that, The cells in the culture medium are animal cells, insect cells, or microbial cells.
6. A cell perfusion culture device, applied to the culture method according to any one of claims 1-5, characterized in that, Includes a drive mechanism, container, first power pump, second power pump, cell culture tank, and controller; The drive mechanism is connected to the container and is used to drive the container to rotate; The container has a first opening located in the bottom center region and a second opening located in the top center region; One end of the first power pump is connected to the first opening through a pipe, and the other end is connected to the cell culture tank through a pipe. It is used to supply material to the container and to transport the concentrated cells back to the cell culture tank by rotating the pump in the opposite direction. The second power pump is connected to the second opening via a pipe and is used for draining liquid and discharging material; The controller is electrically connected to the first power pump, the second power pump, and the drive mechanism, respectively, and is used to control the working status of the first power pump, the second power pump, and the drive mechanism.
7. The apparatus according to claim 6, characterized in that, The first opening and the second opening are connected to the container via a shaft seal.
8. The apparatus according to claim 6, characterized in that, The container is conical, has an internal conical structure, or is cylindrical.
9. The apparatus according to claim 6, characterized in that, The container is made of a temperature- and corrosion-resistant material.
10. The apparatus according to claim 6, characterized in that, The cell culture vessel is a stirred bioreactor; and / or the container is a test tube, centrifuge tube, or culture vessel.