Closed cell culture device with high gas permeability and method

Abstract

A closed cell culture device with high gas permeability and a method. The culture device comprises: a base; a reactor housing, which is arranged above the base and is connected to the base to form a sealed cavity; a gas exchange assembly, which is arranged above the base and is in communication with the sealed cavity; a first liquid exchange assembly, which is arranged above the reactor housing and is connected to the reactor housing; and a second liquid exchange assembly, which is arranged on the side wall of the reactor housing and is in communication with the sealed cavity. A filter is provided in the first liquid exchange assembly, a filter membrane is provided in the filter, and the filter membrane is built in the filter in a folded or stacked manner; and the gas permeation rate of the filter is 5-30 L / min, and the membrane area of the filter membrane is larger than 2700mm 2. The method solves the problem that a bottom silicone breathable membrane will bulge due to negative pressure when a culture medium is extracted, and the problem of a complicated apparatus caused by discharging the culture medium in a positive pressure manner.

Description

A highly permeable cell closed culture device and method Technical Field

[0001] The present invention belongs to the technical field of cell culture, and in particular relates to a highly permeable closed cell culture device and method. Background Art

[0002] Cell culture technology refers to the process of growing and proliferating cells in vitro using appropriate nutrients and gases. With the advancement of pharmaceutical technology, cell culture has become an increasingly essential technique in pharmaceutical research and drug production. Currently, cell culture technology has been gradually applied to a variety of technical fields, including drug screening research, monoclonal antibody production, vaccine production, and cell therapy.

[0003] In the prior art, cells used in T cell therapy, adoptive immunotherapy and adoptive cell therapy are often prepared and stored using conventional methods. However, due to the limited growth space and nutrients of cells, they need to be passaged after reaching a certain number of cell proliferation. It is usually necessary to frequently add culture medium to the cells or separate the cells from the culture medium after a large number of cell proliferation. In this process, it is necessary to increase the number of cell proliferation by culturing using a large amount of cell consumables. However, on the one hand, this operation mode requires repeated opening of the lid, which greatly increases the risk of cell contamination during the cell preparation process and prolongs the harvesting time of the cells. Once the cells are contaminated by bacteria, the cells will stop proliferating and die in large numbers, causing huge losses. On the other hand, the growth of cells requires carbon dioxide and oxygen. The existing culture device has low ventilation efficiency and cannot add a large amount of culture medium at one time, so it is impossible to achieve long-term culture of cells, which ultimately leads to low efficiency of cell culture and a large increase in the cost of cell culture.

[0004] Cell and Gene Therapy (CGT) involves transferring defined genetic material into a patient's specific target cells to modify individual gene expression or repair abnormal genes through gene addition, gene correction, or gene silencing, with the goal of curing disease. According to the American Society of Cell and Gene Therapy (ASCGT), there are over 3,600 clinical trials underway in the global CGT pipeline. To date, 23 gene and cell therapy products have been approved by the US FDA, including game-changing treatments for neuromuscular diseases, hereditary blindness, and certain blood cancers. In China, 726 trials have been conducted, and five drugs have been marketed. CGT is highly targeted, killing cancer cells without harming normal cells. Furthermore, since the cells are derived from the patient's own body, they are immune to immune rejection, have virtually no side effects, and are pain-free. CGT treatment involves removing defective cells from the patient's body, genetically modifying them, growing them in culture, and then reinfusing them into the patient. Conventional cell culture processes utilize conventional cell culture flasks, which contain a limited number of cells per consumable. These flasks require regular uncapping for cell passaging and testing, which can easily lead to cell contamination. This culture method is inherently unsuitable for CGT cell culture. CGT cells should be cultured and propagated in a completely closed system whenever possible to minimize unforeseen contamination from human intervention.

[0005] To address these issues, a highly breathable, fully enclosed cell culture system is urgently needed. The entire process from cell culture to collection occurs within a fully enclosed system, with cells and culture medium transported via pipelines, significantly reducing the risk of contamination.

[0006] Existing invention patent CN113462570A discloses a large-capacity suspension cell culture flask. Specifically, the large-capacity suspension cell culture flask is a fully enclosed operating device with an integrated culture flask design that can reduce the risk of cross-contamination during the cell culture process. It uses an extraction method to exchange culture medium. Because the upper breathable membrane uses a traditional single-layer membrane filter to filter gas, the number of pores per unit area is small and the air permeation rate is low. Although it is suitable for gas exchange during cell culture, it cannot maintain real-time and instantaneous pressure balance inside and outside the flask when extracting culture medium. Because the suction of culture medium will cause negative pressure inside the flask, the bottom silicone breathable membrane will bulge due to the negative pressure during the suction of culture medium, affecting the cells being cultured, and further disturbing the cells near the lower connecting tube and being sucked out along with the waste liquid, thereby reducing the cell culture efficiency and the yield of the cultured cells. When replenishing culture medium, the low air permeation rate of the upper breathable membrane leads to a slow replenishment speed, which affects cell growth.

[0007] To avoid negative pressure within the bottle caused by aspirating culture medium, invention patent CN105392876A discloses a closed system device and method for a gas-permeable cell culture process. The closed system device utilizes pressure to push gas into a cell culture and cell recovery device, thereby draining the culture medium and cultured cells. Specifically, this method uses a positive pressure generated by introducing sterile air into the bottle through an air pump to drain the liquid. However, the pressurization time is difficult to control, requiring appropriate gas flow control and monitoring to determine whether the liquid has been completely drained, which increases the complexity of the system.

[0008] Therefore, in order to solve the above-mentioned technical problems, it is necessary to provide a highly permeable closed cell culture device and method. Technical issues

[0009] How to solve the problem in the prior art that the extraction of culture medium causes the bottom silicone breathable membrane to bulge due to negative pressure, and the problem that the discharge of culture medium by positive pressure causes the equipment to become complicated. Technical Solutions

[0010] In order to solve the above problems, the technical solution of the present invention is:

[0011] The first aspect of the present invention provides a highly permeable closed cell culture device, comprising: a base; a reactor shell, arranged above the base and connected to the base to form a closed cavity; a gas exchange component, arranged above the base and connected to the closed cavity; a first liquid exchange component, arranged above the reactor shell and connected to the reactor shell; a second liquid exchange component, arranged on the side wall of the reactor shell and connected to the closed cavity; wherein a filter is provided in the first liquid exchange component, and a filter membrane is provided in the filter, and the filter membrane is built into the filter in a folded or stacked manner; the gas permeability of the filter is 5-30 L / min, and the membrane area of ​​the filter membrane is greater than 2700 mm 2 .

[0012] Furthermore, the first liquid exchange component also includes: a connecting piece, which is arranged at the top of the reactor shell and connected to the reactor shell; an end cap, which is arranged above the connecting piece and connected to the connecting piece, thereby limiting the communication between the closed cavity and the outside world through the connecting piece; a liquid connecting pipe is passed through the interior of the end cap and connected to the end cap; a gas connecting pipe is passed through the interior of the end cap and connected to the end cap; and a filter is arranged in the gas connecting pipe.

[0013] Furthermore, the pore size of the filter membrane is not greater than 0.22 μm, and the material of the filter membrane is any one of PP, PES, PTFE or NF.

[0014] Furthermore, the gas exchange component includes: a support net, arranged above the base and connected to the base; a sealing ring, arranged above the support net and connected to the support net; a second breathable membrane, arranged between the support net and the sealing ring, and connected to both the support net and the sealing ring.

[0015] Furthermore, the second breathable membrane is a hydrophobic breathable membrane, thereby allowing the gas required for cell growth to pass through and being able to block the flow of culture medium. The second breathable membrane is made of silicone.

[0016] Furthermore, the second liquid exchange component includes: a discharge port, which is provided on the side wall of the reactor shell and communicates with the closed cavity; and a discharge pipe, which is connected to the discharge port.

[0017] Optionally, the culture device further comprises a first gas permeable membrane, which is disposed above the reactor shell and communicates with the sealed cavity.

[0018] Optionally, a partition assembly is arranged inside the reactor shell and connected to the inner wall of the reactor shell, thereby dividing the closed cavity into a first chamber and a second chamber, and the first chamber is arranged above the second chamber; the volume ratio of the first chamber to the second chamber is 9:1.

[0019] Furthermore, the separation component includes: a card slot, which is arranged on the circumference of the inner wall of the reactor shell and is connected to the inner wall of the reactor shell; a card plate, the edge of which is arranged inside the card slot and is connected to the card slot, thereby dividing the cavity inside the reactor shell into the first chamber and the second chamber; an exchange port, which is passed through the card plate, and the first chamber and the second chamber are connected through the exchange port; a semipermeable membrane, which is arranged on the card plate and connected to the card plate, and the semipermeable membrane allows the permeation of substances with a molecular weight of less than 10kDa.

[0020] The present invention also provides a cell culture method using the highly permeable closed cell culture device. The specific steps of the method include:

[0021] S1: add culture medium;

[0022] adding culture medium into the sealed cavity of the reactor shell through the liquid connecting tube;

[0023] S2: seeded cells;

[0024] Then, the cells to be cultured are added into the sealed cavity of the reactor shell through the liquid connecting tube;

[0025] S3: cultured cells;

[0026] Keep the gas exchange components unobstructed throughout the cell culture process to ensure the required gases during cell growth, and place the cells in the incubator for culture. During the culture process, samples can be taken through the liquid connecting tube to monitor changes in nutrients in the culture medium and add missing nutrients.

[0027] S4: draining the culture medium;

[0028] Connect the liquid connecting tube to the waste liquid bottle, and use the peristaltic pump to discharge the waste culture medium in the closed cavity. During the discharge process, the external air passes through the filter to filter out pollutants and then enters the closed cavity, thereby ensuring the pressure balance inside the device;

[0029] S5: Collect cells;

[0030] The cells are collected by the second fluid exchange assembly.

[0031] The present invention also provides another cell culture method, which uses the highly permeable cell closed culture device, and the specific steps of the method include:

[0032] S1: seeded cells;

[0033] After diluting the desired cells with 10% of the volume of the sealed chamber using a culture medium, the cells are added to the second chamber of the reactor housing through a second liquid exchange component;

[0034] S2: add culture medium;

[0035] adding culture medium into the first chamber through the liquid connecting tube, wherein the added culture medium occupies 90% of the volume of the closed chamber;

[0036] S3: cultured cells;

[0037] During the whole process of culturing cells, the first gas permeable membrane and the gas exchange component are kept unobstructed to ensure the required gas during cell growth, and the cells are placed in an incubator for cultivation;

[0038] S4: Change the culture medium;

[0039] When the culture medium needs to be replaced, the waste culture medium is sucked out through the liquid connecting tube. During the suction process, the contaminants are filtered out by the filter and then enter the closed cavity, thereby ensuring the pressure balance inside the device. After the suction is completed, fresh culture medium is input through the liquid connecting tube for cell growth.

[0040] S5: collect concentrated cells;

[0041] Repeat step S4 multiple times to obtain multiple batches of mixed cultured cells, cell secretion products and culture medium in the second chamber, that is, to obtain concentrated cells; connect the first liquid exchange component to the waste liquid bottle to discharge the waste culture medium in the first chamber; mix the cultured cells, cell secretion products and culture medium in the second chamber and suspend them in the culture medium, then open the second liquid exchange component, and transfer and collect the cultured cells, cell secretion products and culture medium through the second liquid exchange component. Beneficial effects

[0042] The highly permeable closed cell culture device and method of the present invention solve the problem in the prior art that the extraction of culture medium causes the bottom silicone breathable membrane to bulge due to negative pressure, and the problem that the discharge of culture medium by positive pressure causes the equipment to be complicated, and has practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG1 is an exploded view of the device of the present invention;

[0044] FIG2 is a diagram of the device of the present invention after assembly;

[0045] FIG3 is a diagram showing the internal structure of the device of the present invention;

[0046] FIG4 is another internal structure diagram of the device of the present invention;

[0047] Explanation of the accompanying drawings: 1. First breathable membrane; 2. Gas exchange assembly; 2-1. Sealing ring; 2-2. Second breathable membrane; 2-3. Support net; 3. First liquid exchange assembly; 3-1. End cap; 3-2. Connecting piece; 3-3. Liquid connecting tube; 3-4. Gas connecting tube, 3-5. Filter, 4. Second liquid exchange assembly; 4-1. Discharge port; 4-2. Discharge pipe; 5. Base; 6. Partition assembly; 6-1. Card slot; 6-2. Card plate; 6-3. Exchange port; 6-4. Semipermeable membrane; 7. Reactor shell; 7-1. First chamber; 7-2. Second chamber. Best Mode for Carrying Out the Invention

[0048] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although exemplary embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present disclosure and to fully convey the scope of the present disclosure to those skilled in the art. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0049] As shown in Figures 1-3, this embodiment provides a highly permeable closed cell culture device, which includes: a base 5, a reactor shell 7, a gas exchange component 2, a first liquid exchange component 3, and a second liquid exchange component 4.

[0050] The reactor shell 7 is disposed above the base 5 and connected to the base 5 , thereby forming a closed space isolated from the external air.

[0051] As shown in Figure 1, the gas exchange assembly 2 is arranged above the base 5 and is connected to the enclosed space or the second chamber 7-2. Specifically, the gas exchange assembly 2 includes: a support mesh 2-3, which is arranged above the base 5 and connected to the base 5; a sealing ring 2-1, which is arranged above the support mesh 2-3 and connected to the support mesh 2-3; and a second breathable membrane 2-2, which is arranged between the support mesh 2-3 and the sealing ring 2-1 and connected to both the support mesh 2-3 and the sealing ring 2-1. The second breathable membrane 2-2 is a hydrophobic breathable membrane. Cells can directly obtain the gas required for cell growth from the second breathable membrane 2-2 at the bottom, thereby increasing the gas transfer rate. The second breathable membrane 2-2 can also block the flow of culture medium. The second breathable membrane 2-2 is made of silicone. During static suspension culture, cells will naturally settle on the second breathable membrane 2-2.

[0052] As shown in Figures 1-3, the first liquid exchange component 3 is arranged above the reactor shell 7, adjacent to the first gas permeable membrane 1, and connected to the reactor shell 7 to realize the delivery and / or extraction of culture medium.

[0053] The first fluid exchange component 3 includes:

[0054] The connecting piece 3-2 is provided at the top of the reactor shell 7 and connected to the reactor shell 7; the top of the connecting piece 3-2 is open, and the outer periphery of the open end is provided with a thread, which is connected to the end cap 3-1 through the thread;

[0055] The end cap 3-1 is arranged above the connecting piece 3-2 and is threadedly connected to the connecting piece 3-2, thereby limiting the closed cavity from being connected to the outside through the connecting piece 3-2; a rubber pad is provided between the end cap 3-1 and the connecting piece 3-2 to ensure the sealing effect.

[0056] The liquid communication tube 3-3 is provided inside the end cap 3-1 and connected to the end cap 3-1, so that the closed cavity is connected to the outside through the communication tube 3-3;

[0057] The gas communication tube 3-4 is provided inside the end cap 3-1, is provided adjacent to the liquid communication tube 3-3, and is connected to the end cap 3-1;

[0058] The liquid connecting tube 3-3 is inserted into and connected to the connecting piece 3-2, connecting the first chamber 7-1 to an external waste culture medium recovery device or a fresh culture medium storage device via the liquid connecting tube 3-3. A peristaltic pump is provided in the connecting pipe to provide suction pressure. The peristaltic pump generates much less pressure than the air pump described in patent CN105392876A and does not damage the interior of the reactor. The liquid connecting tube 3-3 is sealed and closed when not in use. The liquid connecting tube 3-3 can be provided as one, two, or more liquid connecting tubes. When provided as one, culture medium is introduced and withdrawn through one liquid connecting tube 3-3; when provided as two, culture medium can be introduced through one liquid connecting tube 3-3 and withdrawn through the other liquid connecting tube 3-3. One end of the liquid connecting tube 3-3 extends through the end cap 3-1 to facilitate connection to the peristaltic pump's piping, while the other end extends to a height of 10% of the volume of the reactor shell 7.

[0059] The gas connecting tube 3-4 is passed through the interior of the end cap 3-1, is arranged adjacent to the liquid connecting tube 3-3, and is connected to the end cap 3-1; the filter 3-5 is arranged outside the reactor shell 7 and is connected to the gas connecting tube 3-4.

[0060] The filter 3-5 is arranged outside the reactor shell 7 and connected to the gas communication pipe 3-4. The filter 3-5 is provided with a filter membrane, which is built into the filter 3-5 in a folded or stacked manner. The gas permeation rate of the filter 3-5 is 5-30 L / min, and the membrane area of ​​the filter membrane is greater than 2700 mm 2 The pore size of the filter membrane is 0.22 μm, and the material of the filter membrane is any one of PP, PES, PTFE, or NF. When the culture medium or cells are discharged by suction, the external air provides sterile air to the interior of the closed cavity through filters 3-5, thereby balancing the pressure inside the closed cavity.

[0061] Traditional needle filters are of the diaphragm type, with a small filter area and a low air permeability, which can easily lead to negative pressure in the cell culture flask, making liquid transfer difficult and causing the bottom air permeable membrane to bulge. This device uses an external high-permeability filter 3-5. The filter 3-5 has a bacteria-blocking and breathable function, and uses a high-area folded or stacked multi-layer filter membrane structure internally. Since the folded or stacked multi-layer filter membrane has a large membrane area and a large porosity per unit area, the increase in the permeability is not only due to the increase in membrane area, but also the doubling of the number of pores per unit area. Therefore, the gas permeability has a huge advantage over the traditional diaphragm type. The gas permeability of filter 3-5 in this device can reach 5-30L / min, while the maximum gas permeability of the traditional diaphragm filter is only 0.05L / min. Comparison in terms of area: the area of ​​a 25mm diameter diaphragm is 490mm 2 , the membrane area of ​​3-5 filters with the same diameter can reach 2732mm 2 -16200mm 2 During the cell collection process, the device uses a filter 3-5 to greatly improve the instantaneous gas permeability. When the culture medium or cells are discharged by suction, the filter 3-5 quickly provides sterile air to reduce the negative pressure in the bottle caused by the suction of the culture medium, and maintain the pressure balance in the reactor. When the culture medium is sucked, the second gas permeable membrane 2-2 (silicone gas permeable membrane) at the bottom will not bulge due to negative pressure, which will cause cell loss during suction, and prevent the rupture of the gas permeable membrane from causing problems such as leakage of culture medium and contamination. At the same time, the device does not need to be connected to an air pump, and a peristaltic pump can be used directly to pump out the liquid. Moreover, due to the increase in the air permeability area of ​​the filter 3-5, the liquid is discharged at a faster speed, which shortens the operation time. The air directly enters the culture bottle through the filter 3-5, which is relatively simple and convenient to use.

[0062] As shown in FIG1 , the second liquid exchange component 4 is disposed on the side wall of the reactor shell 7 and is in communication with the sealed cavity to enable the introduction and / or discharge of cells.

[0063] Specifically, the second liquid exchange assembly 4 comprises: a discharge port 4-1, located on the sidewall of the reactor housing 7 and communicating with the sealed chamber of the reactor housing 7; and a discharge tube 4-2, located at and connected to the discharge port 4-1. This tube can be connected to a collection bottle to collect cells. The distance from the discharge port 4-1 to the bottom is one-tenth of the height of the effective volume bottle. The discharge tube 4-2 is sealed and closed when not in use.

[0064] A first breathable membrane 1 may also be provided above the reactor shell 7. The breathable membrane 1 is made of PTFE material and is used to increase the gas exchange efficiency during the culture process.

[0065] Preferably, as shown in FIG4 , the device may also be provided with a partition assembly 6 , which is provided inside the reactor shell 7 and connected to the inner side wall of the reactor shell 7 , so that the interior of the reactor shell 7 is divided into a first chamber 7 - 1 and a second chamber 7 - 2 , and the first chamber 7 - 1 is provided above the second chamber 7 - 2 ; the partition assembly 6 provided in this embodiment can prevent cell disturbance caused during the discharge of waste culture medium, and avoid the loss of cells caused by the discharge of waste culture medium; thereby achieving effective separation of culture medium and cultured cells, and being able to discharge waste culture medium and add fresh culture medium in a closed space without affecting cell growth. Fresh culture medium can be added under sterile conditions to ensure long-term uninterrupted culture of cells.

[0066] Furthermore, the partition assembly 6 includes: a slot 6-1, disposed circumferentially along the inner wall of the reactor shell 7 and connected thereto; a plate 6-2, the edge of which is disposed within the slot 6-1 and connected thereto, thereby dividing the interior of the reactor shell 7 into a first chamber 7-1 and a second chamber 7-2. The plate 6-2 can be configured as a single layer or multiple layers; the plate 6-2 can be configured as an annular structure or a mesh structure. An exchange port 6-3 is provided through the plate 6-2, connecting the first chamber 7-1 and the second chamber 7-2. When the plate 6-2 is configured as an annular structure, the middle region of the annular structure serves as the exchange port 6-3; when the plate 6-2 is configured as a mesh structure, the mesh serves as the exchange port 6-3. The area of ​​the exchange port 6-3 should meet the exchange efficiency of the culture medium and should be as large as possible.

[0067] Semipermeable membrane 6-4, when the card board 6-2 is configured as a single layer, the semipermeable membrane 6-4 is fixedly mounted on the card board 6-2 and connected to the card board 6-2. When the card board 6-2 is configured as two or more layers, the semipermeable membrane 6-4 is clamped and fixed between the two or more layers of card boards 6-2. The semipermeable membrane 6-4 of the partition assembly 6 allows nutrients with a molecular weight less than 10kDa to permeate from the first chamber 7-1 to the second chamber 7-2, and metabolic inhibitors to permeate from the second chamber 7-2 to the first chamber 7-1, while ensuring that all target proteins and cells remain in the second chamber 7-2. That is, the semipermeable membrane 6-4 can block cells and antibodies and proteins with larger molecular weights, but the culture medium can be freely exchanged. Preferably, the volume ratio of the first chamber 7-1 to the second chamber 7-2 is 9:1. That is, a partition assembly 6 is set at a position 10% above the bottom of the culture bottle. Since the cells will naturally settle to the bottom of the device during static suspension culture, the first chamber 7-1 is filled with culture medium, providing rich culture medium and nutrient supply; the second chamber 7-2 is filled with 10% volume culture medium and cultured cells, which can form a high-density cell culture area and concentrate the produced antibodies and proteins.

[0068] Another aspect of the present invention provides a method for a highly permeable cell enclosed culture device, which, when the partition component 6 is not provided, can be applied to cell gene therapy operations. The specific steps of the method include:

[0069] S1: add culture medium;

[0070] Adding culture medium into the closed cavity of the reactor shell 7 through the liquid connecting pipe 3-3, wherein the added culture medium occupies 90% of the volume of the closed cavity;

[0071] S2: seeded cells;

[0072] Then, the required cells are diluted with 10% of the volume of the closed cavity culture medium and added into the closed cavity of the reactor shell 7 through the liquid connecting tube 3-3;

[0073] S3: cultured cells;

[0074] During the whole process of culturing cells, the gas exchange component 2 is kept unobstructed to ensure the required gas during cell growth, and the cells are placed in an incubator for cultivation. During the cultivation process, samples can be taken through the liquid connecting tube 3-3 to monitor the changes in the nutrient components in the culture medium and add the missing nutrients.

[0075] S4: draining the culture medium;

[0076] Connect the liquid connecting tube 3-3 to the waste liquid bottle, and use the peristaltic pump to discharge the waste culture medium in the closed cavity. During the discharge process, the external air passes through the filter 3-5 to filter out pollutants and then enters the closed cavity, thereby quickly ensuring the pressure balance inside the device;

[0077] S5: Collect cells;

[0078] The cells are collected by the second fluid exchange component 4 .

[0079] When provided with a partition component 6, the device is not only suitable for operations in the direction of cell gene therapy, but is also suitable for related operations such as concentrating and collecting antibody protein. The second breathable membrane 2-2 and the intermediate semipermeable membrane 6-4 in the device enable the product to break through the culture height limit of traditional culture plates, square bottles, and culture bags. The static culture height of traditional culture plates and square bottles is about 0.3 cm, while the height of culture bags is about 1.0 cm. If the height exceeds the limit, the oxygen and nutrient supply in the culture device will be greatly restricted, and it will not be possible to ensure sufficient supply of oxygen and nutrients required for cell culture. At the same time, the waste gas generated by cell growth cannot be discharged, which ultimately leads to a sharp decrease in cell growth viability and yield. The culture height of the device can reach about 10 cm.

[0080] When culturing cells to produce proteins or antibodies, traditional culture plates and square bottles are generally limited to only about 3-5 days of static culture due to their volume limitations and limited nutrient supply of the culture medium. However, the present device breaks through the culture height limit of traditional culture containers. The second gas permeable membrane 2-2 at the bottom can balance oxygen and carbon dioxide. The semi-permeable membrane 6-4 has an allowable molecular weight of less than 10kDa, which is used to exchange fresh culture medium and culture metabolites with the second cell culture chamber 7-2, while ensuring that all proteins remain in the second chamber 7-2. Users can extend cell culture time to about 10-14 days based on experimental design, greatly extending the cell culture time and producing sufficient proteins and antibodies. Traditional culture containers collect cells and antibodies and proteins, but ultimately only collect all the culture medium, cells, proteins, and antibodies together. However, the present device is equipped with a semi-permeable membrane 6-4 of approximately 10kDa, which can concentrate the produced antibodies and proteins in the second chamber 7-2 of the membrane. Collection requires only about 1 / 10 of the volume of traditional cell culture methods, greatly reducing downstream work content.

[0081] The specific steps of this method include:

[0082] S1: seeded cells;

[0083] After diluting the desired cells with 10% of the volume of the sealed chamber with culture medium, the cells are added to the second chamber 7 - 2 of the reactor housing 7 through the second liquid exchange component 4 ;

[0084] S2: add culture medium;

[0085] Adding culture medium into the first chamber 7-1 through the liquid connecting tube 3-3, wherein the added culture medium occupies 90% of the volume of the closed chamber;

[0086] S3: cultured cells;

[0087] During the whole process of culturing cells, the first gas permeable membrane and the gas exchange component 2 are kept unobstructed to ensure the required gas during the cell growth process, and the cells are placed in an incubator for cultivation;

[0088] S4: Change the culture medium;

[0089] When the culture medium needs to be replaced, the waste culture medium is sucked out through the liquid connecting tube 3-3. During the suction process, the contaminants are filtered out by the filter 3-5 and then enter the closed cavity, thereby quickly ensuring the pressure balance inside the device. After the suction is completed, fresh culture medium is input through the liquid connecting tube 3-3 for cell growth.

[0090] S5: collect concentrated cells;

[0091] Repeat step S4 multiple times to obtain multiple batches of mixed cultured cells, cell secretion products and culture medium in the second chamber 7-2, that is, concentrated cells; connect the first liquid exchange component 3 to the waste liquid bottle to discharge the waste culture medium in the first chamber 7-1; mix the cultured cells, cell secretion products and culture medium in the second chamber 7-2 and suspend them in the culture medium, then open the second liquid exchange component, and transfer and collect the cultured cells, cell secretion products and culture medium through the second liquid exchange component 4.

[0092] This device enables high-density cell culture with adequate nutrient supply and long culture time. The antibodies and proteins produced by cell culture using this device are much higher than those produced by traditional culture methods. For example, the concentration of antibodies produced by hybridomas is around 1-5 mg / mL, which is 50-100 times higher than that of traditional culture methods. Modes for Carrying Out the Invention

[0093] When the separation component 6 is not provided, the method is applicable to cell gene therapy operations. The specific steps of the method include:

[0094] S1: add culture medium;

[0095] Adding culture medium into the closed cavity of the reactor shell 7 through the liquid connecting pipe 3-3, wherein the added culture medium occupies 90% of the volume of the closed cavity;

[0096] S2: seeded cells;

[0097] Then, the required cells are diluted with 10% of the volume of the closed cavity culture medium and added into the closed cavity of the reactor shell 7 through the liquid connecting tube 3-3;

[0098] S3: cultured cells;

[0099] During the whole process of culturing cells, the gas exchange component 2 is kept unobstructed to ensure the required gas during cell growth, and the cells are placed in an incubator for cultivation. During the cultivation process, samples can be taken through the liquid connecting tube 3-3 to monitor the changes in the nutrient components in the culture medium and add the missing nutrients.

[0100] S4: draining the culture medium;

[0101] Connect the liquid connecting tube 3-3 to the waste liquid bottle, and use the peristaltic pump to discharge the waste culture medium in the closed cavity. During the discharge process, the external air passes through the filter 3-5 to filter out pollutants and then enters the closed cavity, thereby quickly ensuring the pressure balance inside the device;

[0102] S5: Collect cells;

[0103] The cells are collected by the second fluid exchange component 4 .

[0104] When provided with a partition component 6, the device is not only suitable for operations in the direction of cell gene therapy, but is also suitable for related operations such as concentrating and collecting antibody protein. The second breathable membrane 2-2 and the intermediate semipermeable membrane 6-4 in the device enable the product to break through the culture height limit of traditional culture plates, square bottles, and culture bags. The static culture height of traditional culture plates and square bottles is about 0.3 cm, while the height of culture bags is about 1.0 cm. If the height exceeds the limit, the oxygen and nutrient supply in the culture device will be greatly restricted, and it will not be possible to ensure sufficient supply of oxygen and nutrients required for cell culture. At the same time, the waste gas generated by cell growth cannot be discharged, which ultimately leads to a sharp decrease in cell growth viability and yield. The culture height of the device can reach about 10 cm.

[0105] When culturing cells to produce proteins or antibodies, traditional culture plates and square bottles are generally limited to only about 3-5 days of static culture due to their volume limitations and limited nutrient supply of the culture medium. However, the present device breaks through the culture height limit of traditional culture containers. The second gas permeable membrane 2-2 at the bottom can balance oxygen and carbon dioxide. The semi-permeable membrane 6-4 has an allowable molecular weight of less than 10kDa, which is used to exchange fresh culture medium and culture metabolites with the second cell culture chamber 7-2, while ensuring that all proteins remain in the second chamber 7-2. Users can extend cell culture time to about 10-14 days based on experimental design, greatly extending the cell culture time and producing sufficient proteins and antibodies. Traditional culture containers collect cells and antibodies and proteins, but ultimately only collect all the culture medium, cells, proteins, and antibodies together. However, the present device is equipped with a semi-permeable membrane 6-4 of approximately 10kDa, which can concentrate the produced antibodies and proteins in the second chamber 7-2 of the membrane. Collection requires only about 1 / 10 of the volume of traditional cell culture methods, greatly reducing downstream work content.

[0106] The specific steps of this method include:

[0107] S1: seeded cells;

[0108] After diluting the desired cells with 10% of the volume of the sealed chamber with culture medium, the cells are added to the second chamber 7 - 2 of the reactor housing 7 through the second liquid exchange component 4 ;

[0109] S2: add culture medium;

[0110] Adding culture medium into the first chamber 7-1 through the liquid connecting tube 3-3, wherein the added culture medium occupies 90% of the volume of the closed chamber;

[0111] S3: cultured cells;

[0112] During the whole process of culturing cells, the first gas permeable membrane and the gas exchange component 2 are kept unobstructed to ensure the required gas during the cell growth process, and the cells are placed in an incubator for cultivation;

[0113] S4: Change the culture medium;

[0114] When the culture medium needs to be replaced, the waste culture medium is sucked out through the liquid connecting tube 3-3. During the suction process, the contaminants are filtered out by the filter 3-5 and then enter the closed cavity, thereby quickly ensuring the pressure balance inside the device. After the suction is completed, fresh culture medium is input through the liquid connecting tube 3-3 for cell growth.

[0115] S5: collect concentrated cells;

[0116] Repeat step S4 multiple times to obtain multiple batches of mixed cultured cells, cell secretion products and culture medium in the second chamber 7-2, that is, concentrated cells; connect the first liquid exchange component 3 to the waste liquid bottle to discharge the waste culture medium in the first chamber 7-1; mix the cultured cells, cell secretion products and culture medium in the second chamber 7-2 and suspend them in the culture medium, then open the second liquid exchange component, and transfer and collect the cultured cells, cell secretion products and culture medium through the second liquid exchange component 4. Industrial Applicability

[0117] The present invention provides a highly permeable closed cell culture device and method. The present invention uses an externally connected highly permeable filter and a high-area folded or stacked multi-layer filter membrane structure inside the filter. Since the folded or stacked multi-layer filter membrane not only increases the membrane area, but also multiplies the number of pores per unit area, the gas permeation rate has a huge advantage over the traditional membrane type, greatly improving the instantaneous gas permeability and reducing the negative pressure in the bottle caused by the suction of the culture medium. When the culture medium is suctioned, the bottom silicone breathable membrane will not bulge due to the negative pressure. At the same time, compared with the positive pressure type device for discharging the culture medium, the present invention does not need to be connected to an air pump, and can directly use a peristaltic pump to pump out the liquid. Moreover, since the filter balances the pressure in the enclosed space, the liquid is discharged at a higher speed, shortening the operation time. The air directly enters the enclosed culture space through the filter, which is relatively simple and convenient to use.

[0118] The present invention is also provided with a partition component, which can further prevent cell disturbance caused by the discharge of waste culture medium, and avoid the discharge of cells with the waste culture medium, resulting in loss; thereby achieving effective separation of culture medium and cultured cells, and can discharge waste culture medium and add fresh culture medium in a closed space without affecting cell growth, ensuring long-term uninterrupted cell culture; and forming a high-density cell culture area, while obtaining concentrated antibodies and proteins, etc.

[0119] This device enables high-density cell culture with adequate nutrient supply and long culture time. The antibodies and proteins produced by cell culture using this device are much higher than those produced by traditional culture methods. For example, the concentration of antibodies produced by hybridomas is around 1-5 mg / mL, which is 50-100 times higher than that of traditional culture methods.

Claims

1. A highly permeable cell closed culture device, characterized in that: include: Base (5); A reactor shell (7) is disposed above the base (5) and connected to the base (5) to form a closed cavity; A gas exchange component (2) is disposed above the base (5) and is in communication with the sealed cavity; A first liquid exchange component (3) is disposed above the reactor shell (7) and connected to the reactor shell (7); A second liquid exchange component (4) is disposed on the side wall of the reactor shell (7) and is in communication with the sealed cavity; The first liquid exchange component (3) is provided with a filter (3-5), the filter (3-5) is provided with a filter membrane, and the filter membrane is built into the filter (3-5) in a folded or stacked manner; the gas permeation rate of the filter (3-5) is 5-30 L / min, and the membrane area of ​​the filter membrane is greater than 2700 mm 2 .

2. A highly air-permeable cell-enclosed culture device according to claim 1, characterized in that: The first fluid exchange component (3) further comprises: A connecting piece (3-2) is arranged at the top end of the reactor shell (7) and connected to the reactor shell (7); An end cap (3-1) is disposed above the connecting piece (3-2) and connected to the connecting piece (3-2), thereby limiting the closed cavity from being connected to the outside through the connecting piece (3-2); The liquid communication tube (3-3) is passed through the interior of the end cap (3-1) and is connected to the end cap (3-1); The gas communication tube (3-4) is passed through the interior of the end cap (3-1) and is connected to the end cap (3-1); a filter (3-5) is arranged in the gas communication tube (3-4).

3. A highly permeable cell-enclosed culture device according to claim 1, characterized in that: The pore size of the filter membrane is no greater than 0.22 μm, and the material of the filter membrane is any one of PP, PES, PTFE or NF.

4. The highly permeable cell-enclosed culture device according to claim 1, characterized in that: The gas exchange component (2) comprises: A supporting net (2-3) is arranged above the base (5) and connected to the base (5); A sealing ring (2-1) is arranged above the supporting net (2-3) and connected to the supporting net (2-3); The second breathable membrane (2-2) is arranged between the supporting net (2-3) and the sealing ring (2-1), and is connected to both the supporting net (2-3) and the sealing ring (2-1).

5. A highly air-permeable cell-enclosed culture device according to claim 4, characterized in that: The second gas permeable membrane (2-2) is a hydrophobic gas permeable membrane, thereby allowing the gas required for cell growth to pass through and being able to block the flow of culture medium. The second gas permeable membrane (2-2) is made of silica gel.

6. The highly air-permeable cell-enclosed culture device according to claim 1, characterized in that: The second fluid exchange component (4) comprises: An outlet (4-1) is arranged on the side wall of the reactor shell (7) and is connected to the sealed cavity; The discharge pipe (4-2) is arranged at the discharge outlet (4-1) and connected to the discharge outlet (4-1).

7. The highly permeable cell enclosed culture device according to claim 1, characterized in that: The culture device further comprises a first gas permeable membrane (1), wherein the first gas permeable membrane (1) is arranged above the reactor shell (7) and is in communication with the closed cavity.

8. The highly air-permeable cell-enclosed culture device according to claim 1, characterized in that: include: A partition assembly (6) is arranged inside the reactor shell (7) and connected to the inner wall of the reactor shell (7), thereby dividing the closed cavity into a first chamber (7-1) and a second chamber (7-2), and the first chamber (7-1) is arranged above the second chamber (7-2); the volume ratio of the first chamber (7-1) to the second chamber (7-2) is 9:

1.

9. The highly permeable cell-enclosed culture device according to claim 8, characterized in that: The partition component (6) comprises: A card slot (6-1) is arranged on the circumference of the inner wall of the reactor shell (7) and is connected to the inner wall of the reactor shell (7); a clamping plate (6-2), the edge of which is arranged inside the clamping slot (6-1) and connected to the clamping slot (6-1), thereby dividing the cavity inside the reactor shell (7) into the first chamber (7-1) and the second chamber (7-2); An exchange port (6-3) is provided through the card plate (6-2), and the first chamber (7-1) and the second chamber (7-2) are connected through the exchange port (6-3); A semipermeable membrane (6-4) is disposed on the cardboard (6-2) and connected to the cardboard (6-2). The semipermeable membrane (6-4) Allows permeation of substances with a molecular weight less than 10 kDa.

10. A cell culture method, using the highly gas permeable cell closed culture device according to claim 1, characterized in that: The specific steps of this method include: S1: add culture medium; adding culture medium into the sealed cavity of the reactor shell (7) through the liquid connecting tube (3-3); S2: seeded cells; Then, the cells to be cultured are added into the sealed cavity of the reactor shell (7) through the liquid connecting tube (3-3); S3: cultured cells; During the whole process of culturing cells, the gas exchange component (2) is kept unobstructed to ensure the required gas during the cell growth process, and the cells are placed in an incubator for culturing; during the culturing process, samples can be taken through the liquid connecting tube (3-3) to monitor the changes in the nutrient components in the culture medium and add the missing nutrient components; S4: draining the culture medium; The liquid connecting tube (3-3) is connected to the waste liquid bottle, and the waste culture medium in the closed cavity is discharged by a peristaltic pump. During the discharge process, the external air passes through the filter (3-5) to filter out pollutants and then enters the closed cavity, thereby ensuring the pressure balance inside the device; S5: Collect cells; The cells are collected by the second fluid exchange component (4).

11. A cell culture method, using the highly gas permeable cell closed culture device according to claim 8, characterized in that: The specific steps of this method include: S1: seeded cells; After diluting the desired cells with 10% of the volume of the closed chamber culture medium, the cells are added to the second chamber (7-2) of the reactor shell (7) through the second liquid exchange component (4); S2: add culture medium; Adding culture medium into the first chamber (7-1) through the liquid connecting tube (3-3), wherein the added culture medium accounts for 90% of the volume of the closed chamber; S3: cultured cells; During the whole process of culturing cells, the first gas permeable membrane (1) and the gas exchange component (2) are kept unobstructed to ensure the necessary gas during the cell growth process, and the cells are placed in an incubator for culturing; S4: Change the culture medium; When the culture medium needs to be replaced, the waste culture medium is sucked out through the liquid connecting tube (3-3), and the pollutants are filtered out through the filter (3-5) during the suction process and then enter the closed cavity, thereby ensuring the pressure balance inside the device; after the suction is completed, fresh culture medium is input through the liquid connecting tube (3-3) for cell growth; S5: collect concentrated cells; Repeat step S4 multiple times to obtain multiple batches of mixed cultured cells, cell secretion products and culture medium in the second chamber (7-2), that is, to obtain concentrated cells; connect the first liquid exchange component (3) to the waste liquid bottle to discharge the waste culture medium in the first chamber (7-1); the cultured cells, cell secretion products and culture medium in the second chamber (7-2) are concentrated; The culture medium is mixed and suspended in the culture medium, and then the second liquid exchange component (4) is opened, and the cultured cells, cell secretion products and culture medium are transferred and collected through the second liquid exchange component (4).

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