Immune cell culture regulation device and culture method

Abstract

The application relates to the technical field of cell culture, in particular to an immune cell culture regulation device and a culture method, which comprise a device box, a ring-shaped cavity is arranged in the device box, a rotating disc is arranged in the ring-shaped cavity in a rotating mode, a plurality of containing cabins are arranged on the rotating disc in a circumferential interval, detachable culture bottles are adaptively installed in the containing cabins, and operation windows corresponding to the positions of the containing cabins are arranged on the device box; the culture bottles are uniformly rotated by the rotating disc, culture liquid in the culture bottles and immune cells reciprocally flow under the action of gravity, a dynamic fluid environment is formed, the problems of cell settlement and agglomeration in static culture are solved, the mass transfer efficiency of nutrients and oxygen is remarkably improved, local metabolic waste accumulation is avoided, the in-vivo physiological environment is closer, and the activity and proliferation efficiency of immune cells are effectively improved.

Description

Technical Field

[0001] This application relates to the field of cell culture technology, and in particular to an immune cell culture regulation device and culture method. Background Technology

[0002] Immune cell culture is a core biotechnology that simulates the in vivo microenvironment to isolate, expand, regulate the function of, and even genetically modify immune cells. The key to the culture process lies in constructing a highly controllable in vitro niche. This relies on well-defined, serum-free, and animal-derived culture media to mitigate the risk of exogenous contamination and ensure batch-to-batch stability.

[0003] More importantly, the precise regulation of cytokines allows for the targeted guidance of immune cells to differentiate into specific functional phenotypes by combining different cytokines, thus meeting various treatment or research needs.

[0004] For example, in a culture device for lymphoma immunotherapy with application number CN202511561446.6, during operation, the switching transmission disc at the bottom drives multiple culture boxes to rotate and shift. Through a unique cam slide rail design at the bottom, the culture boxes are controlled to automatically rise when they reach a specific station and automatically descend when they leave the station. When the culture box rises, the feed pipe at the top automatically inserts into the interface of the culture box, and at the same time, the mechanical structure drives the ball valve on the culture box to open, realizing the injection of nutrient solution or immune stimulating factors. When the culture box descends, the valve automatically closes and resets to a sealed state, ensuring that the culture environment instantly returns to a fully closed state.

[0005] The aforementioned existing technologies reduce the risk of contamination from manual operation and improve the standardization of culture by automating and sealing the culture of immune cells. However, the culture box is still relatively static during operation, while immune cells are in a dynamic circulating environment in the body. Static culture violates their physiological characteristics and is prone to cell sedimentation and aggregation, affecting their proliferative activity and function.

[0006] Meanwhile, the mass transfer efficiency is extremely low in static environments, the nutrients in the culture medium are unevenly distributed, and metabolic waste is prone to accumulate locally and cannot be discharged in time. This non-physiological microenvironment makes it difficult to maintain the stable conditions required for high-density culture, which ultimately severely limits the cell expansion efficiency and clinical-grade quality.

[0007] In light of the above discussion, existing immune cell culture techniques still have potential for improvement in certain aspects and require further optimization. Summary of the Invention

[0008] To address the aforementioned technical problems, this application provides an immune cell culture regulation device and culture method, employing the following technical solution: In a first aspect, an immune cell culture regulation device includes an equipment box, an annular chamber is constructed inside the equipment box, a turntable is rotatably arranged inside the annular chamber, a plurality of receiving chambers are arranged at intervals along the circumference on the turntable, a detachable culture bottle is adapted to be installed in the receiving chamber, and an operation window corresponding to the position of the receiving chamber is opened on the equipment box. The turntable is configured to drive the culture flask to rotate around the turntable axis, so that the culture medium in the culture flask and the immune cells flow and mix back and forth under the action of gravity, simulating the dynamic circulating environment of immune cells in vivo, thereby providing immune cells with in vitro culture conditions close to physiological conditions to maintain the biological activity and functional characteristics of immune cells. The culture flask includes a cylindrical body that can be detachably installed in a housing chamber. Both ends of the cylindrical body are provided with detachable culture dishes. The cylindrical body is provided with a circulation connection part that connects the two culture dishes. The circulation connection part is used to guide the culture medium to form a continuous circulation flow path between the two culture dishes during the rotation of the culture flask.

[0009] Preferably, the circulation connection includes symmetrically arranged collecting hoppers inside the cylinder, and a spiral tube assembly connects the two collecting hoppers.

[0010] Preferably, the spiral tube assembly includes multiple spiral tube layers arranged at intervals along the axial direction of the cylinder. Each spiral tube layer includes multiple spiral guide tubes evenly distributed in the circumferential direction. The spiral guide tubes extend in a spiral shape and connect between two collecting hoppers.

[0011] Preferably, a sealing ring is provided at one end of the collecting hopper, and the culture dish will contact the end of the collecting hopper with the sealing ring, forming a sealed culture cavity between the collecting hopper and the culture dish.

[0012] Preferably, the culture dish includes a dish base that can be detachably installed at both ends of the cylinder, an annular magnetic ring is provided on the inner side of the dish base, a detachable filter plate is magnetically attracted on the annular magnetic ring, and the upper cavity is used to hold immune cells and culture medium. The filter plate divides the dish into an upper chamber and a lower chamber. The filter plate allows waste products generated by immune cell metabolism to pass through and enter the lower chamber, while retaining immune cells and culture medium in the upper chamber to participate in circulation.

[0013] Preferably, the dish holder is provided with a sampling section, which is used to sample immune cells in the upper cavity; The sampling section includes a connector that passes through the dish base, with the upper end of the connector extending to the upper end of the filter plate. The connector has an inlet located at the upper end of the filter plate, and the bottom of the equipment box has a sampling port corresponding to the connector. A piston is installed inside the connector via a spring, and the piston blocks the liquid inlet.

[0014] Preferably, the piston has an outlet corresponding to the inlet cylinder, and the upper end of the piston has a protrusion corresponding to the inlet.

[0015] Preferably, the turntable is equipped with a latch, which is used to secure the culture flask within the containment chamber; The buckle includes guide grooves on the turntable that correspond one-to-one with the receiving chambers. A locking block is pressed into the guide groove by a spring. The locking block has a pin block located at the upper end of the culture bottle in the receiving chamber. The pin block is used to limit the position of the culture bottle.

[0016] Preferably, the pin block is provided with a guide slope.

[0017] Secondly, a method for culturing immune cells includes the following steps: S1: Culture preparation. The turntable rotates in the annular chamber, and the containment chamber passes through the vertical placement station corresponding to the working window in sequence, and the culture bottle is put into the containment chamber to provide a closed environment for culture. S2: During equipment installation, the squeeze pin guide slope is used to push the locking block down along the guide groove to compress the spring. After the culture bottle is fully in place, the spring rebounds to reset the locking block and the pin. The pin presses the upper end of the culture bottle to achieve axial limitation, ensuring that the culture bottle does not loosen or come out, and maintaining dynamic operation stability. S3: Circulating rotation, the turntable drives the culture flask to rotate in a circle, and the two end plates are constantly up and down as the position changes. The culture medium and immune cells are gathered through the collecting funnel under the action of gravity and enter the spiral tube group composed of multi-layer spiral tubes. The flow extends along the spiral path and forms a disturbance, and circulates back and forth between the two end culture plates. S4: Cell culture. The filter disc is fixed to the dish base by a ring magnetic ring, dividing the inner cavity into an upper cavity and a lower cavity. The upper cavity retains immune cells and culture medium to participate in circulation, while small molecule waste generated by metabolism passes through the filter disc and enters the lower cavity for collection under the action of gravity. S5: Sampling and replenishment. Turn the culture bottle to the vertical position, align the connector with the sampling port, use the sampling test tube to lift the piston, compress the spring and open the inlet, so that the liquid in the upper chamber flows into the test tube through the inlet and outlet. After sampling or replenishment, pull out the test tube, and the piston will return to its original position under the action of the spring.

[0018] In summary, this application includes at least one of the following beneficial technical effects: 1. This invention uses a turntable to rotate the culture flask, causing the culture medium and immune cells inside the flask to circulate repeatedly under gravity, forming a dynamic fluid environment. This completely solves the problems of cell sedimentation and aggregation in static culture, significantly improves the mass transfer efficiency of nutrients and oxygen, avoids the accumulation of local metabolic waste, and is closer to the physiological environment in the body, effectively improving the activity and proliferation efficiency of immune cells.

[0019] 2. The culture flask of this invention adopts a multi-seal structure with interference fit between the plate holders at both ends of the cylinder and the sealing ring of the collecting funnel. Combined with the built-in collecting funnel and the multi-layer spiral tube assembly, it forms a directional circulation flow path, which not only ensures that there is no leakage or pollution during the culture process, but also extends the flow path and generates gentle disturbance through the spiral structure, so as to protect the cells from mechanical damage while achieving efficient circulation of the culture medium.

[0020] 3. The present invention has a magnetic filter plate in the dish holder, which can selectively pass through metabolic waste and retain immune cells and culture medium, so as to realize the simultaneous separation of culture and waste, avoid waste backflow and contamination, significantly extend the culture cycle, improve the cleanliness of the culture system, and the magnetic structure is easy to disassemble, clean and replace, making it convenient to use. Attached Figure Description

[0021] Figure 1 This is a structural schematic diagram from the first perspective of the present invention.

[0022] Figure 2 This is a cross-sectional view of the equipment box of the present invention.

[0023] Figure 3 This is a schematic diagram of the structure of the turntable of the present invention.

[0024] Figure 4 This is a schematic diagram of the structure between the turntable and the culture flask of the present invention.

[0025] Figure 5 This is the present invention. Figure 4 Enlarged view of a portion of point A in the middle.

[0026] Figure 6 This is a schematic diagram of the structure of the culture flask of the present invention.

[0027] Figure 7 This is a cross-sectional view of the culture flask of the present invention.

[0028] Figure 8 This is a cross-sectional view of the loop connection part of the present invention.

[0029] Figure 9 This is a schematic diagram of the structure of the loop connection part of the present invention.

[0030] Figure 10 This is a schematic diagram of the structure of the culture dish.

[0031] Figure 11 This is a schematic diagram of the sampling section of the present invention.

[0032] Figure 12 This is a cross-sectional view of the sampling section of the present invention.

[0033] Figure 13 This is a schematic diagram of the piston structure of the present invention.

[0034] Explanation of reference numerals in the attached drawings: 1. Equipment box; 11. Operating window; 2. Annular chamber; 3. Turntable; 31. Receptacle; 4. Buckle; 41. Guide groove; 42. Locking block; 43. Pin block; 44. Guide slope; 5. Culture flask; 51. Cylinder; 52. Collector; 53. Circulation connection; 54. Spiral tube assembly; 55. Spiral tube layer; 56. Spiral conduit; 57. Sealing ring; 58. Culture chamber; 6. Culture dish; 61. Dish holder; 62. Annular magnetic ring; 63. Filter plate; 64. Upper chamber; 65. Lower chamber; 7. Sampling section; 71. Connector; 72. Liquid inlet; 73. Piston; 74. Liquid outlet; 75. Protrusion; 76. Sampling port. Detailed Implementation

[0035] The following is in conjunction with the appendix Figures 1 to 13 This application will be described in further detail.

[0036] This application discloses an immune cell culture regulation device and culture method. A portion of the wall material is extracted by drilling a hole in the wall, and then the compressive strength of the material is tested to determine the compressive strength of the building wall.

[0037] Example 1: Reference Figure 1 , Figure 2 , Figure 3 and Figure 4 The device shown is an immune cell culture regulation device, including a device box 1. The device box 1 has an annular chamber 2. A turntable 3 is rotatably arranged in the annular chamber 2. Multiple containment chambers 31 are arranged circumferentially on the turntable 3. The number of containment chambers 31 can be flexibly set according to the actual culture scale (4 are shown in the figure). A detachable culture bottle 5 is adapted to be installed in the containment chamber 31. The culture bottle 5 is made of transparent sterile material (such as high borosilicate glass or medical-grade PC material) to facilitate observation of the culture status of immune cells in the bottle.

[0038] The equipment box 1 has an operation window 11 corresponding to the position of the receiving chamber 31. The operation window 11 corresponds to only one vertical position of the culture bottle 5. That is, when the turntable 3 is rotated to the designated position, only one culture bottle 5 in the receiving chamber 31 is in a vertical position, and the position of the culture bottle 5 corresponds precisely to the position of the operation window 11. The operator can use the operation window 11 to pick up, put down, and replace the culture bottle 5 in the vertical position, as well as perform temporary observation and sampling during the culture process.

[0039] The rotating configuration of the turntable 3 drives the culture flask 5 to rotate around the axis of the turntable 3, so that the culture medium and immune cells in the culture flask 5 flow back and forth and mix under the action of gravity. When the culture flask 5 rotates with the turntable 3 to the upper part of the annular chamber 2, the culture medium flows to the bottom of the culture flask 5 under the action of gravity; when the culture flask 5 rotates to the lower part of the annular chamber 2, the culture medium flows to the upper part of the culture flask 5 under the action of gravity. This cycle repeats to form a continuous dynamic fluid environment, simulating the dynamic circulating environment of immune cells in vivo, thereby providing immune cells with in vitro culture conditions close to physiological conditions to maintain the biological activity and functional characteristics of immune cells.

[0040] Reference Figure 3 , Figure 4 and Figure 5 As shown, the turntable 3 is driven by a drive mechanism to achieve controllable rotation. Its rotation mode is configured to rotate slowly and uniformly in the same direction, thereby driving the culture bottle 5 installed in the housing 31 to make circular motion around the axis of the turntable 3, and at the same time driving the culture bottle 5 to rotate synchronously with the turntable 3.

[0041] As the culture flask 5 rotates around the axis of the turntable 3, the culture medium and immune cells inside the flask flow and mix repeatedly under the action of gravity. When the culture flask 5 rotates with the turntable 3 to the upper part of the annular chamber 2, the culture medium flows to the bottom of the culture flask 5 under the action of gravity; when the culture flask 5 rotates to the lower part of the annular chamber 2, the culture medium flows to the upper part of the culture flask 5 under the action of gravity. This cycle repeats, forming a continuous dynamic fluid environment that accurately simulates the dynamic circulating physiological environment of immune cells in the body.

[0042] This technology effectively solves the problems of cell sedimentation and aggregation caused by static culture in existing technologies, keeping immune cells in a dispersed and suspended state at all times, promoting full contact between cells and culture medium, nutrients, and oxygen. At the same time, the reciprocating flow of culture medium can improve mass transfer efficiency, avoid uneven distribution of nutrients and local accumulation of metabolic waste, and provide immune cells with in vitro culture conditions that are close to the physiological state in vivo. This effectively maintains the biological activity and functional characteristics of immune cells, and improves the proliferation efficiency and clinical-grade quality of immune cells.

[0043] Reference Figure 5 and Figure 6 As shown, the culture flask 5 is made of transparent and sterile material (such as high borosilicate glass and medical-grade PC material), which makes it easy to observe the culture status of immune cells and the flow of culture medium inside the flask. The whole flask can be detached and installed in the housing 31 of the turntable 3 to achieve stable installation and easy to pick up, put down and replace.

[0044] The turntable 3 is equipped with a latch 4, which is used to restrict the culture flask 5 within the housing chamber 31. This ensures that the culture flask 5 will not fall off or shift from the housing chamber 31 due to centrifugal force, shaking, or other factors when it rotates in a circular motion driven by the turntable 3, thus ensuring the installation stability of the culture flask 5 and ensuring the smooth progress of dynamic circulation culture of immune cells.

[0045] Specifically, the buckle 4 includes a guide groove 41 on the turntable 3 that corresponds one-to-one with the receiving chamber 31. A locking block 42 is provided in the guide groove 41 by a spring sliding. A pin block 43 is constructed on the locking block 42 and located at the upper end of the culture bottle 5 in the receiving chamber 31. The pin block 43 is used to limit the culture bottle 5.

[0046] Under normal conditions, the spring is in a naturally extended state (or slightly compressed state), applying an upward elastic pressure to the locking block 42, pushing the locking block 42 to slide upward along the guide groove 41. At this time, the pin 43 on the locking block 42 moves upward simultaneously, extending into the interior of the receiving compartment 31, and the lower end face of the pin 43 is above the opening of the receiving compartment 31. The pin block 43 is provided with a guide slope 44. When the operator aligns the culture bottle 5 with the opening of the receiving chamber 31 and inserts it downward into the receiving chamber 31, the upper edge of the culture bottle 5 will first contact the guide slope 44 on the pin block 43. Since the guide slope 44 has an inclined angle, the pressure of the culture bottle 5 being inserted downward will be decomposed into a lateral component along the slope. This component pushes the locking block 42 to overcome the elastic pressure of the spring and slide downward along the guide groove 41. At the same time, the spring is compressed and stores energy.

[0047] As the culture flask 5 continues to be inserted downwards until it is completely flush with the inner wall of the receiving chamber 31 (reaching the preset installation position), the upper edge of the culture flask 5 passes the guide slope 44 of the pin block 43, and no longer applies lateral pressure to the pin block 43. Under the action of its own elastic restoring force, the spring pushes the locking block 42 to return to its original position along the guide groove 41. The pin block 43 on the locking block 42 moves upwards simultaneously, and finally the lower end face of the pin block 43 is tightly flush with the upper end face of the culture flask 5, forming a limit on the culture flask 5 from above, locking the culture flask 5 in the receiving chamber 31, and completing the installation and fixing. At this time, the pin block 43 and the inner wall of the receiving chamber 31 work together to prevent the culture flask 5 from coming out upwards. At the same time, the inner wall of the receiving chamber 31 restricts the culture flask 5 from moving left and right and forward and backward, achieving all-round limiting.

[0048] During the rotation of turntable 3, culture flask 5 will be subjected to centrifugal force, causing it to tend to detach from the containment chamber 31 upwards and outwards. At this time, the spring will always apply upward elastic pressure to the locking block 42, so that the lower end face of the pin block 43 will continue to be tightly attached to the upper end face of the culture flask 5, thereby achieving dynamic positioning of the culture flask 5 and preventing it from detaching due to gravity.

[0049] When the operator needs to remove the culture bottle 5, they only need to manually push the locking block 42 forward. The locking block 42 slides to one side along the guide groove 41, and at the same time, the spring is compressed. The pin 43 on the locking block 42 moves synchronously until the lower end face of the pin 43 disengages from the upper end face of the culture bottle 5 and completely exits the containment chamber 31, releasing the upper limit of the culture bottle 5. At this time, the operator can lift the culture bottle 5 upward. After lifting, the operator releases the pressed locking block 42. Under the action of the elastic restoring force, the spring pushes the locking block 42 and the pin 43 upward to reset, returning to the initial standby state, waiting for the next installation and locking of the culture bottle 5.

[0050] Reference Figure 6 , Figure 7 and Figure 8 As shown, the culture flask 5 includes a cylindrical body 51 that can be detachably installed in the housing 31. The cylindrical body 51 is a hollow columnar structure and is the main supporting component of the culture flask 5. Its outer diameter is adapted to the size of the housing 31 and can be stably embedded in the housing 31 to achieve detachable installation.

[0051] The cylinder 51 adopts a sealed design, and both ends of the cylinder 51 are equipped with detachable culture dishes 6. The culture dishes 6 and the ends of the cylinder 51 are connected by a threaded connection to achieve a tight fit between the two and ensure that the culture medium does not leak during the culture process.

[0052] The cylinder 51 is provided with a circulation connection part 53 that connects the two end culture dishes 6. The circulation connection part 53 is used to guide the culture medium to form a continuous circulation flow path between the two end culture dishes 6 during the rotation of the culture bottle 5.

[0053] During installation, immune cells and culture medium are placed in one of the culture dishes 6. Then, the culture dish 6 is tightly connected to the cylinder 51 by interference fit. The assembled culture bottle 5 is then stably embedded into the receiving chamber 31 of the turntable 3 to complete the installation preparation before culture.

[0054] When the turntable 3 drives the culture flask 5 to rotate around the axis of the turntable 3, the culture flask 5 rotates synchronously with the turntable 3, and its spatial position changes continuously (alternating between the upper, lower, and side parts of the annular chamber 2). During this process, the positions of the two culture dishes 6 at both ends of the cylinder 51 will be reversed as the culture flask 5 rotates. Initially, the immune cells and culture medium inside the upper culture dish 6 will flow into the lower culture dish 6 under the influence of gravity through the circulation connection part 53. As the culture flask 5 continues to rotate, the culture dish 6 that was originally at the lower position will gradually rotate to the upper position, and the culture dish 6 that was originally at the upper position will rotate to the lower position, that is, the upper and lower positions of the two culture dishes 6 will be reversed.

[0055] This cycle repeats continuously. As the culture flask 5 rotates, the circulation connection 53 continuously guides the culture medium to flow between the two end culture dishes 6, forming a stable circulation path. This allows the culture medium to be fully mixed with the immune cells, preventing cell sedimentation and aggregation, while also improving mass transfer efficiency. This provides the immune cells with a culture environment that closely resembles the dynamic circulation in vivo, ensuring the biological activity and functional characteristics of the immune cells.

[0056] Reference Figure 8 , Figure 9 and Figure 10 As shown, the circulation connection part 53 includes symmetrically arranged collecting hoppers 52 inside the cylinder 51, which correspond one-to-one with the culture dishes 6 at both ends of the cylinder 51. A spiral tube assembly 54 is connected between the two collecting hoppers 52. The spiral tube assembly 54 is the culture medium delivery channel of the circulation connection part 53, which is connected between the two collecting hoppers 52 to extend the flow path of the culture medium.

[0057] The spiral tube assembly 54 includes multiple spiral tube layers 55 arranged at intervals along the axial direction of the cylinder 51. Each spiral tube layer 55 includes multiple spiral conduits 56 evenly distributed in the circumferential direction. The spiral conduits 56 extend in a spiral shape and are connected between two collecting hoppers 52.

[0058] When the culture flask 5 rotates with the turntable 3 and the two end culture dishes 6 are reversed, the immune cells and culture medium in the initially upper culture dish 6 flow downward under the action of gravity. They first enter the spiral tube group 54 through the corresponding side collecting funnel 52. The culture medium flows slowly along the spiral channel of the spiral tube group 54 and finally flows into the culture dish 6 that is now in the lower position through the other end collecting funnel 52.

[0059] As the culture flask 5 continues to rotate, the two end culture dishes 6 switch positions again, and the above process is repeated. The collecting funnel 52 continuously gathers and guides the culture medium, and the spiral tube assembly 54 continuously delivers the culture medium, ensuring that the circulation path is continuously and stably formed, and realizing the reciprocating circulation of the culture medium between the two end culture dishes 6.

[0060] Furthermore, a sealing ring 57 is provided at one end of the collecting hopper 52. When the culture dish 6 is installed to the end of the cylinder 51 by interference fit, the culture dish 6 will be in close contact with the end of the collecting hopper 52 where the sealing ring 57 is provided. After the sealing ring 57 is pressed, a sealed culture cavity 58 is formed between the collecting hopper 52 and the culture dish 6, which is used to hold immune cells and culture medium and prevent culture medium leakage.

[0061] Blood circulation is not a constant, uniform process: the heart pumps blood intermittently and periodically, and the blood flow velocity fluctuates periodically with relaxation and contraction; constant, uniform rotation only brings a stable and unchanging direction of gravity flow and a fixed fluid flow velocity. The fluid environment is too stable and differs from the physiological environment of pulsating blood flow brought about by the heartbeat. Therefore, it is recommended to change to intermittent start-stop and periodic variable speed rotation.

[0062] Turntable 3 can be configured to rotate at a constant speed, rotate at a periodic speed, or rotate intermittently. In the constant speed mode, the height of the dish holder 61 can be switched smoothly, and the culture medium can circulate slowly along the spiral tube by gravity, reducing fluid shear force. The periodic speed and intermittent start-stop modes can simulate the pulsating blood flow of the heart, creating a periodically fluctuating fluid environment. Operators can freely switch the operation mode of turntable 3 according to the stage of immune cell culture.

[0063] Example 2: Reference Figure 10 , Figure 11 , Figure 12 and Figure 13 As shown, based on Embodiment 1, the culture dish 6 includes a dish base 61 that can be detachably installed at both ends of the cylindrical body 51. An annular magnetic ring 62 is provided on the inner side of the dish base 61. A detachable filter plate 63 is magnetically attracted to the annular magnetic ring 62. The upper cavity 64 is used to hold immune cells and culture medium. The filter plate 63 divides the dish base 61 into an upper cavity 64 and a lower cavity 65. The filter plate 63 allows waste products generated by immune cell metabolism to pass through and enter the lower cavity 65, while retaining immune cells and culture medium in the upper cavity 64 to participate in circulation.

[0064] The annular magnetic ring 62 fixes the filter plate 63 by magnetic attraction, ensuring that the filter plate 63 fits tightly against the inner side of the dish base 61, thus effectively separating the upper cavity 64 and the lower cavity 65. The upper cavity 64 is used to hold immune cells and culture medium and is the main culture area for immune cells, while the lower cavity 65 is used to collect waste products generated by immune cell metabolism, thus separating waste products from the culture system.

[0065] The filter disc 63 has specific selective permeability properties. Its pore size is precisely designed to allow small molecule waste products (such as metabolites and apoptotic fragments) generated by immune cell metabolism to pass through, allowing the waste to smoothly enter the lower cavity 65. At the same time, it can effectively retain immune cells and culture medium, preventing immune cells from entering the lower cavity 65 with the waste, ensuring that immune cells and culture medium are always kept in the upper cavity 64 to participate in subsequent circulation, avoiding the loss of immune cells and ensuring the culture effect.

[0066] Meanwhile, the magnetic detachable design of the filter plate 63 makes it easy for operators to regularly disassemble the filter plate 63, clean the metabolic waste in the lower chamber 65, or replace the filter plate 63 to ensure the filtration effect. It takes into account both the stability of the culture and the convenience of operation, and is compatible with the overall design of the culture bottle 5 to jointly ensure the high-quality culture of immune cells.

[0067] As the culture flask 5 rotates around the axis with the turntable 3, the culture dish 6 rotates synchronously with the turntable 3. The dish seats 61 at both ends will alternately be in the upper and lower positions. At this time, the filter plate 63 is always tightly attached to the inside of the dish seat 61 by the magnetic attraction of the annular magnetic ring 62, maintaining the effective separation between the upper cavity 64 and the lower cavity 65, and ensuring that the filtration function is stable.

[0068] The specific working process is as follows: When the culture dish 6 rotates to the upper position with the turntable 3, the immune cells and culture medium in the upper cavity 64 of the dish seat 61 naturally adhere to the upper surface of the filter plate 63 under the action of gravity. The culture medium solvent, amino acids, inorganic salts, cytokines and other small nutrient molecules, as well as soluble small molecule metabolites such as lactic acid and hydrogen ions; these substances have a small molecular particle size and can permeate and flow bidirectionally between the upper and lower cavities 65. Solid particles such as intact immune cells, cell aggregates, apoptotic fragments, necrotic cell residues, and protein precipitates; the particle size of the above particles is larger than the pore size of the filter plate 63 and will be intercepted by the filter plate 63.

[0069] Small molecule waste products generated by immune cell metabolism (such as metabolites, apoptosis fragments, etc.) will pass through the precise pores of the filter plate 63 with the assistance of gravity and enter the lower chamber 65 for collection. When the culture dish 6 rotates to the lower position with the turntable 3, the immune cells and culture medium in the upper chamber 64 are still retained by the filter plate 63 and will not enter the lower chamber 65 due to the inversion of position or the reversal of gravity. At the same time, the metabolic waste collected in the lower chamber 65 will remain at the bottom of the lower chamber 65 under the action of gravity and will not flow back through the filter plate 63 to the upper chamber 64 to contaminate the culture system.

[0070] Throughout the rotation process, the filter disc 63 maintains a stable selective permeability function, continuously separating immune cells, culture medium and metabolic waste. This ensures that the immune cells and culture medium in the upper cavity 64 can participate in the circulation flow normally, while preventing metabolic waste from spreading with the circulation, thus ensuring a clean culture environment for immune cells and maintaining the biological activity and culture quality of immune cells.

[0071] Example 3: Based on Example 2, a sampling section 7 is provided on the dish holder 61. The sampling section 7 is used to sample the immune cells in the upper cavity 64, facilitating the operator to periodically monitor the culture status of the immune cells (such as proliferation activity, phenotypic differentiation, etc.). The sampling section 7 is also used to replenish the upper cavity 64 with nutrient solution, cytokines, and other substances required for culture, adapting to the nutritional consumption needs of the immune cells during the culture process. When the concentration of nutrient solution in the upper cavity 64 is insufficient, or when it is necessary to add specific cytokines to regulate immune cell differentiation, the operator can open the sealing cap of the sampling section 7 under sterile conditions and inject an appropriate amount of nutrient solution or cytokines into the upper cavity 64 through the sampling section 7 using a sterile pipette tool.

[0072] The detailed sampling unit 7 includes a connector 71 that passes through the dish holder 61, with the upper end of the connector 71 extending to the upper end of the filter plate 63. The connector 71 has an inlet 72 located at the upper end of the filter plate 63. A sampling port 76 corresponding to the connector 71 is located at the bottom of the equipment box 1, and the culture flask 5 placed at the sampling port 76 is in a vertical position. A piston 73 is slidably pressed inside the connector 71 by a spring, and the piston 73 blocks the inlet 72. The piston 73 has an outlet 74 corresponding to the inlet cylinder, and a protrusion 75 corresponding to the inlet 72 is located at the upper end of the piston 73.

[0073] During sampling, culture flask 5 rotates with turntable 3 to a vertical position, at which point dish holder 61 is in a vertical state, and the lower end of connector 71 is precisely aligned with sampling port 76 at the bottom of equipment box 1. The operator prepares the sampling tube in a sterile environment. Insert the sampling tube through the sampling port 76 at the bottom of the equipment box 1 and slowly insert it into the connector 71 until the top of the sampling tube contacts the bottom of the piston 73 inside the connector 71; continue to push the sampling tube upward, the sampling tube applies an upward thrust to the piston 73, the thrust overcomes the elastic pressure of the spring, causing the spring to be further compressed, and the piston 73 moves upward accordingly.

[0074] As piston 73 moves upward, the protrusion 75 at the upper end of piston 73 moves upward synchronously, gradually disengaging from the inlet 72 of connector 71 and releasing the blockage of inlet 72; when piston 73 moves to the designated position, the outlet 74 on piston 73 connects with the inlet 72 of connector 71, forming a complete fluid channel, which passes through the upper cavity 64 of dish 61, through inlet 72 and outlet 74, and enters the sampling test tube.

[0075] At this time, the immune cells and culture medium in the upper cavity 64 of the dish holder 61, under the action of gravity (because the culture flask 5 is in a vertical position), enter the interior of the connector 71 through the inlet 72 at the upper end of the connector 71, and then flow into the sampling test tube through the outlet 74 on the piston 73, completing the sampling operation; due to the filtering effect of the filter plate 63, the metabolic waste in the lower cavity 65 will not enter the upper cavity 64, so the sampled sample contains only immune cells and pure culture medium, without waste contamination.

[0076] After sampling is completed, the sampling tube is pulled out, and the upward pushing force of the sampling tube on the piston 73 disappears. Under the action of its own elastic restoring force, the spring pushes the piston 73 to return to its original position. The protrusion 75 at the upper end of the piston 73 is re-embedded into the liquid inlet 72, re-sealing the liquid inlet 72. At the same time, the sliding seal between the piston 73 and the inner wall of the connector 71 continues to be effective, and the sampling part 7 returns to its normal sealed state, avoiding leakage of culture medium or external contamination. The culture bottle 5 can continue to rotate with the turntable 3 without affecting the subsequent dynamic circulation culture.

[0077] Finally, the present invention also provides a method for culturing immune cells, the method of use of which includes the following steps: S1: Culture preparation. The turntable 3 rotates in the annular chamber 2. Each containment chamber 31 passes through the vertical placement station corresponding to the unique operation window 11 in sequence. The culture flasks 5 containing immune cells and culture medium are assembled by fitting. The sealing rings 57 at both ends of the cylinder 51 and the collection hopper 52 form a sealed culture chamber 58. Then the culture flasks 5 are put into the containment chambers 31 to provide a closed and stable initial environment for dynamic circulation culture.

[0078] S2: During equipment installation, the pressure pin 43 guides the inclined plane 44, pushing the locking block 42 down along the guide groove 41 to compress the spring. After the culture bottle 5 is fully in place, the spring rebounds, causing the locking block 42 and the pin 43 to reset. The pin 43 presses against the upper end of the culture bottle 5 to achieve axial limitation. During the rotation of the turntable 3, the buckle 4 remains locked to counteract centrifugal force, ensuring that the culture bottle 5 does not loosen or come out, and maintaining dynamic operational stability.

[0079] S3: Circulating rotation, the turntable 3 drives the culture flask 5 to rotate in a circle, and the two end plate holders 61 are constantly up and down as their positions change. Under the action of gravity, the culture medium and immune cells are gathered through the collecting funnel 52 and enter the spiral tube group 54 composed of multi-layer spiral conduits 56. The flow extends along the spiral path and forms a disturbance, circulating back and forth between the two end culture dishes 6, avoiding cell sedimentation and aggregation, and improving the efficiency of nutrient and oxygen mass transfer.

[0080] S4: Cell culture. The filter plate 63 is fixed to the dish base 61 by the annular magnetic ring 62, dividing the inner cavity into an upper cavity 64 and a lower cavity 65. The upper cavity 64 retains immune cells and culture medium to participate in circulation, while small molecule waste generated by metabolism passes through the filter plate 63 under the action of gravity and enters the lower cavity 65 for collection. When the culture flask 5 is inverted, the waste in the lower cavity 65 will not flow back, thus continuously maintaining the cleanliness of the culture system and improving cell activity and amplification quality.

[0081] S5: Sampling and replenishment: Rotate culture bottle 5 to a vertical position, align connector 71 with sampling port 76, use sampling test tube to lift piston 73, compress spring and open inlet 72, allowing liquid in upper chamber 64 to flow into test tube through inlet 72 and outlet 74; after sampling or replenishment, pull out piston 73, piston 73 returns to its original position under spring action, protrusion 75 re-seals inlet 72, restoring the seal, without affecting subsequent continuous culture.

[0082] The embodiments described herein are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, all equivalent changes made in accordance with the structure, shape, and principle of the present invention should be covered within the scope of protection of the present invention.

Claims

1. An immune cell culture regulation device, comprising an equipment box (1), characterized in that: The equipment box (1) has an annular chamber (2) inside, and a turntable (3) is rotatably arranged inside the annular chamber (2). Multiple accommodating chambers (31) are arranged circumferentially on the turntable (3). Removable culture bottles (5) are adapted to be installed in the accommodating chambers (31). An operation window (11) corresponding to the position of the accommodating chamber (31) is opened on the equipment box (1). The turntable (3) is configured to drive the culture bottle (5) to rotate around the axis of the turntable (3), so that the culture medium in the culture bottle (5) and the immune cells flow and mix repeatedly under the action of gravity, simulating the dynamic circulation environment of immune cells in the body, thereby providing immune cells with in vitro culture conditions close to physiological state, so as to maintain the biological activity and functional characteristics of immune cells. The culture flask (5) includes a cylindrical body (51) that can be detachably installed in the containing chamber (31). Both ends of the cylindrical body (51) are provided with detachable culture dishes (6). The cylindrical body (51) is provided with a circulation connection part (53) that connects the two culture dishes (6). The circulation connection part (53) is used to guide the culture medium to form a continuous circulation flow path between the two culture dishes (6) during the rotation of the culture flask (5).

2. The immune cell culture regulation device according to claim 1, characterized in that: The circulation connection part (53) includes symmetrically arranged collection buckets (52) inside the cylinder (51), and a spiral tube group (54) is connected between the two collection buckets (52).

3. The immune cell culture regulation device according to claim 2, characterized in that: The spiral tube assembly (54) includes multiple spiral tube layers (55) arranged at intervals along the axial direction of the cylinder (51). Each spiral tube layer (55) includes multiple spiral conduits (56) evenly distributed in the circumferential direction. The spiral conduits (56) extend in a spiral shape and are connected between the two collection buckets (52).

4. The immune cell culture regulation device according to claim 2, characterized in that: The collecting hopper (52) is provided with a sealing ring (57) at one end. The culture dish (6) will contact the end of the collecting hopper (52) provided with the sealing ring (57) to form a sealed culture cavity (58) between the collecting hopper (52) and the culture dish (6).

5. The immune cell culture regulation device according to claim 2, characterized in that: The culture dish (6) includes a dish base (61) that can be detachably installed at both ends of the cylinder (51). An annular magnetic ring (62) is provided on the inner side of the dish base (61). A detachable filter plate (63) is magnetically attracted to the annular magnetic ring (62). The upper cavity (64) is used to hold immune cells and culture medium. The filter plate (63) divides the dish (61) into an upper chamber (64) and a lower chamber (65). The filter plate (63) allows waste products generated by immune cell metabolism to pass through and enter the lower chamber (65), while retaining immune cells and culture medium in the upper chamber (64) to participate in circulation.

6. The immune cell culture regulation device according to claim 5, characterized in that: The dish holder (61) is provided with a sampling section (7), which is used to sample immune cells in the upper cavity (64); The sampling unit (7) includes a connector (71) that passes through the dish base (61). The upper end of the connector (71) extends to the upper end of the filter plate (63). The connector (71) has an inlet (72) located at the upper end of the filter plate (63). The bottom of the equipment box (1) is provided with a sampling port (76) corresponding to the connector (71). A piston (73) is installed inside the connector (71) by means of a spring sliding pressure, and the piston (73) blocks the liquid inlet (72).

7. The immune cell culture regulation device according to claim 6, characterized in that: The piston (73) has an outlet (74) corresponding to the inlet cylinder, and the upper end of the piston (73) has a protrusion (75) corresponding to the inlet (72).

8. The immune cell culture regulation device according to claim 1, characterized in that: The turntable (3) is provided with a buckle (4) for securing the culture bottle (5) within the housing (31); The buckle (4) includes a guide groove (41) on the turntable (3) that corresponds one-to-one with the receiving chamber (31). A locking block (42) is provided in the guide groove (41) by a spring sliding. A pin (43) is constructed on the locking block (42) located at the upper end of the culture bottle (5) in the receiving chamber (31). The pin (43) is used to limit the culture bottle (5).

9. The immune cell culture regulation device according to claim 8, characterized in that: The pin block (43) is provided with a guide slope (44).

10. A cell culture method using any one of the immune cell culture regulation devices as described in claims 1 to 9, characterized in that, Includes the following steps: S1: Culture preparation, the turntable (3) rotates in the annular chamber (2), the container (31) passes through the vertical placement station corresponding to the window in sequence, and the culture bottle (5) is put into the container (31) to provide a closed environment for culture; S2: Equipment installation, squeeze the pin block (43) to guide the inclined surface (44), push the locking block (42) along the guide groove (41) to compress the spring and move down. After the culture bottle (5) is fully in place, the spring rebounds to reset the locking block (42) and the pin block (43). The pin block (43) presses the upper end of the culture bottle (5) to achieve axial limit, ensuring that the culture bottle (5) does not loosen or come out, and maintaining dynamic operation stability; S3: Circulating rotation, the turntable (3) drives the culture bottle (5) to rotate in a circle, and the two end plate holders (61) are constantly up and down as their positions change. Under the action of gravity, the culture medium and immune cells converge through the collecting funnel (52) into the spiral tube group (54) composed of multi-layer spiral conduit (56), extend along the spiral path and form disturbances, and circulate back and forth between the two end culture plates (6); S4: Cell culture, filter plate (63) is fixed to dish base (61) by ring magnetic ring (62), dividing the inner cavity into upper cavity (64) and lower cavity (65). The upper cavity (64) retains immune cells and culture medium to participate in circulation, and small molecule waste generated by metabolism passes through filter plate (63) and enters lower cavity (65) for collection under the action of gravity. S5: Sampling and replenishment. Turn the culture bottle (5) to the vertical position and align the connector (71) with the sampling port (76). Use the sampling test tube to lift the piston (73), compress the spring and open the inlet (72). Let the liquid in the upper chamber (64) flow into the test tube through the inlet (72) and outlet (74). After sampling or replenishment, pull it out. The piston (73) will reset under the action of the spring.

Images