Stirring type bioreactor and application thereof

Abstract

The invention provides a stirring type bioreactor which is composed of a base, a reaction tank, a biological culture bag, a stirring device, a supporting frame and a control device, the base, the stirring device and the supporting frame can be matched with the reaction tanks of different specifications and sizes, so that the sizes of the reaction tanks can be flexibly adjusted, the utilization rate is increased, and the cost is reduced; by improving the shape and the structure of the stirring blades and arranging the double-layer blades, the upper layer is the axial flow blade and is provided with a hollow part, the lower layer is the radial flow blade, the blades are special and work cooperatively, and the effect of the spoiler is combined, so that the influence of shearing force on cells is reduced, and the cell activity and the culture density are improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology equipment and relates to a bioreactor and its application. Background Technology

[0002] In in vitro cell culture, the bioreactor is a key piece of equipment in the entire process, providing a suitable growth environment for the cells to proliferate rapidly and form the desired biological tissue products. Among them, the most widely used is the stirred bioreactor, which mainly consists of a stirring mechanism, monitoring equipment such as temperature and pH monitoring, reaction tanks, and nutrient solution dispensing mechanisms.

[0003] Stirred bioreactors remain the mainstream choice for cell culture due to their high flexibility, excellent mixing and mass transfer performance, reliability, controllability, and scalability. However, stirred bioreactors can easily damage certain fragile cell types, thus limiting their application in the culture of fragile cells. Therefore, it is necessary to improve existing stirred bioreactors to reduce the damage to cells caused by stirring shear forces, adapt them to the culture of more fragile cells, and expand their application range.

[0004] Small stirred bioreactors (5-20L in volume) used for experimental research typically require various tanks of different sizes to accommodate different experimental conditions. This necessitates purchasing different sizes of stirred bioreactor bases and filling control devices to match the tanks, leading to the following problems: 1. High cost and large footprint, causing inconvenience; 2. Each stirred bioreactor requires installation, operation, performance verification, and validation processes, which are time-consuming, labor-intensive, and costly; 3. Batch-to-batch variations exist between different filling control devices; using different sizes of bioreactors in the same experiment can affect the accuracy of experimental results; 4. Control units for different sizes of stirred bioreactors need to be developed individually, which is time-consuming and labor-intensive; 5. Switching between multiple stirred bioreactors of different sizes is prone to errors, delaying research progress. Therefore, there is an urgent need to find a stirred bioreactor that can adapt to tanks of different sizes. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a novel stirred bioreactor, comprising a base, a reaction vessel, a biological culture bag, a stirring device, a support frame, and a control device. The base, stirring device, and support frame can be matched with reaction vessels of different sizes, allowing for flexible adjustment of the reaction vessel size, improving utilization and reducing costs. By improving the shape and structure of the stirring blades, a double-layered blade configuration is implemented: the upper layer is an axial flow blade with perforations, and the lower layer is a radial flow blade. These dedicated blades work synergistically, and combined with the effect of baffles, the impact of shear force on cells is reduced, thereby improving cell viability and culture density.

[0006] On the one hand, the present invention provides a stirred bioreactor, which includes a base and a reaction vessel. The reaction vessel can be replaced with a vessel of different volume specifications, all of which can be combined with the base for use.

[0007] This invention improves the structure of the base and the reaction vessel, enabling the reaction vessel to be adjustable in size. In other words, the base can accommodate various sizes of vessels, offering high adaptability. It is also easy to install and disassemble, facilitating operation. Users only need to purchase one base to use a series of reaction vessels, saving costs.

[0008] By using this stirred bioreactor, the process parameters (such as stirring speed, aeration strategy, temperature, pH control, etc.) can be highly comparable and scalable throughout the entire process from pilot-scale to production by changing the reaction vessel. This reduces development risks and time, improves equipment utilization and flexibility, and lowers costs. Moreover, operators only need to learn and maintain one system, which simplifies operation, reduces human error, and also reduces the complexity of spare parts inventory.

[0009] Furthermore, the volume of the reaction vessel is 5~20L; the upper surface of the base is provided with a protrusion, and the bottom of the reaction vessel is provided with a groove, the protrusion can be combined with the groove to fix the reaction vessel.

[0010] In some designs, the upper surface of the base has an elongated protrusion, and the bottom of the reaction vessel has a groove that matches the protrusion. The reaction vessel and base are installed by pushing the vessel along the matching direction of the protrusion and groove. As the size and volume of the reaction vessel change, the size of the groove at the bottom of the vessel must remain constant to achieve compatibility between different sizes of reaction vessels and the base.

[0011] In some embodiments, the stirred bioreactor provided by this invention is a small-scale bioreactor, which can be flexibly applied to reaction tanks of 5 to 20 L.

[0012] In some embodiments, the 5-20L reaction vessel is a cylindrical vessel with a height of 30-100cm and a cross-sectional diameter of 20-60cm.

[0013] Furthermore, a fixing nut is provided on the protrusion on the surface of the base for locking the reaction vessel after it is joined.

[0014] After the reaction vessel is pushed in along the protrusion on the base surface, in order to prevent displacement during use, the position of the reaction vessel must be locked with a fixing nut.

[0015] In some embodiments, the protrusion has a row of screw holes corresponding to multiple fixing nuts, and the bottom of the reaction vessel also has corresponding screw holes. The appropriate screw hole position can be selected according to the size of the reaction vessel, and then the position of the reaction vessel can be limited and locked by fixing nuts.

[0016] In some embodiments, the number of fixing nuts and screw holes can be one or more, respectively located on the protrusion and the bottom of the reaction vessel, with 1 to 2 nuts on each side of the bottom of the reaction vessel, thereby locking the reaction vessel from different directions on both sides.

[0017] Furthermore, the stirred bioreactor also includes a stirring device, a support frame, and a control device; the support frame is located above the base and includes vertical arms and horizontal arms, the vertical distance of which is adjustable; the control device is connected to the reaction vessel through the support frame.

[0018] The support frame is used to support and place the stirring drive device, as well as other components used to connect and control the reaction vessel, such as the filling system, feeding system, temperature and pH detection devices, etc.

[0019] Understandably, as the size of the reaction vessel changes, the vertical and horizontal arms of the support frame also need to be changed accordingly to match the reaction vessel.

[0020] In some embodiments, the vertical arm of the support frame is provided with an extension track for adjusting the length of the vertical arm.

[0021] Furthermore, the stirring device includes a stirring drive device and a stirring paddle. The stirring paddle is located inside the biological culture bag, and the stirring drive device is located on the cross arm of the support frame and is always directly facing the middle position of the base. The stirring paddle includes a stirring shaft and upper and lower combined blades. The upper layer is provided with axial flow blades, and the lower layer is provided with radial flow blades.

[0022] The stirring drive is located on the cross arm of the support frame and is always directly opposite the center of the base, so that the stirring drive is always located at the center of the cross-section of the reaction vessel. As the volume of the reaction vessel changes from 5L to 20L, the stirring drive is always directly opposite the center of the reaction vessel.

[0023] The shear forces generated during stirring in a stirred bioreactor can affect cell culture, especially for more fragile cells (such as PBMCs). When the shear forces are too high, for example, exceeding the cell's tolerance (e.g., 100 Pa) at a certain rotation speed (e.g., 100 rpm), cell death is highly likely. Therefore, engineering techniques are needed to create a mild environment through low-shear force design, minimizing the shear forces that cells may experience to an appropriate level, allowing cells to maintain high viability and robust proliferation capacity during culture, thereby improving cell culture outcomes.

[0024] This invention divides the stirring paddle into upper and lower layers of blades, which can take advantage of the hydrodynamic characteristics of different types of blades, complement each other, achieve a synergistic effect, and take into account mixing, mass transfer and low shear, so as to create a uniform and mild culture environment for cells.

[0025] Because stirred bioreactors are more conducive to scale-up, it is necessary to solve the problem of culturing fragile cells in stirred bioreactors in order to enable large-scale culture and industrial production of fragile cells in the future. This invention achieves efficient culture of fragile cells by improving the shape and structure of the impeller in a stirred bioreactor.

[0026] In some embodiments, the axial flow impeller includes any one or more of the following: marine propeller, oblique blade turbine propeller, airfoil propeller, spiral ribbon propeller, and oblique blade open turbine propeller. It can generate strong axial flow, pushing the liquid from the stirring axis downward or upward, and then back along the inner wall of the reaction vessel to form a large overall circulation. This can ensure that the concentration and temperature of cells, nutrients, and metabolites in the upper and lower parts of the reaction vessel remain uniform, avoid the formation of "static zones" or concentration gradients, improve mixing efficiency, achieve rapid overall circulation, and ensure that all cells have an equal opportunity to come into contact with oxygen and promote the discharge of metabolic waste such as carbon dioxide.

[0027] In some embodiments, the radial flow impeller includes any one or more of the following: gas dispersion impeller, flat blade turbine impeller, arrow-shaped impeller, concave blade turbine impeller, plate impeller, and anchor impeller. It can generate strong radial flow, that is, the liquid is thrown out in the opposite direction from the center height of the impeller towards the inner wall of the reaction vessel. It can effectively break the bubbles introduced from the bottom of the impeller into smaller microbubbles, greatly increasing the gas-liquid contact area. The powerful radial jet can also flush the bottom of the reaction vessel, preventing cells from depositing due to gravity, and can also achieve efficient oxygen mass transfer.

[0028] The combination of upper and lower blades ensures efficient oxygen mass transfer and provides a smoother fluid field, resulting in lower overall shear force and avoiding shear force problems caused by excessive rotational speed.

[0029] Furthermore, the axial flow blades are marine-type blades with large oblique blades; the radial flow blades are gas dispersion blades with arc-shaped impellers.

[0030] This invention selects the optimal combination of axial flow impellers (naval-style impellers with large oblique blades) and radial flow impellers (gas dispersion impellers with arc-shaped impellers) from different impeller design combinations. This combination achieves synergistic effects, realizes a perfect balance between low shear and high mass transfer, significantly improves cell culture results, and has good scalability.

[0031] Furthermore, the radial flow blade has perforations with a perforation diameter of 0.8~1.6cm and a porosity of 5~20%.

[0032] Studies have shown that adding perforations to the lower radial flow impeller blades can effectively reduce shear forces and minimize damage to fragile cell cultures. This is likely because the gas dispersion impeller generates shear forces during gas dispersion; the perforations optimize the flow field, reduce stirring power and torque, disrupt the formation of large, stable vortices, and promote lateral mixing of fluids. This provides a gentler, more favorable fluid environment for shear-sensitive cells, thereby improving cell activity and product yield.

[0033] The size and porosity of the perforated pores directly affect cell culture outcomes; inappropriate pore size or porosity can lead to cell culture failure. This invention significantly improves the culture density and viability of shear-sensitive cells by selecting the optimal perforated pore size and porosity.

[0034] Furthermore, the stirring drive device and the stirring paddle are magnetically connected; a first magnetic block is provided below the stirring drive device, and the bottom surface of the first magnetic block is connected to a second magnetic block at the upper end of the stirring shaft; a hexagonal groove is provided inward at the middle position of the bottom surface of the first magnetic block, and a hexagonal protrusion that can connect with the hexagonal groove is provided at the middle position of the upper surface of the second magnetic block; the size of the bottom surface of the first magnetic block is greater than or equal to the size of the upper surface of the second magnetic block.

[0035] The cell culture bags of corresponding specifications used in reaction vessels of different volumes are equipped with stirring paddles of corresponding specifications. The area of ​​the second magnetic block of the stirring paddle is smaller than the area of ​​the stirring drive device, thereby ensuring that they can be used in combination. The hexagonal groove of the first magnetic block and the hexagonal protrusion on the second magnetic block ensure the fixed position of the stirring drive device and the stirring paddle, so that the stirring drive device can drive the stirring paddle to rotate more stably.

[0036] When the hexagonal groove and hexagonal protrusion on the first magnetic block are combined, the hexagonal sides can help limit and fix the connection position between the stirring drive device and the stirring paddle, improve the connection stability between the stirring drive device and the stirring paddle, make the rhythm of the stirring process more precise, and prevent micro-slippage from affecting the stirring effect.

[0037] Furthermore, the reaction vessel is provided with a raised baffle plate, and the biological culture bag is provided with a baffle bag that extends into its interior and accommodates the baffle plate. The baffle bag is sealed to the outer wall of the biological culture bag.

[0038] By installing baffles on the inner wall of the reactor, the rotating flow can be transformed into a more ordered axial mixing, breaking the original vortex and forming a complex flow that tumbles up and down and left and right, eliminating dead zones, achieving true homogeneity within the reactor, significantly improving mixing efficiency, increasing mass transfer, and also helping to reduce the impact of shear force on cells, preventing cell deposition, ensuring that all cells are in a uniform suspension state, and facilitating the rational use of energy. In conjunction with the upper and lower layer combined blades, it creates a more ideal growth environment for fragile cells, and no additional protective agents are needed during cell culture, ensuring cell viability and culture density.

[0039] Furthermore, the reaction vessel is provided with baffles at both the upper and lower parts, with the upper baffle extending downwards and the lower baffle extending upwards; the upper baffle can be detached along the inner wall of the reaction vessel.

[0040] Studies have shown that baffles need to be installed at the top and bottom of the reaction vessel. The upper and lower baffles are combined with two layers of impellers to reduce shear force, improve mass transfer, and significantly enhance cell culture results.

[0041] The upper baffle can be removed along the inner wall of the reaction vessel, which helps with the installation and removal of disposable cell culture bags. When the disposable cell culture bag is being fitted onto the lower baffle, the upper baffle can be removed first to prevent affecting the installation of the cell culture bag. After the disposable cell culture bag is installed, the baffle can be fitted onto it from the top to complete the installation.

[0042] Meanwhile, the baffle plate also helps to fix the cell culture bag, preventing it from shifting with the flow of the culture medium and improving batch-to-batch consistency of cell culture results.

[0043] In another aspect, the present invention provides a cell culture method, which uses a stirred bioreactor as described above for culture.

[0044] In some embodiments, the cells are shear-sensitive or fragile cells.

[0045] In some methods, the Pluronic F-68 protectant is not required during the cell culture process.

[0046] When culturing fragile cells in a stirred bioreactor, Pluronic F-68 (a nonionic surfactant) is typically added to protect the cells from fluid shear damage. However, Pluronic F-68 can inhibit the growth rate of some cells and promote foam formation, affecting the cell culture results. The stirred bioreactor provided by this invention minimizes the impact of shear forces while ensuring better mass transfer, making it suitable for stirred culture of fragile cells without the need for Pluronic F-68.

[0047] In some embodiments, the cells include human lymphocytes (PBMCs). Human lymphocytes (PBMCs) are sensitive to physical shear forces and poorly tolerant of environmental fluctuations, making them difficult to culture in stirred bioreactors, thus hindering their large-scale production. The stirred bioreactor provided by this invention enables efficient culture of human lymphocytes (PBMCs) without the need for the addition of Pluronic F-68 protectant to the culture medium, reducing foam formation.

[0048] In some methods, the culture medium for the human lymphocytes PBMCs is RPMI 1640 medium, supplemented with cholesterol and vitamin E, anti-CD3 / CD28 antibody and interleukin-2 (IL-2).

[0049] The stirred bioreactor provided by this invention has the following beneficial effects: (1) The base and support frame can be matched with reaction vessels of different sizes, so that the size of the reaction vessel can be flexibly adjusted, improving utilization and reducing costs; (2) Improve the connection between the stirring drive device and the stirring paddle to make the connection more stable, the stirring rhythm more precise, and effectively prevent micro-slippage. (3) The combination of the upper and lower blades and the selection of the optimal blade shape can ensure efficient oxygen mass transfer and provide a smoother fluid field, resulting in lower overall shear force and avoiding shear force problems caused by excessive rotation speed. (4) Baffles are installed at the top and bottom of the reaction vessel to reduce the impact of shear force on the cells, work in conjunction with the paddle to improve cell viability and increase cell culture density, and can also be used to fix disposable cell culture bags to improve batch-to-batch consistency of cell culture. (5) It enables efficient culture of human lymphocytes PBMCs without the need to add Pluronic F-68 protectant to the culture medium, thus reducing foam formation. Attached Figure Description

[0050] Figure 1 This is a schematic diagram of the overall structure of a stirred bioreactor. Figure 2 This is a structural diagram of the combination of the base, reaction vessel, and support frame. Figure 3 This is a structural diagram of the base and support assembly; Figure 4 This is a structural diagram of the stirring device; Figure 5 This is a structural diagram of the stirring drive device; Figure 6 This is a structural diagram of the stirring paddle inside a disposable biological culture bag; Figure 7 This is a structural diagram of the stirring paddle (tilted angle) inside a disposable biological culture bag; Figure 8 This is a structural diagram of the stirring paddle (lateral angle) inside a disposable biological culture bag; Figure 9 This is a structural diagram of a disposable biological culture bag. Figure 10 A transparent structural diagram of a disposable biological culture bag; Figure 11 This is a cross-sectional view of a stirred bioreactor; Figure 12 Top view of the reaction vessel; Figure 13 The diagram shows the disassembly of the upper baffle plate of the reaction vessel. Detailed Implementation

[0051] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and are not intended to limit it in any way. All features disclosed in the embodiments of the present invention, or all steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0052] Example 1: The stirred bioreactor provided by the present invention The stirred bioreactor provided in this embodiment is as follows: Figures 1-3 The stirred bioreactor 1 includes a base 2 and a reaction vessel 3. The reaction vessel 3 can be replaced with vessels of different volumes, all of which can be combined with and used with the base 2. The volume of the reaction vessel 3 is 5~20L. The upper surface of the base 2 has a protrusion 4, and the bottom 8 of the reaction vessel 3 has a groove 5. The protrusion 4 can engage with the groove 5 to fix the reaction vessel 3. The reaction vessel 3 and the base 2 can be installed by pushing the reaction vessel 3 along the matching direction of the protrusion 4 and the groove 5. As the size and volume of the reaction vessel 3 change, the size of the groove 5 at the bottom 8 of the reaction vessel 3 must remain unchanged to achieve matching of different sizes of reaction vessels 3 with the base 2. The 5~20L reaction vessel 3 is a cylindrical vessel with a height of 30~100cm and a cross-sectional diameter of 20~60cm.

[0053] like Figure 2 and Figure 3 As shown, a fixing nut 6 is also provided on the protrusion 4 on the surface of the base 2 to lock the reaction vessel 3 after it is joined. After the reaction vessel 3 is pushed in along the protrusion 4 on the surface of the base 2, in order to prevent displacement during use, the fixing nut 6 is used to lock the position of the reaction vessel 3. The protrusion 4 is provided with a row of screw holes 7 corresponding to multiple fixing nuts 6, and the bottom 8 of the reaction vessel 3 is also provided with corresponding outer screw holes 30. The appropriate screw hole position can be selected according to the size of the reaction vessel 3, and the position of the reaction vessel 3 is then defined and locked by the fixing nut 6. The number of fixing nuts 6, screw holes 7 and outer screw holes 30 can be one or more. The screw holes 7 and outer screw holes 30 are respectively provided on the protrusion 4 and the bottom 8 of the reaction vessel 3. One or two outer screw holes 30 are provided on each side of the bottom 8 of the reaction vessel 3, so that the reaction vessel 3 can be locked from different directions on both sides.

[0054] like Figure 1 As shown, the stirred bioreactor 1 also includes a stirring device 9, a support frame 10, and a control device 11. The support frame 10 is located above the base 2 and includes a vertical arm 12 and a horizontal arm 13. The horizontal arm 13 is rotatably mounted on the vertical arm 12. The vertical distance of the vertical arm 12 is adjustable, and the lateral distance of the horizontal arm 13 is adjustable. The control device 11 is connected to the reaction vessel 3 through the support frame 10. The support frame 10 is used to support and place the stirring drive device, as well as other components used to connect and control the reaction vessel 3, such as the filling system, feeding system, temperature and pH detection devices, etc. It is understood that as the size of the reaction vessel 3 changes, the vertical arm 12 and the horizontal arm 13 of the support frame 10 also need to be changed accordingly to match the reaction vessel 3. Each vertical arm 12 of the support frame 10 is provided with an extension rail 14 for adjusting the length of the vertical arm 12.

[0055] like Figure 4As shown, the stirring device 9 includes a stirring drive device 15 and a stirring paddle 16. The stirring paddle 16 is located inside the biological culture bag 17, and the stirring drive device 15 is located on the cross arm 13 of the support frame 10. The stirring paddle 16 includes a stirring shaft 18 and upper and lower combined blades. The upper layer has axial flow blades 19, and the lower layer has radial flow blades 20. By dividing the stirring paddle 16 into upper and lower blades, the hydrodynamic characteristics of different types of blades can be utilized to complement each other, achieving a synergistic effect and taking into account mixing, mass transfer, and low shear, creating a uniform and mild culture environment for cells. Since the stirred bioreactor 1 is more conducive to scale-up, in order to enable fragile cells to be successfully cultured on a large scale and industrially produced in the future, it is necessary to solve the culture problem in the stirred bioreactor 1. This embodiment improves the shape and structure of the blades of the stirred bioreactor 1 by adopting a combination of upper and lower blades, which can ensure efficient oxygen mass transfer and provide a smoother fluid field, resulting in lower overall shear force and avoiding shear force problems caused by excessive rotation speed.

[0056] like Figures 6-8 As shown, the upper axial flow impeller 19 is a marine-style impeller 21, equipped with large, arc-shaped oblique blades 22, with the openings of the arc-shaped oblique blades 22 facing downwards. The downward-facing openings of the arc-shaped impeller blades 22 effectively reduce shear force. The lower radial flow impeller 20 is a gas dispersion impeller 23, equipped with arc-shaped impellers 24. This combination can achieve synergistic effects, realizing a perfect balance between low shear and high mass transfer, significantly improving the cell culture effect, and also has good scalability.

[0057] like Figures 6-8 As shown, the lower radial flow impeller 20 has perforations 25, with a pore size of 0.8~1.6cm and a porosity of 5~20% (in this embodiment, the optimal pore size was determined to be 1.1cm, with one row of perforations 25 on each impeller, four per row arranged neatly, and a porosity of approximately 10%). Adding perforations 25 to the lower radial flow impeller 20 effectively reduces shear force and minimizes damage to fragile cell cultures. This is likely because the gas dispersion impeller 21 generates shear force during gas dispersion; the perforations 25 optimize the flow field, reduce stirring power and torque, disrupt the formation of large, stable vortices, promote lateral mixing of fluids, and provide a gentler, more favorable fluid environment for shear-sensitive cells, thereby improving cell activity and product yield. The pore size and porosity of the perforations 25 directly affect cell culture results; inappropriate pore size or porosity can lead to cell culture failure. This embodiment improves the culture density and activity of shear-sensitive cells by screening the pore size and porosity of the 25-hole perforation.

[0058] like Figures 5-8As shown, the stirring drive device 15 and the stirring paddle 16 are magnetically connected. A first magnetic block 26 is located below the stirring drive device 15, and the lower bottom surface 27 of the first magnetic block 26 is connected to a second magnetic block 28 at the upper end of the stirring shaft 18. A hexagonal groove 29 is provided inwardly at the center of the lower bottom surface 27 of the first magnetic block 26, and a hexagonal protrusion 32 that can engage with the hexagonal groove 29 is provided at the center of the upper surface 31 of the second magnetic block 28. The size of the lower bottom surface 27 of the first magnetic block 26 is greater than or equal to the size of the upper surface 31 of the second magnetic block 28. Different sizes of disposable cell culture bags 17 are used in reaction vessels 3, each containing a stirring paddle 16 of a corresponding size. The area of ​​the second magnetic block 28 of the stirring paddle 16 is smaller than the area of ​​the first magnetic block 26 of the stirring drive device 15, thus ensuring that they can all be used interchangeably. The combination of the hexagonal groove 29 and the hexagonal protrusion 32 ensures the fixed position of the stirring drive device 15 and the stirring paddle 16, making the stirring drive device 15 drive the stirring paddle 16 to rotate more stably, improving the connection stability between the stirring drive device 15 and the stirring paddle 16, making the rhythm of the stirring process more precise, and preventing micro-slippage from affecting the stirring effect.

[0059] like Figures 9-12 As shown, the reaction vessel 3 is provided with a raised baffle plate 34. The height of the baffle plate 34 accounts for 1 / 4 of the height of the reaction vessel 3, and the width accounts for 1 / 5 of the diameter of the reaction vessel 3. The disposable biological culture bag 17 (transparent) is provided with a baffle bag 35 that extends into its interior and accommodates the baffle plate 34. The baffle bag 35 is sealed to the outer wall 36 of the disposable biological culture bag 17.

[0060] By installing baffles 34 on the inner wall 37 of the reaction vessel 3, the rotating flow can be transformed into a more ordered axial mixing, breaking the original vortex and forming a complex flow that tumbles up and down and left and right, eliminating dead zones, achieving true homogeneity within the reaction vessel 3, significantly improving mixing efficiency, increasing mass transfer, and also helping to reduce the impact of shear force on cells, preventing cell deposition, ensuring that all cells are in a uniform suspension state, and also facilitating the rational use of energy. In conjunction with the upper and lower layer combined blades, it creates a more ideal growth environment for fragile cells, and no additional protective agents are needed during cell culture, ensuring cell viability and culture density.

[0061] like Figures 12-13As shown, both the upper part 29 and the lower part 33 of the reaction vessel 3 are equipped with baffles 34. Correspondingly, the upper end 38 and the lower end 39 of the disposable biological culture bag 17 are also equipped with baffle bags 35 that match the baffles 34. One side of the baffles 34 in the upper part 29 of the reaction vessel 3 is connected to the side wall 40 and extends downward, while one side of the baffles 34 in the lower part 33 is connected to the side wall 40 and extends upward. The baffles 34 in the upper part 29 can be detached along the inner wall 37 of the reaction vessel 3. When both the upper part 29 and the lower part 33 of the reaction vessel 3 are equipped with baffles 34, the upper and lower baffles 34 are combined with two layers of impellers, resulting in less shear force, better mass transfer effect, and significantly improved cell culture effect. The baffle 34 on the upper part 29 can be removed along the inner wall 37 of the reaction vessel 3 (it can be removed upwards along the baffle groove 40), which facilitates the installation and removal of the disposable cell culture bag 17. When the disposable cell culture bag 17 is being fitted downwards onto the baffle 34 on the lower part 33, the baffle 34 on the upper part 29 can be removed first to prevent it from affecting the installation of the disposable cell culture bag 17. After the disposable cell culture bag 17 is installed, the baffle 34 can be fitted in from above to complete the installation. At the same time, the baffle 34 also helps to fix the cell culture bag 17, preventing the cell culture bag 17 from shifting with the flow of the culture medium, and improving the batch-to-batch consistency of cell culture results.

[0062] Example 2: Effects of different blade shape combinations This embodiment uses the stirred bioreactor provided in Example 1 for cell culture. The cultured cells are human lymphocytes (PBMCs). The specific culture process is as follows: PBMCs were inoculated into RPMI 1640 medium at a density of 1 x 10⁻⁶. 6 Cells / ml were cultured in a medium supplemented with cholesterol, vitamin E, anti-CD3 / CD28 conjugated magnetic beads, and IL-2 solution. The cholesterol concentration was 2 ml / L, vitamin E was 3 ml / L, and IL-2 was 200 IU / mL. The ratio of anti-CD3 / CD28 conjugated magnetic beads to cells was 1:1. RPMI 1640 medium was added to a 5L fermenter at 37°C, CO2 aeration was 5%, dissolved oxygen was 50%, pH was 7.2, and the stirring speed was 50 rpm. The culture time was 10 days, with cell counting and 0.1L of medium added every 2 days.

[0063] The shapes of the paddles in the cell culture bags were designed according to the five cases shown in Table 1. The effects of different paddle shapes on the cell culture effect were investigated, and the cell viability and cell density after culture were detected. The cell viability was detected by flow cytometry using propidium iodide (PI) dye, and the cell density was detected by cell counting. The results are shown in Table 1.

[0064] Table 1. Effects of different blade shapes on cell culture efficiency

[0065] As shown in Table 1, compared with using a single type of blade, the combination of two layers of blades can significantly improve the cell culture effect. It can also be seen that different combinations of blade shapes result in significant differences in culture effect. The preferred combination of blade shapes, with the upper layer being a marine-style blade and the lower layer being a gas dispersion blade, is the most effective for culturing fragile cells. This may be because such a blade combination creates a more suitable culture environment for fragile cells, resulting in higher cell activity and cell density.

[0066] Example 3: Hollow-out design This embodiment uses the stirred bioreactor provided in Example 1 and performs cell culture according to the method provided in Example 2. The cultured cells are human lymphocytes (PBMCs). The impellers in the cell culture bag are designed according to four different cases as shown in Table 2, depending on whether they have perforations. The effect of different perforation settings in the impellers on the cell culture effect is investigated. The perforation diameter is 1.1 cm and the porosity is 21% evenly distributed. The cell viability and cell density after culture are detected, and the results are shown in Table 2.

[0067] Table 2. Effects of different blade perforation designs on cell culture results

[0068] As shown in Table 2, the perforated design of the lower blades helps improve cell culture results, while the perforated design of the upper blades has a negative impact. Compared with the case where only the upper blades are perforated or all blades are perforated, the case where the lower blades are perforated is the most effective for culturing fragile cells. This is likely because the perforated lower blades help reduce the influence of shear forces and provide a gentler mixing fluid environment suitable for the culture of fragile cells. In contrast, the perforated upper blades may affect mass transfer, reduce cell viability, and be detrimental to cell culture.

[0069] Example 4: Spoiler Installation This embodiment uses the stirred bioreactor provided in Example 1 and performs cell culture according to the method provided in Example 2. The cultured cells are human lymphocytes (PBMCs). Depending on whether a baffle is installed in the reaction vessel, four different designs are presented as shown in Table 3. The effect of the baffle placement in different impeller blades on the cell culture effect is investigated. The cell viability and cell density after culture are detected, and the results are shown in Table 3.

[0070] Table 3. Effects of different baffle settings on cell culture results

[0071] As shown in Table 3, the installation of baffles helps improve cell culture results, and the placement of baffles in different parts of the reaction vessel has different effects. Compared with baffles placed only in the upper or lower part of the reaction vessel, the use of baffles in both the upper and lower parts of the reaction vessel is optimal for culturing fragile cells. The resulting complex mixed fluid environment provides a milder and more comfortable fluid environment for the cells, making it more suitable for the culture of fragile cells.

[0072] Example 5: Effect of Pluronic F-68 Protectant This embodiment uses the stirred bioreactor provided in Example 1 and performs cell culture according to the method provided in Example 2. The cultured cells are human lymphocytes (PBMCs). The culture medium is designed according to the four cases shown in Table 4. The effect of different culture media on the cell culture effect is investigated, and the cell viability and cell density after culture are detected. The results are shown in Table 4.

[0073] Table 4. Effects of different culture media on cell culture efficiency

[0074] As shown in Table 4, while adding Pluronic F-68 protectant alone can achieve good cell culture results, it also generates a large number of bubbles, which is not conducive to precise control of the cell culture process. Replacing Pluronic F-68 protectant with cholesterol and vitamin E can significantly improve cell activity and cell density. Therefore, it is preferable to use the method of not adding Pluronic F-68 protectant and adding cholesterol and vitamin E to ensure the effect of fragile cell culture.

[0075] The application of this invention is not limited to this. It can be extended to other applications, such as those related to environmental protection. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of this invention; therefore, the scope of protection of this invention should be determined by the scope defined in the claims.

Claims

1. A stirred bioreactor, characterized in that, It includes a base and a reaction vessel, which can be replaced with a vessel of different volume specifications, all of which can be combined with the base for use.

2. The stirred bioreactor as described in claim 1, characterized in that, The volume of the reaction vessel is 5~20L; the upper surface of the base is provided with a protrusion, and the bottom of the reaction vessel is provided with a groove, the protrusion can be combined with the groove to fix the reaction vessel.

3. The stirred bioreactor as described in claim 1, characterized in that, The base surface is also provided with a fixing nut on the protrusion for locking the reaction vessel after it is joined.

4. The stirred bioreactor as described in claim 1, characterized in that, It also includes a stirring device, a support frame, and a control device; the support frame is located above the base and includes a vertical arm and a horizontal arm, the vertical distance of which is adjustable; the control device is connected to the reaction vessel through the support frame.

5. The stirred bioreactor as described in claim 4, characterized in that, The stirring device includes a stirring drive and a stirring paddle. The stirring paddle is located inside the biological culture bag, and the stirring drive is located on the cross arm of the support frame and is always directly facing the middle of the base. The stirring paddle includes a stirring shaft and two layers of combined blades, with the upper layer having axial flow blades and the lower layer having radial flow blades.

6. The stirred bioreactor as described in claim 5, characterized in that, The axial flow blades are marine-type blades with large oblique blades; the radial flow blades are gas dispersion blades with arc-shaped impellers.

7. The stirred bioreactor as described in claim 6, characterized in that, The radial flow blades have perforations with a perforation diameter of 0.8~1.6cm and a porosity of 5~20%.

8. The stirred bioreactor as described in claim 4, characterized in that, The stirring drive device and the stirring paddle are magnetically connected; a first magnetic block is provided below the stirring drive device, and the bottom surface of the first magnetic block is connected to a second magnetic block at the upper end of the stirring shaft; a hexagonal groove is provided inward at the middle position of the bottom surface of the first magnetic block, and a hexagonal protrusion that can be connected to the hexagonal groove is provided at the middle position of the upper surface of the second magnetic block; the size of the bottom surface of the first magnetic block is greater than or equal to the size of the upper surface of the second magnetic block.

9. The stirred bioreactor as described in claim 8, characterized in that, The reaction vessel is equipped with a raised baffle plate, and the biological culture bag is equipped with a baffle bag that extends into the interior of the biological culture bag and accommodates the baffle plate. The baffle bag is sealed to the outer wall of the biological culture bag.

10. The stirred bioreactor as described in claim 9, characterized in that, The reaction vessel is equipped with baffles at both the upper and lower parts. The upper baffle extends downward and the lower baffle extends upward. The upper baffle can be removed along the inner wall of the reaction vessel.

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