Stirring type cell culture bag with spoiler

Abstract

The invention provides a stirring type cell culture bag with a spoiler, which is characterized in that the spoiler capable of flexibly rotating is arranged in a disposable stirring type cell culture bag and comprises a cylindrical main body and an arc-shaped fan blade integrally extending from the main body, so that a large eddy current generated by stirring is decomposed into finer and more uniform flow, a mixing dead angle is broken, and the mixing efficiency is improved. The generation of a high shear force area is avoided; the structure of the spoiler is improved, and alternate annular bulges and annular grooves are arranged, so that cells and culture media are prevented from entering and blocking a gap between the spoiler and the fixed shaft; meanwhile, appropriate materials are screened to be used for preparing the spoiler and the stirring paddle, so that the shearing force borne by the cells is remarkably reduced while the mixing efficiency is ensured, and the adhesion of the cells or nutrient substances is remarkably reduced, so that the cell viability, the cell density and the cell final yield are improved.

Description

Technical Field

[0001] This invention relates to cell culture devices in the field of biotechnology, specifically to a stirred cell culture bag with a flow-disrupting element. 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] Disposable bioreactors and culture bags are widely used in modern biopharmaceutical and cell therapy fields due to their advantages such as no need for cleaning and sterilization and avoidance of cross-contamination. The stirred cell culture bag is its core component, using an internal agitator to stir the culture medium and provide a uniform nutrient and gas environment for the cells. However, existing stirred culture bags have the following technical challenges: 1. Shear damage: The turbulence and vortices generated by stirring can cause mechanical damage to cells, especially shear-sensitive cells (such as CAR-T cells and stem cells), leading to decreased cell viability and yield; 2. Uneven mixing and dead zones: Simple agitators in flexible bags easily create mixing dead zones, resulting in uneven distribution of nutrients and metabolic waste; 3. The stirring process easily forms vortices, where the liquid rises along the tank wall due to centrifugal force and falls in the central area, increasing shear force and hindering the thorough mixing of cells and nutrients; 4. Cell adsorption: Traditional materials (such as certain plastics or unoptimized silicone) may cause non-specific adsorption of cells or nutrients, affecting cell growth and product recovery.

[0005] While existing technologies include adding baffles to rigid reactors as flow disruptors, these structures are crude and generate high shear forces, which are still unfavorable for the flexible, shear-sensitive environment of culture bags. Simply improving the shape or material of the agitator often fails to balance mixing efficiency with low shear forces. Therefore, there is an urgent need for a novel design of stirred cell culture bags that can systematically solve the above problems. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a stirred cell culture bag with a baffle. By incorporating a flexibly rotating baffle, including a cylindrical body and arc-shaped fan blades extending integrally from the body, the large eddies generated by stirring are decomposed into finer and more uniform flows, breaking up mixing dead zones and avoiding the generation of high shear force regions. The structure of the baffle is improved by incorporating alternating annular protrusions and annular grooves to prevent cells and culture medium from entering and clogging the gap between the baffle and the fixed shaft. Furthermore, suitable materials are selected for preparing the baffle and the stirring paddle, significantly reducing the shear force on cells and significantly reducing cell or nutrient adhesion while ensuring mixing efficiency, thereby improving cell viability, density, and final yield.

[0007] On one hand, the present invention provides a stirred cell culture bag, including a flexible bag body and a stirring paddle disposed within the bag body, wherein at least two flow-disrupting elements are disposed within the flexible bag body; The turbulence-disrupting element is rotatable and located outside the rotation path of the stirring paddle; The spoiler includes a cylindrical body and arc-shaped fan blades extending integrally from the body.

[0008] In stirred bioreactors, without flow disruptors, power is easily wasted in unproductive vortices, leading to low mass transfer and mixing efficiency. With flow disruptors, power is effectively converted into mixing, mass transfer, and heat exchange, creating a more uniform, stable, and controllable optimal growth environment for cells. Therefore, flow disruptors are an indispensable key technology for achieving large-scale, high-density cell culture in stirred bioreactors (such as the production of monoclonal antibodies, viral vectors, and cell therapy products).

[0009] In existing stirred bioreactors, rigid flow-disrupting components (such as baffles) are usually directly installed on the inner wall of the outer rigid reaction vessel. This design not only brings trouble to the design and installation of cell culture bags (the culture bags need to be fitted with corresponding pockets to cover the baffles), but the rigid baffles also bring high shear forces, which have an adverse effect on cell culture.

[0010] This invention features a specially shaped flow-disrupting element placed directly at the bottom of the cell culture bag. This allows the cells to directly contact the flow-disrupting element during cell culture, rather than the culture bag itself which is fitted with a baffle. This results in superior mixing, mass transfer, and heat exchange. Furthermore, the flow-disrupting element is primarily cylindrical with curved fan blades on its outer surface. Unlike rigid baffles that forcibly obstruct the fluid, this design gently guides and divides the fluid through its streamlined profile, breaking down the large eddies generated by stirring into smaller, more uniform microflows. This eliminates mixing dead zones and avoids the formation of high-shear-force regions. Its location outside the stirring paddle path cleverly intervenes in the low-velocity zone below the cell culture bag.

[0011] The improvement of the flow control component provided by this invention is that the flow control component itself can also rotate freely. This is a very ingenious and advanced design that completely changes the "passive blocking" role of the traditional fixed flow control component (baffle) and makes it an "active flow control element", thereby bringing a series of unique hydrodynamic effects and operational advantages.

[0012] Traditional fixed flow disruptors work by interrupting tangential flow, while this flow disruptor achieves a similar but gentler purpose by co-moving with the fluid. It can effectively reduce local shear forces and energy dissipation, change the vortex structure, achieve more uniform mixing, and enable adaptive flow control. When used in cell culture, it can better protect cells, improve mixing uniformity, reduce gradients, reduce clumping and adhesion, and reduce starting torque and energy consumption, thereby significantly improving cell culture results.

[0013] Furthermore, the curvature of the arc-shaped fan blades is adapted to the rotation direction of the stirring paddle, which is used to guide the culture medium to generate gentle axial and radial flow.

[0014] The arc-shaped fan blade of the turbulence-disrupting component provided by this invention is not a simple vertical baffle; it is more like a "guide plate." Its curved surface is carefully designed to smoothly receive the liquid flow from the agitator and change its direction.

[0015] The curvature of the arc-shaped fan blades is "matched" with the rotation direction of the agitator, which means that the curvature of the fan blades is to conform to and guide the mainstream fluid generated by the agitator, thereby achieving more efficient and gentler mixing and mass transfer with less energy consumption.

[0016] Furthermore, the turbulence-disrupting component includes a bushing and a fixed shaft, the bushing being rotatably mounted on the fixed shaft, and the fixed shaft being fixed to the inner wall of the flexible bag.

[0017] Furthermore, the bushing is mounted on the cylindrical body, and a bearing bushing is provided between the bushing and the fixed shaft; the upper end of the fixed shaft is provided with a protruding protrusion, and the upper end of the bushing is provided with a protrusion groove.

[0018] The bearing bushing design reduces friction between the bushing and the fixed shaft, allowing for freer rotation under fluid scouring.

[0019] In some embodiments, the bearing bushing is a ball bearing, which can greatly reduce rotational resistance and allow the flexible agitator to respond more sensitively and freely to fluid movement.

[0020] The combination design of the bump and the bump groove allows the cylindrical body to rotate after being combined with the fixed shaft, without slipping off the fixed shaft.

[0021] Furthermore, the outer interface of the bushing after it is joined with the fixed shaft is provided with alternating annular protrusions and annular grooves to form a tortuous channel.

[0022] There is a gap between the bushing and the fixed shaft. The bearing bushing can also play a sealing role, which can prevent cell culture medium from entering the gap during cell culture. However, if the bearing bushing is in direct contact with the cell culture medium, as the culture time increases, there is still a possibility that the cell culture medium will enter the gap and affect the flow-changing components.

[0023] This invention creates a tortuous channel by setting alternating annular protrusions and annular grooves at the outer interface between the bushing and the fixed shaft. When cells and culture medium attempt to pass through this tortuous path, they encounter multiple throttling and expansion events. Each gap point generates local eddies and pressure losses, making it difficult for the fluid to flow stably throughout the entire tortuous channel. In other words, the tortuous channel creates significant resistance to the passage of cells or nutrients, thus achieving a good anti-clogging effect.

[0024] Furthermore, the number of the turbulence-disrupting components is 2 to 4, which are evenly arranged on the bottom inner side of the flexible bag body; each turbulence-disrupting component is evenly arranged with arc-shaped fan blades around its perimeter, the curvature of the arc-shaped fan blades is opposite to the rotation direction of the stirring paddle, the arc of the arc-shaped fan blades is a quarter circle, and its diameter is consistent with the diameter of the cylindrical body.

[0025] The rotation direction of the curved fan blades refers to the direction in which the fan blades are set. Although the turbulence component rotates on its own axis and its rotation direction is not controlled, the curvature direction of its fan blades can be set. Setting the curvature direction of the fan blades to be opposite to the rotation direction of the agitator can effectively improve the turbulence effect.

[0026] Optimizing the number and length of fan blades can help improve the turbulence effect, which is more conducive to cell proliferation.

[0027] Furthermore, the agitator is made of a first biological material, the turbulence element is made of a second biological material, and the first biological material is different from the second biological material; the hardness of the first biological material is higher than that of the second biological material.

[0028] To achieve efficient and gentle mixing while maximizing the simulation of the physiological environment and protecting shear-sensitive materials (such as cells and proteins), this invention optimizes the hardness of the first biomaterial and the second flexible material. This allows the agitator and the turbulence-disrupting element to bring synergistic advantages through different combinations of hardness, optimizing shear force distribution and protecting sensitive cells.

[0029] The first biomaterial used in the agitator is softer than metal or other rigid materials, yet retains a certain degree of rigidity to ensure mixing efficiency. When it comes into contact with cells or clumps, it provides better cushioning than rigid materials, reducing instantaneous impact damage. The second flexible material, an even softer material, acts as a low-shear force generator. When fluid flows over its soft surface, it generates gentle, high-frequency disturbances that break up any potential cell clumps, ensuring dissolved oxygen supply without damaging individual cells due to excessive force. The combination of these two materials achieves more efficient multi-scale mixing, resulting in higher and more uniform mixing efficiency than flexible agitators with single-hardness properties or purely rigid agitators.

[0030] In tissue engineering or 3D cell culture, the extracellular matrix (ECM) exhibits varying degrees of stiffness and elasticity. Using flexible materials of different stiffnesses to construct the internal environment of a bioreactor can better simulate the complex mechanical microenvironment in vivo, thus promoting cell growth and tissue formation. This invention employs a stirring impeller to propel the entire cell culture bag, combined with a flow-disrupting element to gently handle each delicate cell. In other words, this invention makes the stirring impeller stiffer than the flow-disrupting element, representing an optimal design achieved through careful mechanical calculations and functional allocation.

[0031] Furthermore, the first biomaterial and the second biomaterial are any one or more of PE (polyethylene), PP (polypropylene), PC (polycarbonate), PET (polyethylene terephthalate), and PEK (polyetherketone).

[0032] Furthermore, the first biomaterial is PET (polyethylene terephthalate), and the second biomaterial is PE (polyethylene).

[0033] Studies have shown that combinations of baffles and agitators made from different materials exhibit varying effects in cell culture. This invention screened for optimal first and second biomaterials: PET as the first biomaterial and PE as the second. The second biomaterial has a lower hardness than the first biomaterial. This combination achieves superior synergistic effects, significantly improving cell viability, density, and final yield.

[0034] Furthermore, the surfaces of the turbulence-causing element and the agitator are subjected to plasma or ultraviolet passivation treatment to form a smooth surface layer capable of dynamically binding water molecules.

[0035] The turbulence-disrupting components and stirring paddles provided by this invention are made of specially treated flexible biomaterials, which have no byproducts, are purer, and have higher biocompatibility. They can form an extremely smooth and chemically inert dynamic hydration layer on the outermost layer of the material, minimizing cell or protein adhesion. Furthermore, when fluid flows through, this flexible surface can buffer stress, thereby significantly reducing shear force from both physical and chemical perspectives.

[0036] In another aspect, the present invention provides a cell culture method, which uses a stirred cell culture bag as described above for culturing.

[0037] The present invention has the following beneficial effects: 1. Significantly reduced shear force: The combination of a flow-disrupting component with a specific shape and structure and a low-shear-force material, and the ability of the flow-disrupting component to rotate flexibly under the action of fluid, reduces shear force and improves mixing efficiency, resulting in a synergistic effect of "1+1>2". It works together from both the flow field design and the contact interface to provide an extremely mild growth environment for cells.

[0038] 2. High efficiency in preventing clogging: By setting an annular protrusion and an annular groove at the outer interface of the baffle, cells and culture medium are prevented from entering and clogging the gap between the baffle and the fixed shaft; 3. Improved culture efficiency: Suitable materials were selected for the preparation of the turbulence-inducing components and agitators. The gentle yet effective mixing ensured the uniform distribution of nutrients and dissolved oxygen, resulting in a significant improvement in cell viability and final cell density. 4. Simple and reliable structure: All components are integrated into the disposable bag, which is compact, easy to mass-produce, and fully retains the inherent advantages of disposable technology. Attached Figure Description

[0039] Figure 1 A schematic diagram of the appearance of a disposable stirred cell culture bag; Figure 2 A perspective view of the internal structure of a disposable stirred cell culture bag. Figure 3This is a schematic diagram of the spoiler's structure; Figure 4 Exploded view of the spoiler; Figure 5 This is a schematic diagram of the bushing structure; Figure 6 This is a schematic diagram of a fixed shaft. Figure 7 This is a cross-sectional view of the spoiler; Figure 8 This is a cross-sectional exploded view of the spoiler; Figure 9 This is an enlarged view of the winding passage; Figure 10 This is an enlarged view of the agitator. Detailed Implementation

[0040] To describe the present invention more specifically, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are merely illustrative of how the present invention is implemented and do not limit the specific scope of the present invention. The scope of the present invention is defined in the claims.

[0041] Example 1: The stirred cell culture bag with a turbulence device provided by the present invention The disposable stirred cell culture bag 1 provided in this embodiment, such as Figures 1-10 As shown. According to Figures 1-2As can be seen, the stirred cell culture bag 1 includes a flexible bag body 2 and a stirring paddle 3 disposed within the bag body. The flexible bag body 2 contains at least two flow-disrupting elements 4. These elements 4 are rotatable and located outside the rotation path of the stirring paddle 3. Each flow-disrupting element 4 includes a cylindrical body 5 and an arc-shaped fan blade 6 extending integrally from this body. By directly placing a flow-disrupting element 4 of a specific shape at the bottom 7 inside the cell culture bag 1, the cells directly contact the flow-disrupting element 4 during cell culture, rather than contacting the culture bag fitted with a baffle, thus achieving better mixing, mass transfer, and heat exchange effects. Simultaneously, the flow-disrupting element 4, with its cylindrical body 5 and arc-shaped fan blades 6 on its outer side, gently guides and divides the fluid through its streamlined profile, decomposing the large eddies generated by stirring into finer, more uniform microflows. This breaks up mixing dead zones and avoids the generation of high-shear force regions. Its location outside the stirring paddle path cleverly intervenes in the low-velocity region below the cell culture bag (bottom 7). The improvement of the flow-disrupting element 4 lies in its ability to rotate freely, making it an "active flow control element" that brings a series of unique hydrodynamic effects and operational advantages. By co-moving with the fluid, the flow-disrupting element 4 achieves a similar but gentler effect, effectively reducing local shear forces and energy dissipation, altering the vortex structure for more uniform mixing, and enabling adaptive flow control. In cell culture, it better protects cells, improves mixing uniformity, reduces gradients, and also reduces clumping and adhesion, lowering start-up torque and energy consumption, thus significantly improving cell culture results. The curved fan blade 6 has a curvature direction opposite to the rotation direction of the agitator 3, guiding the culture medium to generate gentle axial and radial flow. While the rotation direction of the flow-disrupting element 4 is not controlled, the curvature direction of its curved fan blade 6 can be set. Setting the curvature direction of the curved fan blade 6 opposite to the rotation direction of the agitator 3 effectively enhances the turbulence effect.

[0042] like Figure 3 As shown, the arc-shaped fan blade 6 of the baffle 4 is not a simple vertical baffle. Its curved surface is carefully designed to smoothly receive the liquid flow from the agitator 3 and change its direction. The curvature direction of the arc-shaped fan blade 6 is adapted to the rotation direction of the agitator 3, which means that the curvature direction of the arc-shaped fan blade 6 is to conform to and guide the mainstream fluid generated by the agitator 3, thereby achieving more efficient and gentler mixing and mass transfer with less energy consumption.

[0043] like Figures 4-6 As shown, the turbulence-disrupting component 4 is rotatably mounted on the fixed shaft 8, which is fixed to the inner wall 9 of the flexible bag body 1.

[0044] Preferably, the cylindrical body 5 of the spoiler 4 has a bushing 10 at its center, which is fitted onto the fixed shaft 8. A bearing bushing 11 is provided between the bushing 10 and the fixed shaft 8. The upper end of the fixed shaft 8 has a protruding protrusion 12, and the upper end of the bushing 10 has a protrusion groove 14 at the assembly position 13. The combination design of the protrusion 12 and the protrusion groove 14 ensures that the cylindrical body 5 can rotate after being combined with the fixed shaft 8, without slipping off the fixed shaft 8. Since the bushing 10 is made of a flexible material and has a certain degree of elasticity, it can be smoothly fitted onto the fixed shaft 8 under a certain pressure, and the protrusion 12 and the protrusion groove 14 can be smoothly positioned and assembled.

[0045] In this embodiment, the bearing bushing 11 is a ball bearing, which can greatly reduce rotational resistance and allow the flexible flow deflector 4 to rotate more freely under fluid scouring, and to respond more sensitively and freely to fluid movement.

[0046] like Figures 7-9 As shown, the outer interface position 16 after the bushing 10 is combined with the fixed shaft 8 is provided with alternating annular protrusions 17 and annular grooves 18, forming a tortuous channel 19.

[0047] A gap 15 exists between the bushing 10 and the fixed shaft 8. The bearing bushing 11 acts as a seal, preventing cell culture medium from entering the gap 15 during cell culture. However, if the bearing bushing 11 directly contacts the cell culture medium, the cell culture medium may still enter the gap and affect the flow-delaying element 4 as the culture time increases. A tortuous channel 19 is formed by alternating annular protrusions 17 and annular grooves 18 at the outer interface 16 between the bushing 10 and the fixed shaft 8. The tortuous channel 19 includes a first serrated flow channel 22 on the outer wall of the fixed shaft 8 and a second serrated flow channel 23 on the inner wall of the bushing 10. Preferably, the first serrated flow channel 22 has a first annular protrusion 24 with a downward-curving head, and the second serrated flow channel 23 has a second annular protrusion 25 with an upward-curving head. When cells and culture medium attempt to pass through this tortuous path, they encounter multiple throttling and expansion events. Each gap point generates local eddies and pressure losses, making it difficult for the fluid to flow stably throughout the entire tortuous channel 19. In other words, the tortuous channel 19 creates a huge resistance to the passage of cells or nutrients, thus achieving a good anti-blockage effect.

[0048] like Figures 3-5 As shown, there are 2 to 4 flow-dispersing components 4, evenly distributed inside the bottom 7 of the flexible bag body 2; each flow-dispersing component 4 has evenly distributed arc-shaped fan blades 6 around its perimeter, the arc 20 of which is a quarter circumference, and its diameter is consistent with the diameter of the cylindrical body 5. By optimizing the number and length of the arc-shaped fan blades 6, the flow-dispersing effect can be improved, thus being more conducive to cell proliferation. Figure 10The blades 26 of the stirring paddle 3 are fan-shaped 27, forming a 180-degree fan-shaped structure. There are two sets of stirring paddles 3, one upper and one lower. Each set of stirring paddles 3 includes three evenly arranged blades 26. The adjacent blades 26 are arranged at a 45-degree angle. The radius 28 of the fan shape 27 of the blades 26 is one-third of the transverse width of the flexible bag body 2.

[0049] like Figure 2 As shown, the base 21 of the fixed shaft 8 of the spoiler 4 is firmly and evenly fixed to the inner wall of the flexible bag body 2 near the bottom 7 by radio frequency welding process.

[0050] Preferably, the impeller 3 (the material of the impeller blades 26 of the impeller 3) is made of a first biomaterial, and the baffle 4 (including the material of the cylindrical body 5 and the arc-shaped fan blades 6) is made of a second biomaterial, and the first biomaterial is different from the second biomaterial; the hardness of the first biomaterial is higher than that of the second biomaterial. Although the first biomaterial used in the impeller 3 is softer than metal or other rigid materials, it still maintains a certain degree of hardness to ensure mixing efficiency. When it comes into contact with cells or clumps, it provides better cushioning than rigid materials, reducing instantaneous impact damage. The baffle 4 uses a softer second flexible material, which is equivalent to a low-shear force generator. When the fluid flows over its soft surface, it generates gentle, high-frequency disturbances that can break up any cell clumps that may form, ensuring the supply of dissolved oxygen, but without damaging individual cells due to excessive force. The combination of the two achieves more efficient multi-scale mixing, with higher and more uniform mixing efficiency than a flexible stirrer of single hardness or a fully rigid stirrer. In the cell culture bag, the impeller 3 is used to drive the overall mixing, and the baffle 4 is also needed to gently handle each fragile "cell". In other words, making the agitator 3 stiffer than the baffle 4 is an optimal design based on careful mechanical calculations and functional allocation. The agitator 3 has two parts, one at the top and one at the bottom, with an aeration disc 29 at the bottom.

[0051] Preferably, the first biomaterial is PET (polyethylene terephthalate), and the second biomaterial is PE (polyethylene). The combined effects of baffles and agitators made from different materials vary in cell culture. This embodiment selected the optimal first and second biomaterials: PET as the first biomaterial and PE as the second. The second biomaterial has a lower hardness than the first biomaterial. This combination achieves better synergistic effects, significantly improving cell viability, density, and final yield.

[0052] Studies have shown that combinations of baffles and agitators made from different materials exhibit varying effects in cell culture. This embodiment screened the optimal first and second biomaterials to achieve better synergistic effects, significantly improving cell viability, density, and final yield.

[0053] Preferably, the surfaces of the turbulence-disrupting components and the agitator are subjected to plasma or ultraviolet passivation treatment. In this embodiment, it is preferable to treat these components with argon plasma after injection molding, with a treatment power of 500W and a treatment time of 2 minutes, so that the surface contact angle is reduced from 110° to below 40°, forming a superhydrophilic dynamic hydration layer, minimizing cell or protein adhesion, and the flexible surface can buffer stress when fluid flows through, thereby significantly reducing shear force from both physical and chemical levels.

[0054] It should also be noted that the flexible bag body 2 is made of PE material, so the second biomaterial of the flow-dispersing component is also made of PE material, making it easier to weld the flow-dispersing component to the flexible bag body 2.

[0055] Experimental Example 2: Cell Culture Method Using a Stirred Cell Culture Bag This embodiment uses the disposable stirred cell culture bag with a baffle provided in Example 1, which is placed inside a reaction vessel to form a bioreactor for cell culture. The reaction vessel provides support and temperature control for the disposable stirred cell culture bag. The cultured cells are human embryonic kidney cells HEK-293. The specific culture process is as follows: HEK-293 was inoculated into FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific) at a density of 0.6 × 10⁻⁶. 6 The culture medium volume was 2L in a 5L fermenter, the temperature was 37℃, the CO2 aeration rate was 8%, the dissolved oxygen content was 40%, the pH was 7.1, the stirring speed was 100 rpm, and the culture time was 5 days. Cell viability and cell density were measured after culture. Cell viability was detected by flow cytometry using propidium iodide (PI) dye, and cell density was measured by cell counting. After 5 days of culture, the cell viability of HEK-293 was 95%, and the cell density was 31.2 × 10⁻⁶ cells / ml. 6 The cell / ml concentration and the minimal cell adhesion on the surfaces of the agitator and turbulence components demonstrate its excellent low adsorption properties.

[0056] Experiment Example 3: The Effect of Rotatable and Non-Rotatable Baffles on Cell Culture Efficiency This embodiment uses the disposable stirred cell culture bag with a flow-disrupting element provided in Example 1. The flow-disrupting element is configured in two ways: 1. Rotatable (Example 1); 2. Fixed and non-rotatable, with all other conditions remaining the same. Human embryonic kidney cells HEK-293 were cultured according to the method described in Example 2, and the effect of different flow-disrupting elements on the culture effect of human embryonic kidney cells HEK-293 was investigated. The test results are shown in Table 1.

[0057] Table 1. Effects of different flow-changing components on cell culture results

[0058] As shown in Table 1, whether the baffle rotates or not has a direct impact on the cell viability and cell density of HEK-293 cells after 5 days of culture. Using a flexible baffle can significantly improve cell culture viability and cell density.

[0059] Example 4: Selection of the number of arc-shaped fan blades on the spoiler This embodiment uses the disposable stirred cell culture bag with a baffle provided in Example 1. The baffle is rotatable, and the arc-shaped fan blades are set according to the six cases shown in Table 2, while all other conditions remain the same. Human embryonic kidney cells HEK-293 are cultured according to the method described in Example 2, and the effect of the number of arc-shaped fan blades in the baffle on the culture effect of human embryonic kidney cells HEK-293 is investigated. The test results are shown in Table 2.

[0060] Table 2. Effect of the number of arc-shaped fan blades on the cell culture effect of the aerodynamic components.

[0061] As shown in Table 2, the number of arc-shaped fan blades on the baffle directly affects the cell viability and cell density of HEK-293 cells after 5 days of culture. Compared with the baffle without arc-shaped fan blades, the addition of arc-shaped fan blades can significantly improve the cell culture effect. This may be because the addition of arc-shaped fan blades helps to improve mass transfer efficiency. At the same time, the optimal number of arc-shaped fan blades is 4 blades evenly arranged around the cylindrical body, at which time the cell viability and cell density are both higher.

[0062] Example 5: The effect of tortuous channel design on cell culture results This embodiment uses the disposable stirred cell culture bag with a flow-disrupting element provided in Example 1. The design of the tortuous channel at the outer interface after the bushing and the fixed shaft are combined is as follows: 1. A tortuous channel is provided, and the first serrated channel has a first annular protrusion with its head bent downwards, and the second serrated channel has a second annular protrusion with its head bent upwards (Example 1); 2. A tortuous channel is provided, and both the first and second annular protrusions are kept horizontal; 3. No tortuous channel, and all other conditions are kept consistent. Human embryonic kidney cells HEK-293 are cultured according to the method described in Example 2, and the effect of the tortuous channel design on the culture effect of human embryonic kidney cells HEK-293 is investigated. The test results are shown in Table 3.

[0063] Table 3. Effects of tortuous channel design on cell culture outcomes

[0064] As shown in Table 3, compared with the case without a tortuous channel, the cell viability and cell density of HEK-293 human embryonic kidney cells were significantly increased after the tortuous channel was set. The reason may be that the tortuous channel ensures that the rotation of the baffle is not affected by the extension of the culture time. Without the tortuous channel, after a period of culture, the culture medium may seep into the gap between the bushing and the fixed shaft, affecting the rotation of the baffle and thus affecting the cell culture effect.

[0065] Meanwhile, when a tortuous channel is set up, bending the head of the first annular protrusion of the first serrated flow channel downward and bending the head of the second annular protrusion of the second serrated flow channel upward can further improve the cell culture effect. The reason may be that the structural design of the first and second annular protrusions can make the tortuous channel more effective in blocking the infiltration of culture medium, thereby helping to further improve cell viability and cell density.

[0066] Example 6: Selection of materials for impellers and turbulence components This embodiment uses the disposable stirred cell culture bag with a baffle provided in Example 1. The materials of the stirring paddle and the baffle are designed according to different combinations shown in Table 4. Among them, PE (polyethylene), PP (polypropylene), PC (polycarbonate), PET (polyethylene terephthalate), and PEK (polyetherketone) are used. The hardness order is PE is the softest, followed by PP, then PC and PEK, and PET is the hardest. Other conditions are kept consistent. Human embryonic kidney cells HEK-293 are cultured according to the method described in Example 2. The effect of different material designs of the stirring paddle and the baffle on the culture effect of human embryonic kidney cells HEK-293 is investigated. Cell adhesion on the surface of the baffle and the stirring paddle is observed under a microscope. The test results are shown in Table 4.

[0067] Table 4. The Influence of Material Design of Agitator and Baffle Components on Cell Culture Efficiency

[0068] As shown in Table 4, using different materials to prepare the flow-dispersing components and stirring paddles significantly affects the cell density and cell viability of HEK-293 human embryonic kidney cells after culture. This is likely because different materials have different hardness, surface charges, and biocompatibility, which directly affect the cell culture effect. The preferred material combinations are groups 1, 3, and 5, which exhibit high cell viability and density, and all achieve excellent low-adsorption characteristics after plasma treatment. Among these, the third material combination is the most preferred for preparing disposable stirred cell culture bags with flow-dispersing components, and it is also the optimal choice from a cost perspective.

[0069] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A stirred cell culture bag, comprising a flexible bag body and a stirring paddle disposed within the bag body, characterized in that: The flexible bag is equipped with at least two flow-disrupting elements. The turbulence-disrupting element is rotatable and located outside the rotation path of the stirring paddle; The spoiler includes a cylindrical body and arc-shaped fan blades extending integrally from the body.

2. The stirred cell culture bag as described in claim 1, characterized in that, The curved surface of the arc-shaped fan blade is adapted to the rotation direction of the stirring paddle, which is used to guide the culture medium to produce a gentle axial and radial flow.

3. The stirred cell culture bag as described in claim 2, characterized in that, The turbulence-disrupting component includes a bushing and a fixed shaft. The bushing is rotatably mounted on the fixed shaft, and the fixed shaft is fixed to the inner wall of the flexible bag.

4. The stirred cell culture bag as described in claim 3, characterized in that, The bushing is mounted on a cylindrical body, and a bearing bushing is provided between the bushing and the fixed shaft; the upper end of the fixed shaft is provided with a protruding protrusion, and the upper end of the bushing is provided with a protrusion groove.

5. The stirred cell culture bag as described in claim 4, characterized in that, The outer interface of the bushing after it is joined with the fixed shaft is provided with alternating annular protrusions and annular grooves, forming a tortuous channel.

6. The stirred cell culture bag as described in claim 5, characterized in that, The number of the turbulence-disrupting components is 2 to 4, which are evenly arranged on the bottom inner side of the flexible bag body; each turbulence-disrupting component is evenly arranged with arc-shaped fan blades around its perimeter, the curvature of the arc-shaped fan blades is opposite to the rotation direction of the stirring paddle, the arc of the arc-shaped fan blades is a quarter circle, and its diameter is the same as the diameter of the cylindrical body.

7. The stirred cell culture bag as described in claim 6, characterized in that, The agitator is made of a first biological material, and the turbulence-disrupting element is made of a second biological material, wherein the first biological material and the second biological material are different; the hardness of the first biological material is higher than that of the second biological material.

8. The stirred cell culture bag as described in claim 7, characterized in that, The first biomaterial and the second biomaterial are any one or more of PE, PP, PC, PET, and PEK.

9. The stirred cell culture bag as described in claim 8, characterized in that, The first biomaterial is PET, and the second biomaterial is PE.

10. The stirred cell culture bag as described in claim 9, characterized in that, The surfaces of the turbulence-inducing components and the agitator are subjected to plasma or ultraviolet passivation treatment to form a smooth surface layer capable of dynamically binding water molecules.

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