Bioreactor and turbulent flow bioreactor

Abstract

The application relates to a kind of biological reaction tank and torrential biological reactor for supporting biological reaction bag, including tank body and disturbing flow piece, wherein the tank body includes bottom wall, taper wall extending upward from bottom wall and column wall extending upward from taper wall;Wherein the disturbing flow piece is protruded to the inner surface of taper wall for abutting the biological reaction bag to make the inner surface of the biological reaction bag protrude inward at the disturbing flow piece. This is beneficial to increase the shear force of the reaction liquid in the bag, so that when platelets are cultured using the torrential biological reactor, the demand for shear rate in the platelet culture process can be met.

Description

Technical Field

[0001] This invention relates to the field of bioreactor technology, and in particular to a bioreactor and a turbulent flow bioreactor. Background Technology

[0002] In vitro platelet culture technology can alleviate the current supply and demand imbalance of platelets. In order to increase the yield of in vitro platelet culture, several bioreactors have been developed at home and abroad to specifically culture platelets. However, they all use microplates, microfluidic systems, and small-volume shake flasks for culture, and there are no bioreactors for large-volume platelet culture.

[0003] The turbulent flow bioreactor is a non-bubbling interface oxygen transfer mechanism. It generates a turbulent flow through mechanical oscillation, which causes the culture medium to repeatedly wash over the inner surface of the culture bag and rapidly dissolve oxygen. The culture medium washes over and carries away the gas, creating nanoscale soluble microbubbles, and the bubbles are constantly mixed, so that each cell can get sufficient oxygen and nutrients to maintain normal cell growth and metabolism.

[0004] Platelets primarily originate from megakaryocytes (MKs) in the bone marrow. MKs mature until their cytoplasm extends long filaments to form proplatelets, which are then sheared into platelets by the blood. This means that platelet culture processes require a high shear rate. While current turbulent flow bioreactors can meet the dissolved oxygen requirements for proplatelet growth in large-volume culture settings, their low shear rate makes it difficult for mature platelets to be flushed into the solution, resulting in a low platelet concentration in the reaction solution and affecting the actual yield of platelet culture. Summary of the Invention

[0005] According to one aspect of this application, a bioreactor is provided for supporting a bioreactor bag, comprising: a tank body and a baffle, wherein the tank body includes a bottom wall, a conical wall extending upward from the bottom wall, and a columnar wall extending upward from the conical wall; wherein the baffle protrudes from the inner surface of the conical wall for abutting against the bioreactor bag to cause the inner surface of the bioreactor bag to bulge inward at the baffle.

[0006] In one embodiment, the spoiler extends from one end of the cone wall near the bottom wall to one end near the column wall.

[0007] In one embodiment, the spoiler extends along the generatrix of the cone wall.

[0008] In one embodiment, the spoiler is a rubber component.

[0009] In one embodiment, the spoiler is detachably connected to the tank.

[0010] In one embodiment, the number of the baffles is multiple and the multiple baffles are evenly distributed along the circumference of the cone wall on the inner surface of the cone wall.

[0011] In one embodiment, the bioreactor also satisfies: 3≤n≤10, where n is the number of the baffles.

[0012] In one embodiment, the bioreactor satisfies: 0.75≤2L1×cosα / (D1-D2)≤1, where L1 is the radial extension length of the turbulence element along the conical wall, D2 is the bottom inner diameter of the conical wall, D1 is the top inner diameter of the conical wall, and α is the inclination angle of the conical wall.

[0013] In one embodiment, the spoiler is tubular and also satisfies: 11.1mm≤d≤35mm, where d is the outer diameter of the spoiler.

[0014] In one embodiment, the tank body satisfies: 140°≤β≤160°, where β is the angle between the bottom wall and the conical wall.

[0015] In summary, this application provides a flow-dispersing element on the conical wall of the bioreactor. After the bioreactor bag is attached to the inside of the bioreactor, the inside of the bag protrudes inward at the flow-dispersing element on the conical surface. This helps to increase the shear force of the reaction liquid inside the bag, so that the shear rate requirement in the platelet culture process can be met when using a turbulent flow bioreactor to culture platelets.

[0016] According to another aspect of this application, this application also provides a turbulent flow bioreactor, comprising: a bioreactor, a bioreactor bag, and a driving device, wherein the bioreactor bag is attached to the inner wall of the bioreactor; wherein the driving device is driven and connected to the bioreactor, and the rotation axis of the driving device is offset from the central axis of the bioreactor.

[0017] In summary, the turbulent flow bioreactor provided in this application achieves turbulent flow through the design of the inverted cone head of the bioreactor and the eccentric rotation motion. Due to its non-bubbling interface oxygen transfer mechanism, it can meet the dissolved oxygen requirements of the reaction solution in the platelet culture process, and has significant advantages under large-volume culture conditions. In addition, by protruding the flow-inducing elements inward on the conical wall of the bioreactor to increase the shear rate in the reaction solution, the turbulent flow bioreactor can provide a reaction solution shear rate that meets the requirements of the platelet culture process. Attached Figure Description

[0018] Figure 1 This application provides a schematic diagram of the structure of a bioreactor; Figure 2 for Figure 1A schematic diagram of the dimensions of the bioreactor shown.

[0019] Figure label: 10. Tank body; 11. Bottom wall; 12. Conical wall; 13. Column wall; 20. Bumper components. Detailed Implementation

[0020] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0021] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0022] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0023] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0024] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0025] It should be noted that when an element is referred to as being "fixed to" or "set on" another element, it can be directly on the other element or there may be an intervening element. When an element is considered to be "connected to" another element, it can be directly connected to the other element or there may be an intervening element. The terms "vertical," "horizontal," "upper," "lower," "left," "right," and similar expressions used herein are for illustrative purposes only and do not represent the only possible implementation.

[0026] Platelets are the enucleated cytoplasm of megakaryocytes in the circulatory system, playing a vital role in regulating coagulation, preventing hemostasis, and in immune inflammatory responses. Currently, most platelets used in blood transfusions come from blood donors, but the increasing demand for blood products is exacerbating the supply-demand imbalance of platelets.

[0027] Platelets are mainly produced from megakaryocytes (MKs) in the bone marrow. MKs mature until their cytoplasm extends long filaments to form proplatelets, which are then washed and sheared into platelets by the blood.

[0028] Currently, humans have made numerous attempts to produce platelets in vitro in order to increase yield. However, the maturation of MKs (platelet-rich plasma cells), preplatelet production, and platelet production are all highly dependent on the microbial culture environment. Most in vitro platelet cultures only simulate biochemical conditions without taking into account the shear physical environment of blood flow. Therefore, for industrial-scale in vitro platelet culture, bioreactors that can simulate the physical environment and monitor the biochemical microenvironment are crucial.

[0029] Currently, several bioreactors have been developed both domestically and internationally specifically for platelet culture. However, these primarily utilize microplates, microfluidic systems, and small-volume shake flasks for cultivation, lacking bioreactors suitable for large-volume platelet culture. Shake flask and microplate cultivation methods cannot provide real-time monitoring of the microbial environment and are limited to small-volume cultivation, making large-scale platelet culture impossible. Therefore, creating a stable physical and microbial environment through bioreactors is crucial for large-scale in vitro platelet culture.

[0030] The turbulent flow bioreactor utilizes a non-bubbling interface oxygen transfer mechanism. Mechanical oscillation generates a turbulent flow, causing the culture medium to repeatedly wash over the inner surface of the culture bag, rapidly dissolving oxygen. The culture medium also carries away gases, creating nanoscale soluble microbubbles that continuously mix, ensuring each cell receives sufficient oxygen and nutrients to maintain normal cell growth and metabolism. Compared to existing oscillating reactors, this reactor employs an inverted truncated conical tank, which offers a larger specific surface area and better mass transfer than cylindrical oscillating reactors.

[0031] Research has found that the physical environment for platelet production mainly depends on fluid patterns and shear force, with the shear rate in the reaction solution being around 30 s⁻¹. -1 ~100s -1 Within a certain range, it can not only promote platelet release and morphological maturation and prevent platelet aggregation, but also cause less damage to platelets. Although current turbulent flow bioreactors can monitor and control the biochemical environment in real time and meet the dissolved oxygen requirements during large-volume platelet culture, the shear rate still cannot meet the requirements of platelet culture processes, resulting in a slow increase in platelet concentration in the reaction solution and affecting the final yield.

[0032] Therefore, it is necessary to provide a bioreactor and a turbulent flow bioreactor that can meet the requirements for large-volume platelet culture.

[0033] Please see Figure 1 and Figure 2 , Figure 1 This is a schematic diagram of the structure of a bioreactor provided in this application. Figure 2 for Figure 1 The diagram shows the structure of the tank body 10 of the bioreactor. The bioreactor provided in this application includes a tank body 10 and a flow-dispersing element 20. The tank body 10 includes a bottom wall 11, a conical wall 12, and a columnar wall 13 connected sequentially from bottom to top. The conical wall 12 extends gradually from the bottom wall 11 to the columnar wall 13. The flow-dispersing element 20 protrudes from the inner surface of the conical wall 12 to abut against a bioreactor bag attached to the tank body 10, causing the inner surface of the bioreactor bag to bulge inward at the flow-dispersing element 20. In this way, by adding the flow-dispersing element 20 to the tank body 10, the shape of the bioreactor bag in the tank body 10 is forced to change, which helps to increase the shear rate range (30s) during the movement of the bioreactor. -1 ~100s -1 The reaction liquid volume ratio is sufficient to fully flush megakaryocytes, which helps to cause proplatelets to fall off completely, thereby increasing platelet production.

[0034] Optionally, in one embodiment provided in this application, the baffle 20 is made of rubber. This is to match the rubber material of the bioreactor bag, which helps to increase the friction between the baffle 20 and the bioreactor bag, reducing the shaking and misalignment of the bioreactor bag in the tank 10. The rubber baffle 20 will also not scratch the bioreactor bag, preventing damage to the bag. Specifically, the baffle 20 can be made of flexible materials such as silicone or polyurethane to further conform fully to the bioreactor bag through deformation.

[0035] Optionally, in one embodiment provided in this application, the number of the flow-dispersing elements 20 is multiple, and the multiple flow-dispersing elements 20 are evenly distributed along the circumference of the cone wall 12 on the inner surface of the cone wall 12. In this way, by setting multiple evenly distributed flow-dispersing elements 20, the liquid surface shape of the reaction liquid in the bag can be maintained as stable as possible while ensuring the flow-dispersing effect. Furthermore, the bioreactor also satisfies: 3≤n≤10, where n is the number of flow-dispersing elements. By restricting the number of flow-dispersing elements 20 to more than three to ensure the flow-dispersing effect, and by restricting the number of flow-dispersing elements 20 to no more than 10 to avoid the flow-dispersing elements 20 at the bottom of the cone wall 12 being too dense, the bioreactor bag is ensured to be fully attached to the cone wall 12, reducing shaking.

[0036] Optionally, in one embodiment provided in this application, the flow-dispersing element 20 extends from one end of the conical wall 12 near the bottom wall 11 to one end near the column wall 13, preferably extending along the generatrix of the conical wall 12. This arrangement helps reduce sliding resistance during the installation of the bioreactor bag, as the bag slides down along the flow-dispersing element 20, ensuring the bioreactor bag smoothly reaches the bottom of the tank 10. It is understood that in other embodiments, if assembly is not considered, the orientation and shape of the flow-dispersing element 20 can be changed to improve the flow-dispersing effect. For example, the flow-dispersing element 20 can be arranged in a spiral upward manner, designed as a spiral or wavy shape, thereby replacing the straight tubing design.

[0037] Optionally, in one embodiment provided in this application, the baffle 20 is detachably connected to the tank 10, which facilitates assembly and replacement of baffles 20 of different sizes or shapes, and facilitates subsequent upgrades of the bioreactor.

[0038] like Figure 1 and Figure 2As shown, in one embodiment provided in this application, the bioreactor satisfies: 0.75≤2L1×cosα / (D1-D2)≤1, where L1 is the radial extension length of the turbulence member 20 along the conical wall 12, D2 is the bottom inner diameter of the conical wall 12, D1 is the top inner diameter of the conical wall 12, and α is the inclination angle of the conical wall 12. By constraining the condition, on the one hand, the flow-disrupting element 20 is prevented from extending to the inner surface of the column wall 13. Since the reaction liquid in the bag tilts as the tank 10 rotates, the linear velocity of the liquid surface is greater closer to the top. If the flow-disrupting element 20 extends to the column wall 13, the bioreactor bag will also have an inward protrusion at the column wall 13. This protrusion will significantly interfere with the liquid surface. When the tank 10 rotates at a high speed, the reaction liquid may even splash at the highest point of the liquid surface. Microscopically, this manifests as an excessively high shear rate in the reaction liquid, which is detrimental to platelet culture. On the other hand, the extension length of the flow-disrupting element 20 is constrained to be at least 3 / 4 of the radially inclined extension length of the cone wall 12. This ensures that the protrusion of the bioreactor bag on the cone wall 12 has a disturbance effect on the reaction liquid, and ensures that the shear rate of the reaction liquid meets the shear rate requirements of the platelet culture process.

[0039] According to some embodiments of this application, the flow-disrupting element 20 is tubular and also satisfies the following condition: 11.1 mm ≤ d ≤ 35 mm, where d is the outer diameter of the flow-disrupting element. Firstly, the tubular flow-disrupting element 20 can fully conform to the bioreactor bag without scratching the bag. Secondly, by constraining the diameter range of the flow-disrupting element 20, users can adaptively select different sizes of flow-disrupting elements 20 according to the needs of different culture volumes. When the diameter of the flow-disrupting element 20 is too small, its disturbance ability on the reaction solution is insufficient, failing to obtain the shear rate required for platelet culture. When the diameter of the flow-disrupting element 20 is too large, its disturbance ability on the reaction solution is too strong, which may lead to platelet damage due to excessive shear rate.

[0040] According to some embodiments of this application, the bioreactor also satisfies: 140°≤β≤160°, where β is the angle between the bottom wall 11 and the conical wall 12.

[0041] According to another aspect of this application, this application also provides a turbulent bioreactor, including a bioreactor tank, a bioreactor bag, and an eccentric drive device. The bioreactor bag is attached to the inner wall of the bioreactor tank, and the drive device is connected to the bioreactor tank, with the rotation axis of the drive device being offset from the central axis of the bioreactor tank.

[0042] The following will describe, in conjunction with specific embodiments, the actual impact of the bioreactor provided in this application on cell shear rate during platelet culture.

[0043] First, this application designs a turbulent flow bioreactor with a culture volume of 2.5L, and designs a comparative example of this type of turbulent flow bioreactor without the installation of the baffle 20. The basic design parameters of the turbulent flow bioreactor in this embodiment are shown in Table 1, wherein the baffle 20 is a soft rubber straight tube.

[0044] Table 1

[0045] CFD simulation tests were conducted on the turbulent flow bioreactors in Example 1 and the comparative example at the designed rotational speed. The average shear rate of the reaction liquid in Example 1 was found to be 15.95 s⁻¹. -1 The average shear rate of the reaction solution in the comparative example was 15.25 s⁻¹. -1 The comparison shows that the average shear rates of the two cases are similar. Furthermore, this application also measured the volume percentage of the reaction solution within different shear rate ranges, and the results are shown in Table 2.

[0046] Table 2

[0047] As shown in the table, in the 2.5L turbulent bioreactor provided in Example 1 of this application, due to the addition of the baffle 20, the volume percentage of the reaction liquid in the low shear rate range (0~20) decreased by 20.12% compared with the comparative example without the baffle 20, while the volume percentage of the reaction liquid in the medium-low shear rate range (20~100) increased by 15.32%. This indicates that the turbulent bioreactor provided in this application can not only meet the microbial chemical environment for platelet culture, but also form a physical shear environment suitable for platelet culture by adding multiple baffles 20 to the conical wall 12 of the tank 10. This can promote platelet detachment during the collection stage of platelet culture, facilitate collection, and help improve the yield of platelet in vitro culture process. Although the volume of the reaction liquid in the medium-high shear rate range (100~200) increased by 2.96%, and the volume of the reaction liquid in the high shear rate range (above 200) increased by 1.85%, the volume of the reaction liquid in these two ranges was small in Example 1, and the effect on platelets was negligible.

[0048] Furthermore, this application also designed three different designed culture volumes of turbulent flow bioreactors (CUR2.5L, CUR50L, CUR300L) for platelet culture, realizing the scale-up technology of platelet stabilization process from a small volume of 2.5L to a large volume of 300L. The number of flow-disrupting elements 20 in each case is four, and the specific design parameters (design rotation speed, diameter d of flow-disrupting element 20, length L1 of flow-disrupting element 20 extending radially along the cone wall, inner diameter D1 of the top of the cone wall 12, inner diameter D2 of the bottom of the cone wall 12, and inclination angle α of the cone wall 12) are shown in Table 3 below.

[0049] Table 3

[0050] Based on the design parameters, the proportion of the spoiler length on the conical surface, 2L1×cosα / (D1-D2), in the three embodiments are 0.76, 0.91, and 0.95, respectively.

[0051] Based on the CFD simulation results, the volume proportions of the reaction liquid in different shear rate ranges in Examples 1, 2, and 3 are shown in Table 4.

[0052] Table 4

[0053] As shown in Table 4, in all three embodiments, the volume ratio of the reaction liquid in the low-to-medium shear rate range (20~100) exceeded 30%, and even exceeded 40% in Embodiment 3. This indicates that the turbulent bioreactors provided in Embodiments 1, 2, and 3 of this application can meet the requirements of platelet culture process.

[0054] It is worth noting that the volume percentage of the reaction solution in each shear rate range in Table 4 above can also be regarded as the degree of influence of different shear rate ranges on platelet culture in the entire reaction solution. In Example 3, although the influence of the medium-high shear rate range (100~200) reached 13.28%, the influence of the medium-low shear rate range (20~100) was larger, reaching 46.69%, which dominated the shear rate distribution in the entire Example 3. Therefore, in Example 3, the platelet concentration in the reaction solution reached the target concentration in a shorter time, which is beneficial to shorten the culture time and reduce the influence of the medium-high and high shear rate ranges in the reaction solution on platelet culture.

[0055] The above data show that in a turbulent bioreactor, when the bioreactor meets the constraints of the conditional formula provided in this application, it can meet the physical shear environment requirements of platelet culture process under different design culture volume conditions. In particular, under large-volume culture conditions, the preferred shear rate range (20~100) of platelet culture has a dominant influence on the reaction solution, which is beneficial to shorten the culture time and thus increase the final yield.

[0056] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0057] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A bioreactor for supporting a bioreactor bag, characterized in that, include: The tank body includes a bottom wall, a conical wall that gradually expands upward from the bottom wall, and a columnar wall that extends upward from the conical wall; and A flow-disrupting element protrudes from the inner surface of the cone wall to abut against the bioreactor bag, causing the inner surface of the bioreactor bag to bulge inward at the flow-disrupting element. The flow-disrupting element extends from one end of the cone wall near the bottom wall to one end near the column wall. The bioreactor satisfies: 0.75≤2L1×cosα / (D1-D2)≤1, where L1 is the radial extension length of the turbulence element along the conical wall, D2 is the bottom inner diameter of the conical wall, D1 is the top inner diameter of the conical wall, and α is the inclination angle of the conical wall.

2. The bioreactor according to claim 1, characterized in that, The agitator extends along the generatrix of the cone wall.

3. The bioreactor according to claim 1, characterized in that, The aerodynamic component is made of rubber.

4. The bioreactor according to claim 1, characterized in that, The number of the baffles is multiple, and the multiple baffles are evenly distributed along the circumference of the cone wall on the inner surface of the cone wall.

5. The bioreactor according to claim 4, characterized in that, The bioreactor also satisfies: 3≤n≤10, where n is the number of the turbulence-inducing components.

6. The bioreactor according to any one of claims 1 to 5, characterized in that, The aerodynamic component is tubular and also satisfies the following condition: 11.1mm≤d≤35mm, where d is the outer diameter of the aerodynamic component.

7. The bioreactor according to any one of claims 1 to 5, characterized in that, The tank body satisfies: 140°≤β≤160°, where β is the angle between the bottom wall and the conical wall.

8. A turbulent flow bioreactor, characterized in that, include: The bioreactor as described in any one of claims 1 to 7; A bioreactor bag is attached to the inner wall of the bioreactor. A drive device is connected to the bioreactor, and the rotation axis of the drive device is offset from the central axis of the bioreactor.

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