Perfusion bioreactor and system for large scale cell culture

Abstract

A bioreactor system for culturing cells is provided. The bioreactor system includes a vessel comprising an inlet, an outlet, and an internal cavity disposed between the inlet and the outlet and for perfusion of a cell culture medium therethrough. The system further includes a cell culture substrate disposed in the internal cavity in a fixed bed configuration, the surface of the cell culture substrate having a surface area for culturing cells thereon during operation of the bioreactor system. By adjusting the surface area of the cell culture substrate, the bioreactor system can be scaled up or down depending on the number of cells to be cultured.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 535,904, filed August 31, 2023, pursuant to 35 USC § 119, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This disclosure generally relates to apparatus, systems, and methods for culturing cells in a fixed-bed perfusion bioreactor. More specifically, this disclosure relates to fixed-bed bioreactor systems that can be efficiently scaled up for different culture scales, and methods for assembling said systems and culturing cells using these systems. Background Technology

[0004] In the bioprocessing industry, large-scale cell culture is required to produce hormones, enzymes, antibodies, vaccines, and cell therapy agents. Most cells used in bioprocessing are adhesion-dependent, meaning they need to adhere to a surface to grow and function. Traditionally, adherent cell culture is performed on two-dimensional (2D) cell attachment surfaces incorporated into a variety of container forms, such as T-flasks, Petri dishes, cell factories, cell stack culture containers, roller flasks, and HYPERStack® containers. These methods can have significant drawbacks, including difficulty in achieving sufficiently high cell densities for large-scale production of therapeutic agents or cells.

[0005] Alternative methods have been proposed to increase the volumetric density of cultured cells. These methods include microcarrier culture in stirred tanks. In this method, cells attached to the surface of the microcarriers are subjected to continuous shear stress, which significantly impacts proliferation and culture performance. Another example of high-density cell culture systems is the hollow fiber bioreactor, in which cells may form large three-dimensional aggregates as they proliferate in the interspatial fiber space. However, cell growth and performance are severely inhibited due to nutrient deficiency. To mitigate this problem, these bioreactors are made very small and are not suitable for large-scale manufacturing.

[0006] Another example of a high-density culture system for adherent-dependent cells is a packed-bed bioreactor system. For example, packed-bed bioreactor systems containing a packed bed of support or matrix systems for cell retention have been previously disclosed in U.S. Patents 4,833,083, 5,501,971, and 5,510,262. The packed-bed matrix is ​​typically made of porous particles or polymer nonwoven microfibers as a substrate. Such bioreactors function as recirculating flow-through bioreactors. A significant problem with these bioreactors is the non-uniformity of cell distribution within the packed bed. For example, the packed bed acts as a depth filter, where cells are primarily captured in the inlet region, resulting in a gradient distribution of cells during the seeding step. Furthermore, the random fiber packing leads to non-uniform flow resistance and cell capture efficiency across the packed bed cross-section. For example, the culture medium flows rapidly through areas with low cell packing density and slowly through areas with higher resistance due to a larger number of retained cells. This creates a channeling effect, where nutrients and oxygen are delivered more efficiently to areas with lower volumetric cell density, while areas with higher cell density remain under suboptimal culture conditions. Another significant drawback of the packed bed systems disclosed in the prior art is the inability to efficiently collect intact, viable cells at the end of the culture process. U.S. Patent No. 9,273,278 discloses a bioreactor design to improve the efficiency of cell recovery from a packed bed during the cell collection step. This design is based on loosening the packed bed matrix and agitating or stirring the packed bed particles to allow the porous matrix to collide, thus detaching the cells. However, this method is time-consuming, labor-intensive, and can lead to significant cell damage, thereby reducing overall cell viability.

[0007] Roller flasks offer several advantages, such as ease of handling and the ability to monitor cells on the attachment surface. However, from a production perspective, their main disadvantage is the low surface area to volume ratio, while roller flask configurations require a significant amount of manufacturing space. Various methods have been employed to increase the surface area available for adherent cells in roller flask form. Some solutions have been implemented in commercially available products, but room for improvement remains to further enhance roller flask productivity. Traditionally, roller flasks are single-structure products manufactured using a blow molding process. This ease of manufacturing makes roller flasks economically viable in the bioprocessing industry. While some modifications to roller flasks can increase the usable surface area for cell culture without altering the manufacturing process, these modifications only result in minor increases in surface area. Other modifications to the roller flask design significantly increase manufacturing complexity, rendering them uneconomical in the bioprocessing industry. Therefore, there is a need for roller flasks that offer increased surface area and higher bioprocessing productivity while using the same blow molding process for manufacturing.

[0008] While existing platforms can be used to manufacture viral vectors for early clinical trials, a platform is still needed to produce high-quality products in larger quantities to achieve later-stage commercial manufacturing scale. Specifically, a platform that can be efficiently and effectively scaled for cell culture at various scales is required. Using a single platform for different culture scales offers numerous advantages, saving users time and resources on culture and enhancing the potential performance of cells that may be "used to" growing on certain platforms and therefore exhibit healthier culture conditions when using a single platform for different scales. Most commercially available systems offer limited surface area, and these systems sometimes cannot be scaled well to meet manufacturing requirements. However, operating multiple small systems in parallel quickly becomes difficult due to increased footprint and the need to manage other components such as pumps, controllers, and piping.

[0009] Some commercially available bioreactor systems can be scaled up to larger surface areas. Such systems can be scaled up by increasing the reactor diameter and process flow rate while maintaining a constant linear flow rate through the culture bed. The height of the reactor may be limited by factors such as the need for uniform cell seeding and the need to provide a suitable environment across the entire height range (e.g., providing sufficient oxygen to all cells, not just those closest to the inlet).

[0010] Therefore, there is a need for bioreactor systems that can provide a scalable platform and achieve uniform fluid flow, cell distribution, and mass flow rate. Summary of the Invention

[0011] According to one embodiment of this disclosure, a fixed-bed bioreactor system is provided. The bioreactor system includes a container comprising an inlet, an outlet, and a cavity, the cavity being disposed between the inlet and the outlet and for perfusing cell culture medium. The system also includes a cell culture substrate disposed in the cavity in a fixed-bed configuration, the cell culture substrate having a surface area for culturing cells on the surface during operation of the bioreactor system. By adjusting the surface area of ​​the cell culture substrate, the bioreactor system can be scaled up or down as needed to culture the required number of cells. Attached Figure Description

[0012] Figure 1A A schematic cross-sectional view of a bioreactor system of a first dimension according to an embodiment is shown.

[0013] Figure 1B A schematic cross-sectional view of a bioreactor system of a second size according to an embodiment is shown.

[0014] Figure 1CA schematic cross-sectional view of a third-dimensional bioreactor system according to an embodiment is shown.

[0015] Figure 2 This shows a schematic cross-sectional view of a bioreactor system according to some embodiments.

[0016] Figure 3A This shows a plan view of the flow distribution mechanism according to an embodiment.

[0017] Figure 3B This shows a cross-sectional view of an orifice in a fluid distribution mechanism according to an embodiment.

[0018] Figure 3C Display according to embodiments Figure 3A A plan view of the fluid distribution mechanism, with the shaded area showing the concentric ring pattern formed by the orifices.

[0019] Figure 4A This shows a schematic cross-sectional view of a bioreactor system with an integrated bubble trap according to an embodiment.

[0020] Figure 4B Display according to embodiments Figure 4A A close-up view of the integrated bubble trap in the image.

[0021] Figure 5 Displays mass flow rate data of the fluid distribution mechanism according to an embodiment.

[0022] Figure 6A The results of a computational fluid dynamics (CFD) simulation of the geometry of the first pressure chamber according to an embodiment are shown.

[0023] Figure 6B The results of a computational fluid dynamics (CFD) simulation of the geometry of the second pressure chamber according to the embodiment are shown.

[0024] Figure 6C show Figure 6A A close-up view of the CFD simulation shown.

[0025] Figure 6D show Figure 6B A close-up view of the CFD simulation shown.

[0026] Figure 7 This is a display based on the embodiment in Figures 6A to 6D A graph showing the change in the number of cells per unit area in a CFD simulation.

[0027] Figure 8 This is a velocity distribution diagram at the outlet of each orifice ring in the fluid distribution mechanism according to an embodiment.

[0028] Figure 9 This illustrates the guide rod assembly and alignment features of a cell culture substrate for a bioreactor according to an embodiment.

[0029] Figure 10 This is an illustration of a two-layer mesh cell culture substrate according to an embodiment, wherein the substrate has a central cut to fit... Figure 9 The guide rod assembly.

[0030] Figure 11 This is an illustration of a variable height guide rod and a cell culture substrate assembly according to an embodiment.

[0031] Figure 12 According to the embodiments Figure 11 Cross-sectional view of the component. Detailed Implementation

[0032] Various embodiments of this disclosure will be described in detail with reference to the accompanying drawings (if any). The various embodiments mentioned do not limit the scope of the invention, which is limited only by the scope of the appended claims. Furthermore, any examples set forth in this specification are not limiting and are merely examples of many possible embodiments of the claimed invention.

[0033] Embodiments of this disclosure relate to cell culture bioreactor systems, and methods for assembling said bioreactor system and culturing cells using said bioreactor system. According to various aspects of embodiments of this disclosure, a bioreactor system is provided, characterized by an improved single-stage open pressure chamber flow distribution system that ensures uniform velocity, mass, and cell distribution within the reactor. Previous solutions, at best, could only provide uniform velocity distribution and may require graded or multi-stage distribution methods, or multiple branching channels.

[0034] The embodiments also include fluid distribution mechanisms in the inlet and / or outlet pressure chambers of the bioreactor, which are incorporated into a bubble trap (i.e., a buffer to prevent bubbles that inadvertently enter the reactor from interfering with the cell culture process). Previous solutions may have relied on separate and / or external bubble traps.

[0035] As another aspect of this disclosure, the bioreactor disclosed herein is symmetrical in both the axial and radial directions. To achieve radial symmetry, a central guide rod design can be used according to some embodiments. The guide rod ensures the correct orientation of the cell culture substrate during assembly and, independent of the orientation of individual cell basal layers, distinguishes two distinct layers through cuts in the substrate material. Using a centrally located guide rod provides symmetry and simplifies the fabrication and assembly of bioreactor components. Previous solutions used guide rods offset to one side of the reactor, close to the reactor wall, and in some cases, to achieve alignment of specific mesh layers, it may be necessary to assemble the mesh cell basal layers with alternating orientations by flipping every other layer.

[0036] According to some preferred embodiments, a bioreactor system is provided that is capable of increasing the surface area of ​​a cell culture substrate from 20 m² to... 2 Expanded to 100 m 2 For example, a so-called "pilot scale" platform system may include a surface area of ​​20... m 2 50 m 2 and 100 m 2 Bioreactors. However, the embodiments are not limited to these surface area sizes. According to an embodiment, the bioreactor system can be scaled up to 500 m². 2 With 1000 m 2 The surface area between them. Bioreactors of different sizes may differ only in the height of the bioreactor (and correspondingly, the height of the cell culture substrate inside the reactor), while other aspects (such as fluid distribution mechanisms, bioreactor width, and other design features) may remain the same in size. Unless otherwise stated, "bioreactor height" or "reactor height" may refer to the height of the vessel itself, the height of the fixed bed substrate inside the vessel, or both. According to some embodiments, bioreactors of different sizes may even maintain the same process conditions (such as flow rate) by only changing the height of the reactor. In addition to the geometry optimized for uniform cell distribution, the bioreactor system of this disclosure has additional features not shown in other systems, such as an integrated bubble trap and guide rods that keep the cell substrate bed assembly ordered and symmetrical.

[0037] like Figure 1A-1C As shown, the bioreactor system of this disclosure can be scaled to different sizes simply by scaling the heights h1, h2, and h3 of the reactor, respectively. For example, Figure 1A The bioreactor system 100a can have a surface area, for example, about 20. m 2The substrate 102a, Figure 1B The bioreactor system 100b can have a surface area of, for example, about 50. m 2 The substrate 102b, and Figure 1C The bioreactor system 100c can have a surface area, for example, about 100. m 2 The substrate is 102b. These figures are shown by way of example only, and embodiments include different surface areas. Keeping other design aspects the same, simply scaling the height of the fixed bed simplifies the fabrication of the bioreactor system and achieves consistent performance across different scales. For example, while embodiments of this disclosure are not limited to any particular construction of the bioreactor vessel, some embodiments include multi-component reactor assemblies comprising container sidewalls surrounding the fixed bed cell culture substrate and other components (e.g., dispenser plates, end caps, inlets / outlets, etc.) at both ends of the fixed bed. A system that can scale cell culture scale simply by scaling the container height means that only the container sidewalls (and correspondingly, the amount of cell substrate contained within the container) need to be scaled, while other components (e.g., dispenser plates, end caps, fixtures, inlets / outlets, tubing, etc.) can remain unchanged and can be used in reactors of any size. This greatly simplifies the fabrication, assembly, and operation of the system.

[0038] Figure 2 Additional features of the bioreactor system 110 according to embodiments of the present disclosure are shown. The bioreactor system 110 includes a container 111 with sidewalls defining an interior 112 having a cell culture substrate 114. Further details of the substrate will be discussed below. The container 111 also includes an inlet 116 and an outlet 118, respectively designed for the inflow and outflow of materials (e.g., cells, culture media, etc.) from the interior of the container 111. The bioreactor system 110 may also include a guide rod 120, centrally located on the cell culture substrate 114. The guide rod 120 can be used to simplify the assembly and arrangement of the cell substrate 114. Between the inlet 116 and the cell culture substrate 114 is an inlet pressure chamber 122 and an inlet fluid distribution mechanism 124, or inlet distributor plate. The bioreactor system 110 may also include an outlet fluid distribution mechanism 126, or outlet distributor plate, and an outlet pressure chamber 128.

[0039] Embodiments of the bioreactor system disclosed herein include a cylindrical body housing a fixed bed of cell culture medium. In some examples used herein for illustrative purposes, the cylindrical body has an inner diameter of approximately 11.5 inches, but the principles disclosed herein also apply to other sizes. The bioreactor system can be considered to have an axial direction and a radial direction, wherein the axial direction is parallel to the height of the bioreactor. The axial direction can also be considered to be parallel to the general direction of fluid flow through the bioreactor (e.g., from the inlet through the fixed bed to the outlet). The radial direction is perpendicular to the axial direction. To uniformly seed cells in the radial direction, the incoming culture medium is diverted from the inlet and diffused across the entire width of the mesh bed. This is achieved by a fluid distribution plate in the inlet pressure chamber of the bioreactor. Various aspects of embodiments of this disclosure may include one or more of the following three performance characteristics for an advantageous distribution plate: (1) producing a uniform velocity distribution, (2) producing a uniform mass flow rate, and (3) producing uniform cell distribution. As used in this paper, uniformity is defined as the deviation from the mean. For example, uniform velocity distribution means that the axial velocity component of the culture medium as it changes radially through the bioreactor deviates from the mean axial velocity by less than 10%.

[0040] The dispenser plate is configured to provide a sufficiently large back pressure to the incoming cell culture medium, forcing it to diffuse substantially radially before entering the mesh bed axially. Theoretically, a porous material could be used here, as the permeability can be selected to provide the necessary back pressure while offering numerous very small pores. However, porous materials are likely to trap all cells and become clogged, making them unsuitable for this application. A discrete form of porous material is a perforated plate with many individual pores. The more pores and the smaller each pore is, the better. However, if the pores are too small and / or too few pores are present, the velocity on the cells and the corresponding shear forces can actually be detrimental to the process. Furthermore, the dispenser plate must be fabricated and processing tolerances must be considered. This paper uses a perforated plate with a total open area of ​​approximately 1%. This is achieved through O(500) pores with a diameter of 3 / 64 inch (approximately 1.19 mm). Maximizing the total open area while maintaining acceptable performance is likely desirable, as this would reduce the spacing between the individual pores and decrease the velocity in each pore. According to various aspects of the embodiments described herein, the pore pattern is designed to ensure equal mass flow rates through the mesh bed. For example, in various aspects of the embodiments, it is designed such that each orifice can provide mass flow to the mesh layer or cellular substrate directly above it. There are several ways to pave a circle into equal areas and distribute the orifices. For example, the distributor plate can be divided into several rings, and the number of orifices in each ring can be calculated based on a constant ratio between the area of ​​the ring and the total area of ​​the orifices in that ring. Furthermore, the width of each ring is approximately equal to the spacing between the orifices within each ring.

[0041] According to the embodiments, cell culture medium is delivered into the reactor from one side. In the embodiments shown herein, a single central inlet is considered, but embodiments also include multiple inlets. A well-designed single-stage fluid distribution plate and inlet pressure chamber are sufficient to satisfy all three uniformity requirements mentioned above (i.e., uniform velocity distribution, uniform mass flow rate, and uniform cell distribution). Compared to multi-stage methods where cell culture medium diffuses radially through two or more perforated plates, this method is more compact and also reduces the number of recirculation zones, which can lead to undesirable side effects such as preferential cell concentration and cell trapping before reaching the cell substrate fixation bed. The embodiments described herein reduce the impact of these recirculation zones by optimizing the geometry of the inlet pressure chamber (including the distance between the reactor inlet and the distribution plate).

[0042] Figure 3A This illustration shows an example of an inlet fluid distribution mechanism according to various aspects of an embodiment. The fluid distribution plate 200 of this example has a perforation pattern 202, thereby ensuring uniform velocity and mass flow rates. The figure shows 16 rings, totaling 659 perforations, each with a diameter of 3 / 64 inch. Figure 3B As shown, each orifice 202 can have a chamfer on its downstream side 206 facing the fixed bed substrate, thus minimizing the contact area between the fluid distribution plate 200 and the adjacent cell culture substrate. Figure 3C In order to better illustrate the hole pattern, the sixteen rings 208 of hole 202 are highlighted as alternating shaded areas.

[0043] Figure 4A and 4B An example of a bioreactor 400 is shown, the reactor having an inlet distribution plate 402 with an integrated bubble trap 404. For clarity, Figure 4B A close-up view of one side of the inlet distribution plate 402 and the bubble trap 404 is shown. Bubbles are a well-known problem in bioreactors because they negatively impact the process by hindering cell attachment to the substrate or by clogging pores in the distributor plate, causing uneven culture medium delivery. Ideally, bubbles should never enter the reactor, and external inline bubble traps can be used with the bioreactor. However, such external bubble traps complicate the system, especially since these traps are typically single-use devices and must be sterilized with the reactor before use, or they must be cleaned in place. Understanding this, distributor plates with auxiliary functions for collecting and removing bubbles can be designed. According to embodiments, such as... Figure 4A and 4BAs shown, the bottom or upstream surface 406 of the dispenser plate is conical. The buoyancy of the bubbles forces them to reach the highest point of surface 406, causing them to travel upwards and outwards toward the outside of the inlet dispenser plate 402. The dispenser plate 402 is designed to be thickest at the center and gradually thins toward the outer edge. Additionally, grooves 408 are provided around the periphery of the dispenser plate 402. Therefore, bubbles are guided upwards by the conical surface 406 away from the center of the dispenser plate 402 and then captured in the grooves 408, where they do not interfere with the cell culture process. Here, bubbles can be siphoned off or simply retained in the grooves 408. To siphon off bubbles, a bubble trap outlet 410 in fluid communication with the grooves 408 can be provided. The size of the grooves 408 will determine how many bubbles can be collected before siphoning them off. This conical surface 406, together with the surface tension of the bubbles, prevents bubbles from entering the cell culture substrate.

[0044] Other aspects of embodiments of this disclosure include a central alignment rod within a bioreactor for assembling a cell substrate within the reactor. An embodiment of this alignment rod will be discussed with reference to a woven cell culture substrate further discussed below. Specifically, this will be discussed with reference to a bioreactor with stacked mesh layers for a cell culture substrate fixation bed. Experiments have shown that axial distribution of cells in the bioreactor is improved when the orientation of the mesh substrate layers alternates at 45-degree angles with adjacent mesh layers. That is, if each mesh disc is made of fibers extending perpendicularly to each other, alternating 45-degree angles are achieved by placing a mesh layer with fibers extending at 0 and 90 degrees, and then placing a second mesh layer with fibers extending at 45 and 135 degrees on top of the first layer. This ensures that adjacent layers do not interlock and prevent cells from passing through the bed. Previous designs have explored using guide rods located on one side of the reactor to align these mesh layers to the correct orientation. For this purpose, each layer has a characteristic cutout with 22.5-degree symmetry, which determines its orientation. During mesh bed assembly, every other layer is then flipped to create a 45-degree offset. However, this is a cumbersome and error-prone process, especially since it is difficult to automate. Another drawback of this early design is that the asymmetrical placement of the guide rods against the sidewalls of the reactor disrupts the internal symmetry of the bioreactor.

[0045] Therefore, embodiments of this disclosure include an improved guide rod concept that simplifies and enhances the assembly process while maintaining the symmetry within the bioreactor. Instead of relying on flipping every other mesh layer to ensure correct orientation, the proposed design cuts the alignment feature itself at a defined angle, thereby fixing the orientation of the mesh layers by the feature of the guide rod. One of the simplest implementations of this design is the guide rod 900, which has a cross-shaped cross-section 902 located at the radial center of the bioreactor, while also providing structural strength, such as… Figure 9 As shown in the diagram, the 45-degree orientation offset is achieved by processing the mesh layers in two stacks 910 and 911, each stack having features 912 and 913 cut out at a 45-degree angle relative to the mesh layer orientation (see Figure 1). Figure 10 The image shows that the first stack has a feature cut at 0 degrees, and the second stack has a feature cut at 45 degrees. The only remaining process step is to stagger the two stacks. Because this type of alignment feature does not rely on flipping individual mesh layers, it is not limited to the orientation offset of two mesh layers between alternating layers. The alignment feature can be cut at any angle, and therefore it is also possible to produce pseudo-random mesh components in a controlled and repeatable manner.

[0046] Another consideration for mesh components is compaction, i.e., the number of layers per unit height in the bioreactor. The variability in mesh thickness can vary by up to 10%, so a layer-based approach will result in different mesh bed heights, or, when forced to reach the same height, different amounts of pressure (i.e., mesh compaction). According to embodiments of this disclosure, this problem can also be addressed using guide rods 1100 and another fluid distributor plate 1102 at the top of the mesh, allowed to slide freely along the guide rods, such as... Figure 11 and 12 As shown in the diagram, the weight of the distributor plate will ensure that any number of mesh layers have the same amount of compaction. Alternatively, a method based on mesh layer weight / height can be implemented using different numbers of mesh layers, and the guide rod height can be fixed to ensure a well-defined volume within the bioreactor.

[0047] Example

[0048] The following examples are shown to illustrate various aspects of embodiments of this disclosure. These examples are intended to illustrate certain aspects and advantages of the embodiments herein, but should not be construed as limiting the embodiments of this disclosure to the examples shown and discussed.

[0049] Figure 7 This graph shows the mass flow rate as a function of radial distance from the center of a pair of inlet fluid distributor plates. Example 1 is an inlet fluid distributor plate according to an embodiment of this disclosure (and as...). Figures 3A-3C As shown in the figure and described above, Example 1 is an alternative distributor plate, while Example 2 is an alternative distributor plate. As shown in the figure, the distributor plate of Example 1 exhibits a more uniform mass flow rate across the entire radius of the distributor plate, and its mass flow rate fluctuation is significantly reduced compared to Example 2. Specifically, the mass flow rate fluctuation between adjacent rings in Example 1 is reduced by more than 30% compared to Example 2. This indicates that the orifice spacing and design discussed above do indeed contribute to improving the mass flow rate characteristics.

[0050] Figures 6A-6D This paper presents the results of computational fluid dynamics (CFD) simulations performed on two pressure chamber geometries according to embodiments of the present disclosure. Specifically, the distance between the inlet and the distributor plate was varied to examine the effect of preferential cell concentration on cell distribution in a substrate-fixed bed. Figure 6A In the middle, the distance from the inlet to the bottom of the dispenser plate is 0.625 inches, while... Figure 6B In this case, the distance is increased to 2.125 inches. Figure 6C and 6D This indicates the rate of change of the culture medium and the presence or absence of the recirculation zone. In these examples, assuming uniform cell seeding at the inlet, increasing the distance between the inlet and the dispenser plate will eliminate the effect of preferential cell concentration on cell distribution in the fixed-bed substrate. The increased distance (from...) pushes the recirculation zone radially outward. Figure 6C Zone #5 to Figure 6D (Zone #8 in the middle). These dimensions are just examples, but they show that increasing the distance from the inlet to the dispenser plate has a positive effect on cell distribution and uniformity in the fixed bed, and it is believed that increasing the radial distance of the recirculation also plays a role in this regard.

[0051] Figure 7 Display by Figures 6A-6D The figure shown is a graph of the simulation results. This graph shows that when the distance between the inlet and the dispenser plate is only 0.625 inches, the recirculation zone allows more cells to pass through the inner ring. When the distance between the inlet and the plate is increased to 2.125 inches, the cell distribution becomes more uniform.

[0052] Regarding the bubble trap discussed above, the gradual narrowing of the lower surface of the inlet distributor plate will affect the flow characteristics, such as... Figure 8 As shown. Figure 8 As shown in the figure, the velocity reaching the outlet of the perforated rings (each ring is indicated by a zone number in the figure) increases with increasing zone number or distance from the center of the inlet plate. A thinner distributor plate will generate less back pressure, and therefore more fluid will be directed to the outer perforated rings. However, advantageously, this will counteract the boundary layer that naturally forms at the reactor wall. The velocity distribution can also be precisely controlled by adjusting the cone angle. Figure 8 As shown, the distance from the inlet to the plate also has a slight effect, with a larger distance causing a slightly higher speed.

[0053] The fixed-bed substrate according to the embodiments will now be described in more detail. Different types of packed-bed bioreactors have been used in conventional large-scale cell culture bioreactors. Typically, these packed beds contain a porous matrix to retain adherent or suspended cells and support growth and proliferation. The packed-bed matrix provides a high surface area to volume ratio, thus allowing for higher cell densities than in other systems. The packed bed typically acts as a depth filter, where cells are physically trapped or entangled in the fibers of the matrix. However, because the cell inoculum flows linearly through the packed bed, the cells experience uneven distribution within the packed bed. Therefore, for example, a higher cell density is present at the inlet region of the bioreactor, while the cell density is significantly lower at the outlet portion of the bioreactor. This uneven distribution of cells within the packed bed significantly hinders the scalability of such bioreactors in bioprocess manufacturing.

[0054] Another problem encountered with existing packed-bed bioreactors is the channeling effect. Due to the randomness of the nonwoven fibers used as fillers, the local fiber density is non-uniform at any given cross-section of the packed bed. The culture medium flows faster in areas of low fiber density (higher bed permeability) and much slower in areas of high fiber density (lower bed permeability). This non-uniform perfusion of the culture medium in the packed bed creates a channeling effect. This manifests itself as a significant nutrient and metabolite gradient, negatively impacting overall cell culture and bioreactor performance. Cells located in areas of low culture medium perfusion will starve and often die due to nutrient deficiency or metabolite poisoning. Cell collection is another problem encountered when using bioreactors with nonwoven fiber-filled scaffolds. Because the packed bed acts as a depth filter, cells released at the end of the cell culture process are trapped within the packed bed, resulting in extremely low cell recovery rates. This severely limits the use of these bioreactors in bioprocesses that use live cells as products.

[0055] This disclosure includes embodiments of cell growth substrates and / or packed bed systems for adherent cells, which enable the simple and efficient scaling up of cells or cell-derived products (e.g., proteins, antibodies, viral particles) to any practically feasible production scale. In one embodiment, a substrate is provided having structurally defined surface regions for adherent cell attachment and proliferation, good mechanical strength, and forming a highly uniform, multi-layered, interconnected fluid network when assembled in a packed bed or other bioreactor. In certain embodiments, mechanically stable, non-degradable woven meshes can be used to support adherent cell production. Such substrates enable uniform cell seeding and efficient collection of cells or other products from the bioreactor. Furthermore, embodiments of this disclosure support cell culture to obtain confluent monolayers or multilayers of adherent cells on the disclosed substrate and can avoid the formation of 3D cell aggregates with restricted nutrient diffusion and increased metabolite concentrations. The structurally defined substrates of one or more embodiments enable complete cell recovery from a packed bed in a bioreactor and continuous cell collection. In another embodiment of this disclosure, a cell culture method is provided for the bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors using a bioreactor with a substrate.

[0056] As used in this article, “structural determination” means that a component has a non-random, ordered structure that follows a determined structural design.

[0057] In one or more embodiments, a cell culture substrate is provided that supports adherent-dependent cells to attach and proliferate at high volumetric density. The substrate can be assembled and used in a bioreactor system (e.g., a perfused back-bed bioreactor) and provides uniform cell distribution during the inoculation step while preventing the formation of large and / or uncontrollable cell aggregates within the substrate or packed bed. Therefore, the substrate eliminates diffusion limitations during bioreactor operation. Additionally, the substrate enables easy and efficient cell collection from the bioreactor.

[0058] The matrix can be formed from a substrate material having a thin or sheet-like structure with a first side and a second side spaced apart by a relatively small thickness. In other words, the sheet-like substrate is thinner relative to the width and / or length of the first and second sides of the substrate. Additionally, multiple pores or openings are formed through the thickness of the substrate. The substrate material between the openings has a certain size and geometry, thereby allowing cells to adhere to the surface of the substrate material as if it were a two-dimensional (2D) surface, while also allowing sufficient fluid to flow around the substrate material and through the openings. In some embodiments, the substrate is a polymer-based material and can be formed as a molded polymer sheet; a full-thickness perforated polymer sheet; multiple filaments fused into a mesh layer; or multiple filaments woven into a mesh layer. The physical structure of the matrix has a high surface area to volume ratio for culturing adherent cells. According to various embodiments, the matrix can be arranged or filled in a bioreactor in certain ways to achieve uniform cell seeding, uniform culture medium perfusion, and efficient cell collection.

[0059] The embodiments of this disclosure can realize a practically scaled viral vector platform, which can produce approximately 10 viral vectors per batch. 15 To about 10 18 Viral genomes are generated at a scale of one or more viral genomes. For example, in some embodiments, the viral genome yield can be approximately 10... 15 To about 10 16 One viral genome or batch, or approximately 10 per batch 16 To about 10 19 One viral genome, or approximately 10 per batch 16 Up to 10 18 One viral genome, or approximately 10 per batch 17 To about 10 19 One viral genome, or approximately 10 per batch 18 To about 10 19 One viral genome, or approximately 10 per batch 18 One or more viral genomes. A “batch” can refer to a single cell culture operation in a single bioreactor vessel. Due to the scalability of the embodiments herein, the bioreactor vessels and the contained cell culture substrates can be appropriately scaled to achieve these yields. This scalability benefits from the structural determinism of the cell culture substrates, which enables uniform cell seeding and culture, uniform culture medium flow rate, and / or uniform collection. In some embodiments, a batch may include the use of multiple bioreactor vessels in coordinating cell culture operations.

[0060] Furthermore, the embodiments disclosed herein not only enable cells to attach and grow on a cell culture substrate, but also enable the collection of live cultured cells. The inability to collect live cells is a significant drawback of current platforms, making it difficult to build and maintain a sufficient number of cells to achieve production capacity. According to one aspect of embodiments of this disclosure, live cells can be collected from a cell culture substrate, comprising between 80% and 100% live cells, or between about 85% and about 99% live cells, or between about 90% and about 99% live cells. For example, in the collected cells, at least 80% are live cells, at least 85% are live cells, at least 90% are live cells, at least 91% are live cells, at least 92% are live cells, at least 93% are live cells, at least 94% are live cells, at least 95% are live cells, at least 96% are live cells, at least 97% are live cells, at least 98% are live cells, or at least 99% are live cells. Cells can be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0061] A cell culture substrate can be a woven mesh layer made of a first plurality of fibers extending in a first direction and a second plurality of fibers extending in a second direction. The woven fibers of the substrate form multiple openings. The size and shape of the openings can vary based on the weave type (e.g., the number, shape, and size of the filaments; the angle between intersecting filaments, etc.). The openings can be defined by a certain width or diameter. On a macroscopic scale, the woven mesh can be characterized as a two-dimensional sheet or layer. However, close observation of the woven mesh reveals that it forms a three-dimensional structure due to the undulations of the intersecting fibers.

[0062] The woven mesh may comprise monofilaments or multifilaments of polymer fibers. In one or more embodiments, the monofilament fibers may have a diameter ranging from about 10 µm to about 1000 µm. At the microscopic level, due to the scale comparison of the fibers to cells (e.g., the fiber diameter is larger than that of a cell), the surface of the monofilament fibers exhibits a regular 2D surface for adherent cells to attach and proliferate. Such fibers are woven into meshes with defined patterns and a certain amount of structural stiffness. The fibers may be woven into meshes having openings ranging from about 10 µm × 10 µm to about 100 µm × 100 µm or to about 1000 µm × 1000 µm. These ranges of filament diameters and opening diameters are examples of some embodiments but are not intended to limit the possible characteristic dimensions of the meshes according to all embodiments.

[0063] The substrate mesh can be made from monofilaments or multifilaments of polymeric materials compatible with cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. The mesh substrate can have different structural patterns or weaving methods, including, for example, knitting, warp knitting, or braiding (e.g., plain weave, twill weave, Dutch weave, five-needle weave).

[0064] It may be necessary to modify the surface chemistry of the mesh filaments to provide the desired cell adhesion properties. Such modifications can be made through chemical treatment of the polymeric material of the mesh or by transplanting cell-adhesion molecules onto the filament surface. Alternatively, a thin layer of a biocompatible hydrogel exhibiting cell-adhesion properties, including, for example, collagen or Matrigel®, can be coated onto the mesh. Alternatively, cell-adhesion properties can be imparted to the surface of the mesh filaments using treatment methods utilizing various types of plasma, process gases, and / or chemical reagents known in the industry.

[0065] High flow resistance uniformity is achieved throughout the matrix or packed bed by using a culture substrate with a sufficiently rigid structure. According to various embodiments, the substrate can be deployed in single-layer or multi-layer configurations. This flexibility eliminates diffusion limitations and ensures uniform delivery of nutrients and oxygen to cells attached to the substrate. Furthermore, the substrate has no cell-trapping areas in the packed bed configuration, allowing for complete collection of highly viable cells at the end of culture. The substrate also enables uniform filling of the packed bed and allows for direct scaling-up from process development units to large-scale industrial bioprocessing units. The ability to collect cells directly from the packed bed eliminates the need to resuspend the substrate in agitated or mechanically vibrating containers. Moreover, the high packing density of the cell culture substrate enables high bioprocess productivity in manageable volumes at an industrial scale.

[0066] As discussed herein, cell culture substrates can be used within bioreactor containers according to one or more embodiments. For example, the substrate can be used in packed-bed bioreactor configurations or other configurations within a three-dimensional culture chamber. However, the embodiments are not limited to three-dimensional culture spaces, and the substrate can be considered for use in configurations considered as two-dimensional culture surfaces, where one or more layers of the substrate are laid flat, such as in a flat-bottomed culture dish, to provide a cell culture substrate. Due to contamination concerns, the container can be a disposable container that can be discarded after use.

[0067] According to one or more embodiments, a packed-bed bioreactor system for culturing cells is provided, wherein the cell culture substrate is used within a culture chamber of a bioreactor vessel. For example, embodiments include a cell culture system comprising a bioreactor vessel having a cell culture chamber within its interior cavity. Within the cell culture chamber is a cell culture substrate made of multiple substrate layers. The multiple substrate layers may comprise multiple separable and distinct substrate layers arranged in a stacked manner, and may also comprise an integral cell culture substrate in which multiple layers are attached together. The substrate layers are stacked such that a first side or a second side of the substrate layer faces a first side or a second side of an adjacent substrate layer. The bioreactor vessel has an inlet at one end for introducing culture medium, cells, and / or nutrients into the culture chamber, and an outlet at the opposite end for removing culture medium, cells, or cell products from the culture chamber. By allowing the substrate layers to be stacked in this manner, the system can be easily scaled up without negatively impacting cell attachment and proliferation, because the stacked substrates have a defined structure and allow for efficient fluid flow.

[0068] In one or more embodiments, the flow resistance and bulk density of a packed bed can be controlled by staggering the stacking of substrate layers with different geometries. Specifically, the mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the fluid flow resistance of the packed bed configuration. By staggering the stacking of meshes of different sizes and geometries, fluid resistance in specific sections of the bioreactor can be controlled. This allows for more uniform liquid perfusion in the packed bed bioreactor. This repeating pattern can continue until the entire bioreactor is filled with mesh. These are merely examples and are for illustrative purposes only, and are not intended to limit possible combinations. In practice, various combinations of meshes of different sizes can yield different distributions of bulk density and flow resistance on the cell growth surface. For example, packed bed towers with regions of different volumetric cell densities (e.g., a series of regions producing a low / high / low / high density isodense pattern) can be assembled by staggering the use of meshes of different sizes.

[0069] As discussed above, embodiments of this disclosure include bioreactor containers capable of cell seeding, culturing, transfection, and / or collection using a cell culture substrate within the container, and capable of operation at different production scales. Bioreactors according to embodiments of this disclosure enable end-users to operate bioprocess experiments at 1× to 10× scales using the same bioreactor unit. The simple scalability model of these embodiments enables the transfer of bioprocesses from research to process development to production scale within a single system. This flexibility in bioreactor capacity configuration will save costs and time in process optimization and validation within the 1× to 10× scale range. Some aspects of the embodiments will also allow end-users to seed and collect cells at the same predetermined flow rate without the need for re-optimization during bioreactor scaling.

[0070] The substrate according to embodiments may be non-woven or woven, such as a woven PET substrate. However, in some embodiments, the substrate may be non-woven. The substrate may include multiple layers of substrate material stacked together, or substrate material in rolls or spirals.

[0071] In one or more embodiments, the cell culture medium is secured to the culture chamber by a fixing mechanism. The fixing mechanism may secure a portion of the cell culture medium to the wall of the culture chamber surrounding the medium, or to the wall of one end of the culture chamber. In some embodiments, the fixing mechanism adheres a portion of the cell culture medium to a member extending through the culture chamber, such as a member extending parallel to or perpendicular to the longitudinal axis of the culture chamber. However, in one or more other embodiments, the cell culture medium may be contained within the culture chamber without being fixedly attached to the wall of the culture chamber or bioreactor vessel. For example, the medium may be contained within the boundaries of the culture chamber or other structural members within the culture chamber to keep the medium within a predetermined area of ​​the bioreactor vessel, without requiring the medium to be fixedly secured to these boundaries or structural members.

[0072] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. According to one or more embodiments described in this disclosure, the culture chamber is capable of containing cell culture media and substrates.

[0073] In a rolling flask configuration, the bioreactor vessel can be operatively attached to a means for moving the bioreactor vessel about its central longitudinal axis. For example, the bioreactor vessel can rotate about its central longitudinal axis. Rotation can be continuous (e.g., continuous in one direction) or discontinuous (e.g., intermittent rotation in a single or alternating direction, or oscillating in a reciprocating rotation direction). During operation, the rotation of the bioreactor vessel causes movement of cells and / or fluid within the chamber. This movement can be considered as movement relative to the chamber walls. For example, when the bioreactor vessel rotates about its central longitudinal axis, gravity can keep the fluid, culture medium, and / or unattached cells facing the lower portion of the chamber. However, in one or more embodiments, the cell culture medium is substantially fixed relative to the vessel and thus rotates with the vessel. In one or more other embodiments, the cell culture medium may be unattached and can move freely relative to the vessel to a desired angle as the vessel rotates. Cells may attach to the cell culture medium, and the movement of the vessel allows the cells to be exposed to the cell culture medium or liquid, as well as oxygen or other gases within the culture chamber.

[0074] By using cell culture substrates according to embodiments of the present disclosure, such as matrices including woven or mesh substrates, roller bottle containers will have increased surface area available for adherent cells to attach, proliferate, and function. Specifically, using a mesh substrate woven from monofilament polymer material within the roller bottle can increase the surface area to about 2.4 to about 4.8 times or about 10 times that of a standard roller bottle. As discussed herein, each monofilament strand of the mesh substrate can itself serve as a 2D surface for adherent cell attachment. Furthermore, multiple layers of mesh can be arranged within the roller bottle, increasing the total available surface area to about 2 to 20 times that of a standard roller bottle. Therefore, existing roller bottle facilities and processes, including cell seeding, culture medium replacement, and cell collection, can be modified by adding the improved cell culture substrates disclosed herein with minimal impact on existing operational infrastructure and processing steps.

[0075] The bioreactor vessel optionally includes one or more outlets that can be attached to inlet and / or outlet devices. Liquids, culture media, or cells can be supplied to or removed from the chamber through these one or more outlets. A single port in the vessel can be used as both an inlet and an outlet, or multiple ports can be provided as dedicated inlets and outlets.

[0076] The embodiments are not limited to the container rotating about a central longitudinal axis. For example, the container can rotate about an axis that is not at the center of the container. In addition, the axis of rotation can be a horizontal axis or a vertical axis.

[0077] definition

[0078] "Totally synthetic" or "completely synthetic" refers to cell cultures composed entirely of synthetic source materials and containing no animal-derived or animal-origin materials, such as the surface of microcarriers or culture containers. The disclosed totally synthetic cell cultures eliminate the risk of foreign contamination.

[0079] The terms “include”, “includes”, or similar terms mean that something is included but not limited to; that is, inclusive rather than exclusive.

[0080] "User" means anyone who uses the systems, methods, articles or kits disclosed herein, and includes anyone who cultures cells to collect cells or cell products, or uses cells or cell products cultured and / or collected in accordance with the examples herein.

[0081] When describing embodiments of this disclosure, the term "about" is used to modify, for example, the amount, concentration, volume, processing temperature, processing time, yield, flow rate, pressure, viscosity, and similar values ​​and ranges thereof of a component in a composition, or the size and similar values ​​and ranges thereof of a component, to refer to variations in numerical quantities that may occur, for example, due to: typical measurement and processing procedures used to prepare materials, compositions, complexes, concentrates, components, articles, or formulations; unintentional errors in these procedures; differences in the manufacture, source, or purity of starting materials or components used to carry out the method; and similar considerations. The term "about" also covers amounts that differ due to aging of a composition or formulation having a particular initial concentration or mixture, and amounts that differ due to mixing or processing of a composition or formulation having a particular initial concentration or mixture.

[0082] The terms “optional” or “optionally” mean that the event or situation described below may or may not occur, and the description includes instances where the event or situation occurs as well as instances where the event or situation does not occur.

[0083] Unless otherwise stated, the indefinite articles “a” or “an” and their corresponding definite articles “the” as used herein mean at least one species, or one or more species.

[0084] Abbreviations well known to those skilled in the art may be used (e.g., “h” or “hrs” for hour, “g” or “gm” for gram, “mL” for milliliter, and “rt” for room temperature, “nm” for nanometer, and similar abbreviations).

[0085] The specific and preferred values ​​and ranges disclosed regarding components, ingredients, additives, dimensions, conditions, and similar aspects are for illustrative purposes only; they do not exclude other defined values ​​or other values ​​within the defined range. The systems, kits, and methods disclosed herein may include any or any combination of the values, specific values, more specific values, and preferred values ​​described herein, including explicit or implicit intermediate values ​​and ranges.

[0086] Unless otherwise expressly stated, no method described herein is intended to be construed as requiring its steps to be performed in a particular order. Therefore, no particular order is intended to be implied unless a method claim actually describes the order in which the steps are followed, or unless the claims or specification otherwise specifically state that the steps are limited to a particular order.

[0087] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope or spirit of the disclosed embodiments. Since modifications, combinations, sub-combinations, and variations of the disclosed embodiments incorporated into the spirit and intent of the embodiments will be apparent to those skilled in the art, the disclosed embodiments should be construed as including all contents within the scope of the appended claims and their equivalents.

[0088] As discussed above, aspects of one or more embodiments include a culture medium inlet configured to supply at least one of cells, cell culture medium, and other components to the lumen of a bioreactor vessel, and a culture medium outlet configured to withdraw at least one of cells or other biological reagents, cell culture medium, and cells or biological byproducts from the lumen during or after cell culture. Additionally, as discussed herein, for a bioreactor contents collection procedure, the culture medium outlet may be configured to supply pressurized fluid to a compartment during collection operations, while the culture medium inlet is configured to withdraw at least one of cells, cell culture medium, and cell byproducts from the lumen during collection operations. The bioreactor system is configured to fill the lumen with pressurized fluid through the culture medium outlet to force at least one of cells, cell culture medium, and cell byproducts out through the culture medium inlet. Therefore, a compartment may also be referred to herein as a collection volume, meaning a space or volume pressurized or filled with fluid in some manner to perform at least a portion of the collection procedure.

[0089] According to some embodiments, the collection solution may include one or more components that help collect and / or release cells from a cell culture substrate. Examples include Accutase® or TrypLE®, but those skilled in the art will recognize alternative collection agents that can be used.

[0090] Other advantages of the embodiments described herein are also apparent. For example, in some embodiments, the cell culture medium substrate extends uninterruptedly within the inner diameter of the bioreactor. This is far simpler than the complex flow channels and paths employed in alternative methods. The flow through the fixed bed is unidirectional, or can be characterized as a piston flow, which again simplifies operation and improves performance, including, for example, cell seeding and collection. Thus, all layers of the packed bed are perfused with the culture medium with the same efficiency, and the flow rate or flux through or through said layers is also the same.

Claims

1. A bioreactor system for culturing cells, the bioreactor system comprising: A container comprising an inlet, an outlet, and a cavity, the cavity being disposed between the inlet and the outlet and configured to allow perfusion of cell culture medium; and A cell culture substrate is disposed in the cavity in a fixed-bed configuration. The cell culture substrate includes a surface with a certain surface area, which is configured to culture cells on the surface during operation of the bioreactor system. The bioreactor system is configured to scale up or down as needed by adjusting the surface area of ​​the cell culture substrate.

2. The bioreactor system of claim 1, wherein the container has a certain height in the direction from the inlet to the outlet, and Configuring the bioreactor system to scale up includes adjusting the height of the container.

3. The bioreactor system according to claim 1 or claim 2, wherein the bioreactor system is configured to scale without adjusting the width of the container, the width of the container being in a direction perpendicular to the height of the container.

4. The bioreactor system according to any one of the preceding claims, wherein the bioreactor system is configured to scale up without adjusting the flow rate of cell culture medium through the lumen.

5. The bioreactor system according to any one of the preceding claims, wherein the bioreactor system is configured to scale up according to different numbers of cells to be cultured, thereby culturing the different numbers of cells at the same flow rate.

6. The bioreactor system according to any one of the preceding claims, the bioreactor system further comprising an inlet fluid distribution mechanism between the inlet and the cell culture substrate.

7. The bioreactor system of claim 6, wherein the bioreactor system is configured to scale up without altering the inlet fluid distribution mechanism.

8. The bioreactor system of claim 6 or claim 7, wherein the inlet fluid distribution mechanism is configured to distribute fluid flowing into the container via the inlet across the entire width of the cavity.

9. The bioreactor system according to any one of claims 6 to 8, wherein the container includes an inlet pressure chamber, the inlet pressure chamber including the inlet fluid distribution mechanism.

10. The bioreactor system according to any one of claims 6 to 9, wherein the inlet fluid distribution mechanism is configured to produce at least one of the following on the fixed bed configuration of the cell culture substrate: (i) uniform velocity distribution, (ii) uniform mass flow rate and (iii) uniform cell distribution.

11. The bioreactor system according to any one of claims 6 to 10, wherein the inlet fluid distribution mechanism comprises a plurality of holes through which fluid flows from the inlet side of the inlet fluid distribution mechanism to the cell base side of the inlet fluid distribution mechanism.

12. The bioreactor system of claim 11, wherein the plurality of orifices comprises a plurality of orifice rings concentrically arranged around the center of the inlet fluid distribution mechanism.

13. The bioreactor system of claim 12, wherein the width of each of the plurality of porous rings is approximately equal to the spacing between the pores within the rings.

14. The bioreactor system according to any one of claims 6 to 13, wherein the inlet fluid distribution mechanism comprises a single-stage distribution mechanism consisting of a single perforated plate.

15. The bioreactor system according to any one of claims 6 to 10, wherein the inlet fluid distribution comprises a distributor plate, the distributor plate comprising a first surface, a second surface, and a plurality of holes extending through the thickness of the distributor plate from the first surface to the second surface. The first surface includes a plurality of annular regions covering the surface of the first surface, each of the plurality of annular regions including a hole ring of the plurality of holes.

16. The bioreactor system of claim 15, wherein each of the plurality of annular regions comprises an annular region surface area (SA) as defined by the following equation. ar ): SADDLE ar = π ( R 2 -r 2 ) Where R is the outer radius of the annular region, and r is the inner radius of the annular region. Each of the plurality of annular regions contains a total hole area, which includes the sum of the areas of all the plurality of holes within the annular region. SA of one of the multiple annular regions ar The ratio to the total hole area is equal to the ratio of any other annular region among the plurality of annular regions.

17. The bioreactor system according to any one of the preceding claims, the bioreactor system further comprising a bubble trap disposed in the container.

18. The bioreactor system of claim 17, further comprising at least one of an inlet pressure stabilizing chamber and an outlet pressure stabilizing chamber, wherein the inlet pressure stabilizing chamber and the outlet pressure stabilizing chamber comprise a perforated distributor plate, and the bubble trap is disposed on the perforated distributor plate of the inlet pressure stabilizing chamber and the outlet pressure stabilizing chamber.

19. The bioreactor system according to any one of the preceding claims, wherein the perforated distributor plate includes a tapered surface on the upstream side of the perforated distributor plate, the tapered surface rising toward the periphery of the perforated distributor plate.

20. The bioreactor system of claim 19, wherein the conical surface surrounds the center of the perforated distributor plate.

21. The bioreactor system of claim 19 or claim 20, wherein the conical surface is contained at or near the low point of the perforated distributor plate.

22. The bioreactor system according to any one of claims 18 to 21, wherein the perforated dispenser plate includes a groove at or near the periphery of the perforated dispenser plate, the groove being configured to at least temporarily confine air bubbles.

23. The bioreactor system of claim 22, further comprising a bubble outlet in the groove, the bubble outlet being configured to siphon bubbles out of the groove.

24. The bioreactor system according to any one of the preceding claims, wherein the surface area is about 1 m². 2 Approximately 3000 m 2 .

25. The bioreactor system according to any one of the preceding claims, wherein the surface area is about 10 m². 2 Approximately 200 m 2 Approximately 20 m 2 Approximately 100 m 2 Approximately 100 m 2 Approximately 500 m 2 or about 500 m 2 Approximately 1000 m 2 .

26. The bioreactor system according to any one of the preceding claims, wherein the surface area can range from about 10 m² 2 Expanded to approximately 200 m 2 From approximately 100 m 2 Expanded to approximately 500 m 2 From approximately 500 m 2 Expanded to approximately 1000 m 2 Or from about 10 m 2 Expanded to approximately 1000 m 2 .

27. The bioreactor system of claim 25, wherein the surface area can range from about 20 m² 2 Expanded to approximately 50 m 2 Expanded to approximately 100 m 2 Expanded to approximately 500 m 2 And expanded to approximately 1000 m 2 .

28. The bioreactor system according to any one of the preceding claims, wherein the surface area is capable of being expanded from a first size to a second size, the second size being about 5 times or about 10 times the first size.

29. The bioreactor system according to any one of the preceding claims, wherein the cell culture substrate comprises a plurality of porous discs arranged in a stacked manner, each of the plurality of porous discs comprising a surface configured to culture cells thereon.

30. A fluid distribution plate comprising: A first side, a second side opposite to the first side, a thickness separating the first side and the second side, and a plurality of holes extending from the first side to the second side through the thickness, and A bubble trap is disposed on the first side, the bubble trap comprising a conical surface on the first side that rises toward the periphery of the fluid distribution plate.

31. The fluid distribution plate of claim 30, wherein the tapered surface surrounds the center of the first side of the fluid distribution plate.

32. The fluid distribution plate according to claim 30 or claim 31, wherein the tapered surface is contained at a low point near the center of the fluid distribution plate.

33. The fluid distribution plate of claim 30, wherein the fluid distribution plate includes a groove at or near the periphery of the fluid distribution plate, the groove being configured to at least temporarily confine air bubbles.

34. The fluid distribution plate of claim 33, further comprising a bubble outlet in the groove, the bubble outlet being configured to siphon bubbles out of the groove.

35. The fluid distribution plate according to any one of claims 30 to 34, wherein the plurality of holes comprises a plurality of hole rings arranged concentrically around the center of the fluid distribution plate.

36. The fluid distribution plate of claim 35, wherein the width of each of the plurality of perforated rings is approximately equal to the spacing between the holes within the rings.

37. The fluid distribution plate according to any one of claims 30 to 36, wherein the first side comprises a plurality of annular regions covering a surface of the first side, each of the plurality of annular regions comprising an annular ring of the plurality of holes.

38. The fluid distribution plate of claim 37, wherein each of the plurality of annular regions comprises an annular region surface area (SA) as defined by the following equation. ar ): SADDLE ar = π (R 2 -r 2 ) Where R is the outer radius of the annular region, and r is the inner radius of the annular region. Each of the plurality of annular regions contains a total hole area, which includes the sum of the areas of all the plurality of holes within the annular region. SA of one of the multiple annular regions ar The ratio to the total hole area is equal to the ratio of any other annular region among the plurality of annular regions.

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