Perfusion bioreactors and systems 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 interior cavity disposed between the inlet and the outlet and for perfusing cell culture media therethrough. The system also includes a cell culture substrate disposed in the interior cavity in a fixed-bed configuration, the cell culture substrate having a surface having a surface area upon for culturing cells thereon during operation of the bioreactor system. The bioreactor system can scale up or down with a desired number of cells to be cultured by adjusting the surface area of the cell culture substrate.

Description

PERFUSION BIOREACTORS AND SYSTEMS FOR LARGE SCALE CELL CULTURECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application Serial No. 63 / 535,904 filed on August 31, 2023, the content of which is relied upon and incorporated herein by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] This disclosure generally relates to apparatuses, systems, and methods for culturing cells in a fixed-bed perfusion bioreactor. In particular, the present disclosure relates to fixed- bed bioreactor systems that are efficiently scalable across culture sizes, and methods of assembling such system and of culturing cells using such systems.BACKGROUND

[0003] In the bioprocessing industry, large scale cultivation of cells is performed for purposes of the production of hormones, enzymes, antibodies, vaccines and cell therapies. A significant portion of the cells used in bioprocessing are anchorage dependent, meaning the cells need a surface to adhere to for growth and functioning. Traditionally, the culturing of adherent cells is performed on two-dimensional (2D) cell-adherent surfaces incorporated in one of a number of vessel formats, such as T-flasks, petri dishes, cell factories, cell stack vessels, roller bottles, and HYPERStack® vessels. These approaches can have significant drawbacks, including the difficulty in achieving cellular density high enough to make it feasible for large scale production of therapies or cells.

[0004] Alternative methods have been suggested to increase volumetric density of cultured cells. These include microcarrier cultures performed in stir tanks. In this approach, cells that are attached to the surface of microcarriers are subject to constant shear stress, resulting in a significant impact on proliferation and culture performance. Another example of a high- density cell culture system is a hollow fiber bioreactor, in which cells may form large three- dimensional aggregates as they proliferate in the interspatial fiber space. However, the cellsgrowth and performance are significantly inhibited by the lack nutrients. To mitigate this problem, these bioreactors are made small and are not suitable for large scale manufacturing.

[0005] Another example of a high-density culture system for anchorage dependent cells is a packed bed bioreactor system. For example, packed bed bioreactor systems that contain a packed bed of support or matrix systems to entrap the cells have been previously disclosed U.S. Patent Nos. 4,833,083; 5,501,971; and 5,510,262. Packed bed matrices usually are made of porous particles as substrates or non-woven microfibers of polymer. Such bioreactors function as recirculation flow-through bioreactors. One of the significant issues with such bioreactors is the non-uniformity of cell distribution inside the packed bed. For example, the packed bed functions as depth filter with cells predominantly trapped at the inlet regions, resulting in a gradient of cell distribution during the inoculation step. In addition, due to random fiber packaging, flow resistance and cell trapping efficiency of cross sections of the packed bed are not uniform. For example, medium flows fast though the regions with low cell packing density and flows slowly through the regions where resistance is higher due to higher number of entrapped cells. This creates a channeling effect where nutrients and oxygen are delivered more efficiently to regions with lower volumetric cells densities and regions with higher cell densities are being maintained in suboptimal culture conditions.Another significant drawback of packed bed systems disclosed in a prior art is the inability to efficiently harvest intact viable cells at the end of culture process. U.S. Patent No. 9,273,278 discloses a bioreactor design to improve the efficiency of cell recovery from the packed bed during cells harvesting step. It is based on loosening the packed bed matrix and agitation or stirring of packed bed particles to allow porous matrices to collide and thus detach the cells. However, this approach is laborious and may cause significant cells damage, thus reducing overall cell viability.

[0006] Roller bottles have several advantages such as ease of handling, and ability to monitor cells on the attachment surface. However, from a production standpoint, the main disadvantage is the low surface area to volume ratio while the roller bottle configuration occupies a large area of manufacturing floor space. Various approaches have been used to increase the surface area available for adherent cells in a roller bottle format. Some solutions have been implemented in commercially available products, but there remains room forimprovement to increase roller bottle productivity even further. Traditionally, a roller bottle is produced as a single structure by a blow-molding process. Such manufacturing simplicity enables economic viability of roller bottles in bioprocessing industry. Some roller bottle modifications to increase the available surface area for cell culturing can be achieved without changing manufacturing process, however only marginal increase of modified roller bottle surface area is obtained. Other modifications of the roller bottle design add significant complexity to manufacturing processes making it economically unviable in the bioprocessing industry. It is desirable therefore to provide roller bottle with increased surface area and bioprocessing productivity, while using the same blow-molding process for its manufacturing.

[0007] While manufacturing of viral vectors for early-phase clinical trials is possible with existing platforms, there is a need for a platform that can produce high-quality product in greater numbers in order to reach late-stage commercial manufacturing scale. In particular, there is a need for platforms that can efficiently and effectively scale across cell cultures of various sizes. Using a single platform across different culture sizes can have many advantages, not only in terms of time and resources for users performing the culture, but also in terms of the potential performance of cells, which may be “used to” growing on certain platforms and thus may exhibit healthier cultures when used across scales of a single platform. Most commercially available systems are limited in the surface area that they can provide, and such systems sometimes do not scale well to manufacturing requirements.However, operating multiple small-scale systems in parallel quickly becomes difficult due to the increased footprint and the demand on other components, such as pumps, controllers, and the tubing management.

[0008] There are a few commercially available bioreactor systems that do scale to large surface areas. Such systems may scale by increasing the reactor diameter and process flow rates, keeping the linear flow velocity through the culture bed constant. The height of the reactors may be limited by the need to uniformly seed cells, as well as to provide a habitable environment throughout the entire height (e.g., enough oxygen has to be provided to all cells and not just to cells closest to the inlet).

[0009] Thus, there is a need for bioreactor systems that enable scalable platforms while also enabling uniform fluid flow, cell distributions, and mass flow.SUMMARY

[0010] According to an embodiment of this disclosure, a fixed-bed bioreactor system is provided. The bioreactor system includes a vessel comprising an inlet, an outlet, and an interior cavity disposed between the inlet and the outlet and for perfusing cell culture media therethrough. The system also includes a cell culture substrate disposed in the interior cavity in a fixed-bed configuration, the cell culture substrate having a surface having a surface area upon for culturing cells thereon during operation of the bioreactor system. The bioreactor system can scale up or down with a desired number of cells to be cultured by adjusting the surface area of the cell culture substrate.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A shows a schematic cross-section view of a bioreactor system of a first size, according to embodiments.

[0012] Figure IB shows a schematic cross-section view of a bioreactor system of a second size, according to embodiments. .

[0013] Figure 1C shows a schematic cross-section view of a bioreactor system of a third size, according to embodiments.

[0014] Figure 2 shows a schematic cross-section view of a bioreactor system, according to some embodiments.

[0015] Figure 3A shows a plan view of a flow distribution mechanism, according to embodiments.

[0016] Figure 3B shows a cross-section view of a hole in the flow distribution mechanism, according to embodiments.

[0017] Figure 3C shows a plan view of the flow distribution mechanism of Figure 3 A with shading to show a concentric ring pattern of holes, according to embodiments.

[0018] Figure 4A shows a schematic cross-section view of a bioreactor system with an integrated bubble trap, according to embodiments.

[0019] Figure 4B shows a close-up of the integrated bubble trap from Figure 4A, according to embodiments.

[0020] Figure 5 shows mass flow rate data for a flow distribution mechanism, according to embodiments.

[0021] Figure 6A shows the result of a computational fluid dynamics (CFD) simulation for a first plenum geometry, according to embodiments.

[0022] Figure 6B shows the result of a computational fluid dynamics (CFD) simulation for a second plenum geometry, according to embodiments.

[0023] Figure 6C shows a close-up view of the CFD simulation shown in Figure 6A.

[0024] Figure 6D shows a close-up view of the CFD simulation shown in Figure 6B.

[0025] Figure 7 is a graph showing variation in cell number per band area for the CFD simulations in Figures 6A-6D, according to embodiments.

[0026] Figure 8 is a graph of the velocity profile at the exit of each ring of holes in the flow distribution mechanism, according to embodiments.

[0027] Figure 9 shows a guide rod assembly and alignment feature for cell culture substrates of a bioreactor, according to embodiments.

[0028] Figure 10 is an illustration of two layers of mesh cell culture substrate with a cutout in the center to match the guide rod assembly of Figure 9, according to embodiments.

[0029] Figure 11 is an illustration of a variable height guide rod and cell culture substrate assembly, according to embodiments.

[0030] Figure 12 is a cross-section view of the assembly of Figure 11, according to embodiments.DETAILED DESCRIPTION

[0031] Various embodiments of the disclosure will be described in detail with reference to drawings, if any. Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments of the claimed invention.

[0032] Embodiments of this disclosure are directed to cell culture bioreactor systems, and methods of assembling and of culturing cells using such bioreactor systems. According toaspects of embodiments of this disclosure, bioreactor systems are provided that feature an improved, single-stage open plenum flow distribution system that ensures uniform velocity, mass and cell distribution in the reactor. Previous solutions may only provide, at best, uniform velocity distributions, and may either require a staged or multi-stage distribution approach or several bifurcating channels.

[0033] Embodiments also include flow distribution mechanisms in the inlet and / or outlet plenum(s) of the bioreactors that incorporate a bubble trap (i.e., a buffer for air bubbles that unintentionally enter the reactor, preventing them from interfering with the cell culture process). Previous solutions may depend on a separate and / or external bubble trap.

[0034] As a further aspect of embodiments of the present disclosure, bioreactors disclosed herein are symmetric in the axial as well as in the radial direction. To achieve symmetry in the radial direction, a central guide rod design can be used, according to some embodiments. The guide rod ensures proper orientation of the cell culture substrate during assembly and does not rely on the orientation of individual cell substrate layers, but differentiates between two different layers via cut-outs in the substrate material. Using a center-located guide rod provides symmetry and eases manufacture and assembly of the bioreactor components. Previous solutions have used a guide rod offset to one side of the reactor, near the reactor walls, and, in some cases, may have required the mesh cell substrate layers to be assembled in alternate orientations by flipping every other layer for particular mesh layer alignments.

[0035] According to some preferred embodiments, a bioreactor system that is capable of scaling of cell culture substrate surface areas from 20 m2to 100 m2are provided. For example, a so-called “pilot scale” platform system could include bioreactors with surface areas of 20 m2, 50 m2, and 100 m2. However, embodiments are not necessarily limited to those surface area sizes. According to embodiments, the bioreactor system can be scaled up to surface areas between 500 m2and 1000 m2. Different scales of bioreactor can differ only in bioreactor height (and, correspondingly, the height of the cell culture substrate inside the reactor), while other aspects (e.g., the flow distribution mechanism, width of the bioreactor, and other design features) can remain the same across sizes. Unless otherwise stated, reference to the “bioreactor height” or “height of the reactor” can refer to the height of the vessel itself, the height of the fixed bed substrate within the vessel, or both. According tosome embodiments, even process conditions (like flow rate) can remain the same across bioreactor sizes, while only the height of the reactor. Besides an optimized geometry for uniform cell distribution, the bioreactors systems of this disclosure can have additional features not shown in other systems, such as an integrated bubble trap and a guide rod that keeps the cell substrate bed assembly ordered and symmetric.

[0036] As shown in Figures 1A-1C, the bioreactor system of this disclosure can scale across different size by only scaling the height hl, h2, and h3, respectively, of the reactor. For example, the bioreactor system 100a of Figure 1A can have a substrate 102a with a surface area of, for example, about 20 m2, the bioreactor system 100b of Figure IB can have a substrate 102b with a surface area of, for example, about 50 m2, and the bioreactor system 100c of Figure 1 C can have a substrate 102b with a surface area of, for example, about 100 m2. These numbers are shown as examples, only, and embodiments include different surface areas. By only needing to scale the height of the fixed bed while other design aspect remain the same, manufacturing of the bioreactor system can be streamlined and consistent performance across scales can be obtained. For example, although embodiments of this disclosure are not limited to any particular construction for the bioreactor vessel, some embodiments include a multi-component reactor assembly that includes a vessel side wall surrounding the fixed-bed cell culture substrate and other components on either end of the fixed bed (e.g., distributor plates, end caps, inlets / outlets, etc.). A system which only requires scaling the vessel height to scale the cell culture means only the vessel side wall (and, correspondingly, the amount of cell substrate contained within) needs to be scaled, while the other components (e.g., distributor plates, endcaps, fixtures, inlets / outlets, tubing, etc.) can remain the same and be used for any sized reactor. This greatly simplifies manufacturing, assembling, and operating the system.

[0037] Figure 2 shows additional features of a bioreactor system 110 according to embodiments of this disclosure. The bioreactor system 110 includes a vessel 111 with a sidewall that defines an interior cavity 112 having a cell culture substrate 114. Additional details of the substrate are discussed below. The vessel 111 also includes an inlet 116 and an outlet 118 designed for flowing materials (e.g., cells, culture media, etc.) into and out of, respectively, the interior cavity of the vessel 111. The bioreactor system 110 may also includea guide rod 120, shown in the center of the cell culture substrate 114. The guide rod 120 can be used to ease assembly and arrangement of the cell substrate 114. Between the inlet 116 and the cell culture substrate 114 is an inlet plenum 122 and an inlet flow distribution mechanism 124, or inlet distributor plate. The bioreactor system 1 10 can also include an outlet flow distribution mechanism 126, or outlet distributor plate, and an outlet plenum 128.

[0038] Embodiments of the bioreactor systems of this disclosure include a cylindrical body that holds the cell culture substrate fixed bed. In some examples used herein for illustration purposes, the cylindrical body has an interior diameter of about 11.5 inches, although the principles disclosed herein are applicable to other sizes. The bioreactor system can be considered to have an axial direction and a radial direction, where the axial direction is parallel to the height of the bioreactor. The axial direction can also be considered parallel to the general fluid flow direction through the bioreactor (e.g., from the inlet, through the fixed bed, and to the outlet). The radial direction is perpendicular to the axial direction. In order to seed cells uniformly in the radial direction, the incoming media is diverted from the inlet and spread out over the entire mesh bed width. This is achieved by a flow distribution plate in the inlet plenum of the bioreactor. Aspects of embodiments of this disclosure can include one or more of the following three performance characteristics for an advantageous distribution plate: (1) generating uniform velocity profile, (2) generating uniform mass flow, and (3) generating uniform cell distribution. As used herein, uniformity is defined as the deviations from the mean, e.g., uniform velocity profile means the axial velocity component of the media through the bioreactor as function of radial position deviates from the mean axial velocity by less than 10%.

[0039] The distributor plate is configured to provide a large enough back pressure to the incoming cell media to force it to spread out radially substantially before entering the mesh bed axially. Theoretically, a porous material could be used here as the permeability could be selected to provide the necessary back pressure, and at the same time provide many, very small pores. However, a porous material would most likely capture all the cells and clog up and is therefore not an option for this application. The discrete version of a porous material is a perforated plate, with many individual holes. The more holes and the smaller each hole, the better. However, if the holes are too small and / or there are too few of them, the velocity andcorresponding shear forces on the cells can actually be harmful to the process. Moreover, the distribution plate must be fabricated and machining tolerances have to be considered. A perforated plate with around 1% total open area is used herein for illustration. This is achieved by 0(500) holes with a diameter of 3 / 64 inches (about 1.19 mm). Maximizing the total open area while maintaining acceptable performance may be desired, because it will decrease the spacing between individual holes and lower the velocity in each hole. According to aspects of embodiments herein, the hole pattern is designed in such a way that equal mass flow through the mesh bed is guaranteed. For example, in aspects of embodiments, it is designed such that each hole provides the mass flow for the mesh or cell substrate directly above it. There are several ways to tile a circle in equal areas and distribute the holes. For example, the distributer plate can be divided into several rings and the number of holes in each ring can be calculated based on a constant ratio between ring area and the total area of the holes in that ring. Moreover, the width of each ring is roughly equal to the spacing of holes within each ring.

[0040] According to embodiments, the cell media is delivered into the reactor from one side of the reactor. In embodiments shown herein, a single, central inlet is contemplated, although embodiments include multiple inlets, as well. A properly designed single-stage flow distribution plate and inlet plenum is sufficient to fulfill all three above-mentioned uniformity requirements (i.e., uniform velocity profile, uniform mass flow, and uniform cell distribution). Compared to a multi-stage approach, where the incoming cell media is spread out radially incrementally via two or more perforated plates, this has the advantage of being more compact while also reducing the number of recirculation zones, which can lead to undesirable side-effects, such as preferential cell concentrations and cell capturing before reaching the cell substrate fixed bed. Embodiments herein reduce the effects of such recirculation zones by optimizing the geometry of the inlet plenum, including the distance between the reactor inlet and the distribution plate.

[0041] Figure 3A shows an example of an inlet flow distribution mechanism, according to aspects of embodiments. This example of a flow distribution plate 200 with a pattern of holes 202 that ensures uniform velocity and mass flow. Shown are sixteen rings with a total of 659 holes, each with a diameter of 3 / 64 of an inch. As shown in Figure 3B, each hole 202 canhave a chamfer on the downstream side 206 that faces the fixed bed substrate, which minimizes the contact area between the flow distribution plate 200 and the adjacent cell culture substrate. In Figure 3C, the sixteen rings 208 of holes 202 are highlighted as alternating shaded regions to better illustrate the hole pattern.

[0042] Figures 4A and 4B show an example of a bioreactor 400 with an inlet distribution plate 402 with an integrated bubble trap 404. Figure 4B shows a closeup of one side of the inlet distribution plate 402 and bubble trap 404, for clarity. Bubbles are a known problem for bioreactors as they can negatively affect the process either by hindering cells from attaching to the substrate, or by causing non-uniformities in the media delivery by blocking holes in the distributor plate. Ideally, bubbles would never enter the reactor, and external, in-line bubble traps can be used with the bioreactors. However, such external bubble traps can dramatically complicate the system, especially because these bubble traps are generally either single-use devices and get sterilized with the reactor before use, or they have to be cleaned in place. Knowing this, the distributor plate can be designed with the secondary function of collecting and removing bubbles. According to embodiments, as shown in Figures 4A and 4B, the bottom or up-stream surface 406 of the distributor plate is tapered. The buoyancy of the bubbles will force them to the highest point of the surface 406, which will cause the bubbles to travel upward and outward toward the outside of the inlet distributor plate 402. The distributor plate 402 was designed such that its thickest in the center and tapers off towards the outer edges. Further, a groove 408 is provided at the perimeter of the distributor plate 402. Thus, bubbles will be guided up and away from the center of the distributor plate 402 by the tapered surface 406 and then trapped in the groove 408 where they cannot interfere with the cell culture process. From there, bubbles can either be siphoned off or simple remain in the groove 408. To siphon away the bubbles, a bubble trap outlet 410 can be provided in fluid communication with the groove 408. The size of the groove 408 will determine how many bubbles can be collected before they have to be siphoned off. This tapered surface 406, together with the surface tension of the bubbles, prevents bubbles from entering the cell culture substrate bed.

[0043] Further aspects of embodiments of this disclosure include a central alignment rod within the bioreactor to assembly the cell substrate within the reactor. Embodiments of thisalignment rod will be discussed in reference to the woven cell culture substrate discussed further below. In particular, this will be discussed with reference to bioreactors having stacked layers of mesh used for the cell culture substrate fixed bed. Experiments have shown that axial cell distributions in bioreactors is improved when the orientation of the mesh substrate layers are alternated at a 45-degree angle with respect to adjacent mesh layers. That is, if each mesh disk is made of fibers running perpendicular to each other, the alternating 45- degree angle is achieved by placing one mesh layer with fibers running at 0 degrees and 90 degrees, and then placing on top of the first layer a second mesh layer with fibers running at 45 degrees and 135 degrees. This ensures adjacent layers do not interlock and block cells from passing through the bed. Previous designs explored using of a guide rod located on one side of the reactor to align these mesh layers in the correct orientations. To do so, each layer had a feature cut-out with a 22.5 degree symmetry that determines its orientation. During the mesh bed assembly, every other layer is then flipped over to create the 45-degree offset. However, this is a tedious process and prone to mistakes, especially because this process is difficult to automate. Another downside of this earlier design is that the symmetry inside the bioreactor is broken by having a guide rod placed asymmetrically against the side wall of the reactor.

[0044] Accordingly, embodiments of this disclosure include an improved guide rod concept that makes the assembly process easier and more reliable, while also maintaining symmetry inside the bioreactor. Instead of relying on flipping over every other mesh to ensure proper orientation, the proposed design cuts the alignment feature itself at a defined angle, such that mesh orientation is fixed by the features of the guide rod. One of the simplest implementations of such a design, that also provides structural strength, is a guide rod 900 with a cross-sectional shape of a cross 902 located at the radial center of the bioreactor, as shown in Figure 9. A 45-degree orientation offset is achieved by processing the mesh in two stacks 910, 911, each with the feature 912, 913 cut out at 45 degrees with respect to the mesh direction (see Figure 10 showing a first stack with a feature cut out at 0 degrees and a second stack with a feature cut out at 45 degrees). The only remaining process step is interlacing the two stacks. Since this type of alignment feature does not rely on flipping over individual layers of mesh, it is also not limited to two mesh orientation offsets between alternatinglayers. The alignment feature can be cut at any angle, therefore a controlled and repeatable way of generating a pseudo-random mash assembly is also possible.

[0045] Another consideration of the mesh assembly is the 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 number-of-layers-based approach will result in either different mesh bed heights, or, when forced into the same height, different amounts of pressure (i.e., compaction of the mesh). According to embodiments of this disclosure, this issue can also be addressed with a guide rod 1100 and another flow distributor plate 1102 on top of the mesh that is allowed to slide freely along the guide rod, as shown in Figures 11 and 12. The weight of the distributor plate would ensure the same amount of compaction for any number of mesh layers.Alternatively, a mesh weight / height-based approach with a varying number of mesh layers can be implemented and the guide rod height can be fixed to ensure a clearly defined volume inside the bioreactor.

[0046] Examples

[0047] The following examples are shown to illustrate aspects of embodiments of this disclosure. These examples are considered illustrative of certain aspects and advantages of embodiments herein, but should not be considered to limit embodiments of this disclosure to the examples shown and discussed.

[0048] Figure 7 shows a graph of the mass flow rate as a function of radial distance from the center of a pair of inlet flow distributor plates. Example 1 is an inlet flow distributor plate according to embodiments of this disclosure (and as shown in Figures 3A-3C and as described above), while Example 2 is an alternative distributor plate. As shown in the graph, the distributor plate of Example 1 shows more even mass flow across the radius of the distributor plate, with greatly reduced mass flow fluctuations compared to Example 2. Specifically, the mass flow fluctuations were reduced by more than 30% between adjacent rings in Example 1 as compared to Example 2. This demonstrates that the hole spacing and design discussed above does contribute to improved mass flow characteristics.

[0049] Figures 6A-6D show the results of computational fluid dynamics (CFD) simulations performed for two plenum geometry designs according to embodiments of this disclosure. Specifically, the distance between the inlet and the distributor plate was varied toexamine the impact on preferential cell concentrations on the cell distribution in the substrate fixed bed. In Figure 6A, the distance from the inlet to the underside of the distributor plate was 0.625 inches, and in Figure 6B the distance was increased to 2.125 inches. Figures 6C and 6D show the velocity magnitude of media and the presence of absence of recirculation bands. In these examples, increasing the distance between the inlet and the distributor plate eliminated the impact of preferential cell concentrations on the cell distribution in the fixed bed substrate, assuming uniform cell seeding at the inlet. The recirculation band was push outward radially with the increased distance (from band #5 in Figure 6C to band #8 in Figure 6D). These dimensions are examples only, but demonstrate that increasing the inlet-to- distributor-plate distance can positively effect cell distribution and uniformity in the fixed bed, and increasing the radial distance of the recirculation is believed to play a part in this.

[0050] Figure 7 shows a plot of results from the simulations shown in Figures 6A-6D. This graph shows that with a distance of just 0.625 inches between the inlet and distributor plate, the recirculation zones cause more cells passing through the inner rings. When the inlet-to- plate distance is increased to 2. 125 inches, there is a much more uniform distribution of cells.

[0051] Regarding the bubble trap discussed above, the tapering of the lower surface of the inlet distributor plate will effect flow characteristics, as shown in Figure 8. As shown in the graph of Figure 8, the velocity at the exit of reach hole ring (each ring being indicated by a band number in the graph) increases as the band number, or distance from the center of the inlet plate, increases. The thinner distributor plate will produce less back pressure and more flow is thus directed toward the outer rings of holes. Advantageously, however, this will counteract the naturally forming boundary layer at the reactor walls. The velocity profile can also be precisely controlled with the angle of the taper. As shown in Figure 8, the inlet-to- plate distance also has a slight impact, with the larger distance resulting in slightly higher velocities.

[0052] More details of the fixed bed substrate according to embodiments will now be described. In conventional large-scale cell culture bioreactors, different types of packed bed bioreactors have been used. Usually these packed beds contain porous matrices to retain adherent or suspension cells, and to support growth and proliferation. Packed-bed matrices provide high surface-area-to-volume ratios, so cell density can be higher than in the othersystems. The packed bed often functions as a depth filter, where cells are physically trapped or entangled in fibers of the matrix. However, because of linear flow of the cell inoculum through the packed bed, cells are subject to heterogeneous distribution inside the packed-bed. Thus, for example, there are higher cell densities at the inlet region of the bioreactor and significantly lower cell densities at the outlet part of the bioreactor. This non-uniform distribution of the cells inside of the packed-bed significantly hinders scalability of such bioreactors in bioprocess manufacturing.

[0053] Another problem encountered in packed bed bioreactors disclosed in prior art is the channeling effect. Due to random nature of packed nonwoven fibers, the local fiber density at any given cross section of the packed bed is not uniform. Medium flows quickly in the regions with low fiber density (high bed permeability) and much slower in the regions of high fiber density (lower bed permeability). Non-uniform media perfusion across the packed bed creates channeling effect. It manifests itself in development of significant nutrient and metabolite gradients that negatively impact overall cell culture and bioreactor performance. Cells located in the regions of low media perfusion will starve and very often die from the lack of nutrients or metabolite poisoning. Cell harvesting is yet another problem encountered when bioreactors packed with non-woven fibrous scaffolds are used. Due to packed-bed functions as depth filter cells that are released at the end of cell culture process are entrapped inside the packed bed, and cell recovery is very low. This significantly limits utilization of such bioreactors in bioprocesses where live cells are the products.

[0054] The present disclosure includes embodiments of a cell growth matrices and / or packed-bed systems for anchorage dependent cells that enable easy and effective scale-up to any practical production scale for cells or cell derived products (e.g., proteins, antibodies, viral particles). In one embodiment, a matrix is provided with a structurally defined surface area for adherent cells to attach and proliferate that has good mechanical strength and forms a highly uniform multiplicity of interconnected fluidic networks when assembled in a packed bed or other bioreactor. In particular embodiments, mechanically stable, non-degradable woven meshes can be used to support adherent cell production. Uniform cell seeding of such a matrix is achievable, as well as efficient harvesting of cells or other products of the bioreactor. In addition, the embodiments of this disclosure support cell culturing to achieveconfluent monolayer or multilayer of adherent cells on disclosed matrix, and can avoid formation of 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. The structurally defined matrix of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of bioreactor. In another embodiment of the present disclosure, a method of cell culturing is provided using bioreactors with the matrix for bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0055] As used herein, “structurally defined” means that a component has a non-random, ordered structure, following a defined structural design.

[0056] In one or more embodiments, a cell culture matrix is provided that supports attachment and proliferation of anchorage dependent cells in a high volumetric density format. The matrix can be assembled and used in a bioreactor system, such as a perfused back bed bioreactor, and provide uniform cell distribution during the inoculation step, while preventing formation of large and / or uncontrollable cell aggregates inside the matrix or packed bed. Thus, the matrix eliminates diffusional limitations during operation of the bioreactor. In addition, the matrix enables easy and efficient cell harvest from the bioreactor.

[0057] The matrix can be formed with a substrate material having a thin or sheet-like construction having first and second sides separated by a relatively small thickness. In other words, the thickness of the sheet-like substrate is small relative to the width and / or length of the first and second sides of the substrate. In addition, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is of a size and geometry that allows cells to adhere to the surface of the substrate material as if it were a two-dimensional (2D) surface, while also allowing adequate fluid 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 polymer sheet with openings punched through the thickness; a number of filaments that are fused into a meshlike layer; or a plurality of filaments that are woven into a mesh layer. The physical structure of the matrix has a high surface-to-volume ratio for culturing anchorage dependent cells. According to various embodiments, the matrix can be arranged or packed in a bioreactor incertain ways to obtain uniform cell seeding, uniform media perfusion, and efficient cell harvest.

[0058] Embodiments of this disclosure can achieve viral vector platforms of a practical size that can produce viral genomes on the scale of about 1015to about 1018or more viral genomes per batch. For example, in some embodiments, the viral genome yield can be about 1015to about 1016viral genomes or batch, or about 1016to about 1019viral genomes per batch, or about 1016- 1018viral genomes per batch, or about 1017to about 1019viral genomes per batch, or about 1018to about 1019viral genomes per batch, or about 1018or more viral genomes per batch. A “batch” can mean a single cell culture run of a single bioreactor vessel. Because of the scalability of the embodiments herein, a bioreactor vessel and the contained cell culture substrate can be appropriately scaled to achieve these yields. This scalability is aided by the structurally defined nature of the cell culture substrate, which provides uniform cell seeding and culturing, uniform media flow, and / or uniform harvesting. In some embodiments, a batch can include multiple bioreactor vessels used in a coordinate cell culture operation.

[0059] In addition, the embodiments disclosed herein enable not only cell attachment and growth to a cell culture substrate, but also the viable harvest of cultured cells. The inability to harvest viable cells is a significant drawback in current platforms, and it leads to difficulty in building and sustaining a sufficient number of cells for production capacity. According to an aspect of embodiments of this disclosure, it is possible to harvest viable cells from the cell culture substrate, including between 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, of the cells that are harvested, at least 80% are viable, at least 85% are viable, at least 90% are viable, at least 91% are viable, at least 92% are viable, at least 93% are viable, at least 94% are viable, at least 95% are viable, at least 96% are viable, at least 97% are viable, at least 98% are viable, or at least 99% are viable. Cells may be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0060] The cell culture substrate can be a woven mesh layer made of a first plurality of fibers running in a first direction and a second plurality of fibers running in a second direction. The woven fibers of the substrate form a plurality of openings. The size and shapeof the openings can vary based on the type of weave (e.g., number, shape and size of filaments; angle between intersecting filaments, etc.). An opening can be defined by a certain width or diameter. A woven mesh may be considered, on a macro-scale, a two-dimensional sheet or layer. However, a close inspection of a woven mesh reveals a three-dimensional structure due to the rising and falling of intersecting fibers of the mesh.

[0061] The woven mesh can be comprised of monofilament or multifilament polymer fibers. In one or more embodiments, a monofilament fiber may have a diameter in a range of about 10 pm to about 1000 pm. On a microscale level, due to the scale of the fiber compared to the cells (e.g., the fiber diameters being larger than the cells), the surface of monofilament fiber is presented as regular 2D surface for adherent cells to attach and proliferate. Such fibers are woven into a mesh that has a defined pattern and a certain amount of structural rigidity. Fibers can be woven into a mesh with openings ranging from about 10 pm x 10 pm, to about 100 pm x 100 pm, or to about 1000 pm x 1000 pm. These ranges of the filament diameters and opening diameters are examples of some embodiments, but are not intended to limit the possible feature sizes of the mesh according to all embodiments.

[0062] The substrate mesh can be fabricated from monofilament or multifilament fibers of polymeric materials compatible in cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide. Mesh substrates may have a different structure patterns or weaves, including, for example knitted, warp-knitted, or woven (plain weave, twilled weave, Dutch weave, five needle weave).

[0063] The surface chemistry of the mesh filaments may need to be modified to provide desired cell adhesion properties. Such modifications can be made through the chemical treatment of the polymer material of mesh or grafting cell adhesion molecules to the filament surface. Alternatively, meshes can be coated with thin layer of biocompatible hydrogels that demonstrate cell adherence properties, including, for example, collagen or Matrigel®. Alternatively, surfaces of filament fibers of the mesh can be rendered with cell adhesive properties through the treatment processes with various types of plasmas, process gases, and / or chemicals known in the industry.

[0064] By using a structurally defined culture matrix of sufficient rigidity, high-flow- resistance uniformity across the matrix or packed bed is achieved. According to various embodiments, the matrix can be deployed in monolayer or multilayer formats. This flexibility eliminates diffusional limitations and provides uniform delivery of nutrients and oxygen to cells attached to the matrix. In addition, the matrix lacks any cell entrapment regions in a packed bed configuration, allowing for complete cell harvest with high viability at the end of culturing. The matrix also delivers packaging uniformity for the packed bed, and enables direct scalability from process development units to large-scale industrial bioprocessing unit. The ability to directly harvest cells from the packed bed eliminates the need of resuspending a matrix in a stirred or mechanically shaken vessel. Further, the high packing density of the cell culture matrix yields high bioprocess productivity in volumes manageable at the industrial scale.

[0065] As discussed herein, the cell culture substrate can be used within a bioreactor vessel, according to one or more embodiments. For example, the substrate can be used in a packed bed bioreactor configuration, or in other configurations within a three-dimensional culture chamber. However, embodiments are not limited to a three-dimensional culture space, and it is contemplated that the substrate can be used in what may be considered a two- dimensional culture surface configuration, where the one or more layers of the substrate lay flat, such as within a flat-bottomed culture dish, to provide a culture substrate for cells. Due to contamination concerns, the vessel can be a single-use vessel that can be disposed of after use.

[0066] A packed-bed bioreactor system for culturing cells is provided, according to one or more embodiments, in which the cell culture matrix is used within a culture chamber of a bioreactor vessel. For example, embodiments include a cell culture system that includes a bioreactor vessel having a cell culture chamber in the interior cavity of the bioreactor vessel. Within the cell culture chamber is a cell culture matrix that is made from multiple substrate layers. The multiple substrate layers can include a plurality of separable and distinct substrate layers that are arranged in a stack, but can also include an integral cell culture matrix in which multiple layers are affixed together. The substrate layers are stacked with the first or second side of a substrate layer facing a first or second side of an adjacent substrate layer.The bioreactor vessel has an inlet at one end for the input of media, cells, and / or nutrients into the culture chamber, and an outlet at the opposite end for removing media, cells, or cell products from the culture chamber. By allowing stacking of substrate layers in this way, the system can be easily scaled up without negative impacts on cell attachment and proliferation, due to the defined structure and efficient fluid flow through the stacked substrates.

[0067] In one or more embodiments, flow resistance and volumetric density of the packed bed can be controlled by interleaving substrate layers of different geometries. In particular, mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the fluid flow resistance in packed bed format. By interlaying meshes of different sizes and geometries, one can control fluidic resistance in specific portion of bioreactor. This will enable better uniformity of liquid perfusion in packed bed bioreactor. Such repetition pattern may continue until the full bioreactor is packed with mesh. These are examples only, and used for illustrative purposes without intending to be limiting on the possible combinations. Indeed, various combinations of meshes of different sizes are possible to obtain different profiles of volumetric density of cells growth surface and flow resistance. For example, a packed bed column with zones of varying volumetric cells densities (e.g., a series of zones creating a pattern of low / high / low / high, etc. densities) can be assembled by interleaving meshes of different sizes.

[0068] As discussed above, embodiments of this disclosure include bioreactor vessels capable of performing cell seeding, culture, transfection, and / or harvesting using a cell culture substrate within the vessels, and capable of operating a different production scales. A bioreactor according to the embodiments of this disclosure enables an end user to run bioprocess experiments on lx to lOx scale using the same bioreactor unit. The simple scalability model of these embodiments enables bioprocess transfer from research to process development to production scale within one system. Such flexibility in the configuration of the bioreactor capacity will produce savings of cost and time of process optimization and validation in the lx to lOx scale range. Aspects of some embodiments will also allow end users to seed and harvest cells at the same predefined flow rates without the need for reoptimization during scale-up of the bioreactor.

[0069] The substrate according to embodiments can be non-woven or woven, such as a woven PET substrate. However, the substrate can be non-woven in some embodiments. The substrate can include multiple layers of substrate material in a stacked arrangement, or a roll or spiral of substrate material.

[0070] In one or more embodiments, the cell culture matrix is secured within the culture chamber by a fixing mechanism. The fixing mechanism may secure a portion of the cell culture matrix to a wall of the culture chamber that surrounds the matrix, or to a chamber wall at one end of the culture chamber. In some embodiments, the fixing mechanism adheres a portion of the cell culture matrix to a member running through the culture chamber, such as member running parallel to the longitudinal axis of the culture chamber, or to a member running perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture matrix may be contained within the culture chamber without being fixedly attached to the wall of the chamber or bioreactor vessel. For example, the matrix may be contained by the boundaries of the culture chamber or other structural members within the chamber such that the matrix is held within a predetermined area of the bioreactor vessel without the matrix being fixedly secured to those boundaries or structural members.

[0071] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. The culture chamber is capable of containing a cell culture matrix and substrate according to one or more of the embodiments described in this disclosure.

[0072] In the roller bottle configuration, the bioreactor vessel may be operably attached to a means for moving the bioreactor vessel about a central longitudinal axis of the vessel. For example, the bioreactor vessel may be rotated about the central longitudinal axis. The rotation may be continuous (e.g., continuing in one direction) or discontinuous (e.g., an intermittent rotation in a single direction or alternating directions, or oscillating in back and forth rotational directions). In operation, the rotation of the bioreactor vessel causes movement of cells and / or fluid within the chamber. This movement can be considered relative with respect to the walls of the chamber. For example, as the bioreactor vessel rotates about its central longitudinal axis, gravity may cause the fluid, culture media, and / or unadhered cells to remain toward a lower portion of the chamber. However, in one or more embodiments, the cell culture matrix is essentially fixed with respect to the vessel, and thus rotates with thevessel. In one or more other embodiments, the cell culture matrix can be unattached and free to move to a desired degree relative to the vessel as the vessel rotates. The cells may adhere to the cell culture matrix, while the movement of the vessel allows the cells to receive exposure to both the cell culture media or liquid, and to oxygen or other gases within the culture chamber.

[0073] By using a cell culture matrix according to embodiments of this disclosure, such as a matrix including a woven or mesh substrate, the roller bottle vessel is provided with an increased surface area available for adherent cells to attach, proliferate, and function. In particular, using a substrate of a woven mesh of monofilament polymer material within the roller bottle, the surface area may increase by of about 2.4 to about 4.8 times, or to about 10 times that of a standard roller bottle. As discussed herein, each monofilament strand of the mesh substrate is capable of presenting itself as 2D surface for adherent cells to attach. In addition, multiple layers of mesh can we arranged in roller bottle, resulting in increases of total available surface area ranging from about 2 to 20 times that of a standard roller bottle. Thus, existing roller bottle facilities and processing, including cell seeding, media exchange, and cell harvesting, can be modified by the addition of the improved cell culture matrix disclosed herein, with minimal impact on existing operation infrastructure and processing steps.

[0074] The bioreactor vessel optionally includes one or more outlets capable of being attached to inlet and / or outlet means. Through the one or more outlets, liquid, media, or cells can be supplied to or removed from the chamber. A single port in the vessel may act as both the inlet and outlet, or multiple ports may be provided for dedicated inlets and outlets.

[0075] Embodiments are not limited to the vessel rotation about a central longitudinal axis. For example, the vessel may rotate about an axis that is not centrally located with respect to the vessel. In addition, the axis of rotation may be a horizonal or vertical axis.Definitions

[0076] “Wholly synthetic” or “fully synthetic” refers to a cell culture article, such as a microcarrier or surface of a culture vessel, that is composed entirely of synthetic source materials and is devoid of any animal derived or animal sourced materials. The disclosed wholly synthetic cell culture article eliminates the risk of xenogeneic contamination.

[0077] ‘ ‘Include,” “includes,” or like terms means encompassing but not limited to, that is, inclusive and not exclusive.

[0078] ‘ ‘Users” refers to those who use the systems, methods, articles, or kits disclosed herein, and include those who are culturing cells for harvesting of cells or cell products, or those who are using cells or cell products cultured and / or harvested according to embodiments herein.

[0079] “About” modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, viscosities, and like values, and ranges thereof, or a dimension of a component, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example: through typical measuring and handling procedures used for preparing materials, compositions, composites, concentrates, component parts, articles of manufacture, or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term “about” also encompasses amounts that differ due to aging of a composition or formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture.

[0080] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0081] The indefinite article “a” or “an” and its corresponding definite article “the” as used herein means at least one, or one or more, unless specified otherwise.

[0082] Abbreviations, which are well known to one of ordinary skill in the art, may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” for gram(s), “mL” for milliliters, and “rt” for room temperature, “nm” for nanometers, and like abbreviations).

[0083] Specific and preferred values disclosed for components, ingredients, additives, dimensions, conditions, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The systems, kits, andmethods of the disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values described herein, including explicit or implicit intermediate values and ranges.

[0084] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that any particular order be inferred.

[0085] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the disclosed embodiments. Since modifications, combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the embodiments may occur to persons skilled in the art, the disclosed embodiments should be construed to include everything within the scope of the appended claims and their equivalents.

[0086] As discussed above, aspects of one or more embodiments include the media inlet being configured to supply at least one of cells, cell culture media, and other components to the interior cavity of the bioreactor vessel, and the media outlet is configured to withdraw at least one of cells or other biological agents, cell culture media, and cellular or biological byproducts from the interior cavity during or after cell culture. In addition, as discussed herein, to perform a harvest procedure of the contents of the bioreactor, the media outlet can be configured to supply pressurized fluid to the spacer section during the harvesting operation, and the media inlet is configured to withdraw at least one of cells, cell culture media, and cell by-products from the interior cavity during the harvesting operation. The bioreactor system is configured to fill the interior cavity with the pressurized fluid via the media outlet to force out at least one of cells, cell culture media, and cell by-products through the media inlet. Accordingly, the spacer section may also be referred to herein as the harvesting volume, meaning it is a space or volume that is pressurized or filled with fluid via some pressurization, in order to perform at least part of the harvest procedure.

[0087] According to some embodiments, the harvest solution can contain one or more components that help to harvest cells and / or release them from the cell culture substrate.Examples include Accutase® or TrypLE®, but a person of ordinary skill in the art would understand alternative harvesting agents that can be used.

[0088] Further advantages of the embodiments herein are also clear. For example, in some embodiments, the cell culture substrate extends uninterrupted across diameter of the interior cavity of the bioreactor. This is simpler than complex flow channels and pathways employed in alternatives. The flow through the fixed bed is unidirectional, or can be characterized as plug flow, which again simplifies operation and improves performance, including for example, cell seeding and harvesting. As a result, all of the layers of packed bed are being perfused with same efficiency and flow or flux of media across or through the layer.

Claims

What is claimed:

1. A bioreactor system for culturing cells, the bioreactor system comprising: a vessel comprising an inlet, an outlet, and an interior cavity disposed between the inlet and the outlet and configured for perfusing cell culture media therethrough; and a cell culture substrate disposed in the interior cavity in a fixed-bed configuration, the cell culture substrate comprising a surface having a surface area upon configured for culturing cells thereon during operation of the bioreactor system, wherein the bioreactor system is configured scale up or down with a desired number of cells to be cultured by adjusting the surface area of the cell culture substrate.

2. The bioreactor system of claim 1, wherein the vessel comprises a height in the direction from the inlet to the outlet, and wherein the bioreactor system being configured to scale comprises adjusting the height of the vessel.

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

4. The bioreactor system of any of the preceding claims, wherein the bioreactor system is configured to scale without adjusting a flow rate of cell culture media through the interior cavity.

5. The bioreactor system of any of the preceding claims, wherein the bioreactor system is configured to scale to different numbers of cells to be cultured at a same flow rate for the different numbers of cells to be cultured.

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

7. The bioreactor system of claim 6, wherein the bio reactor system is configured to scale without changing the inlet flow distribution mechanism.

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

9. The bioreactor system of any of claims 6-8, wherein the vessel comprises an inlet plenum comprising the inlet flow distribution mechanism.

10. The bioreactor system of any of claims 6-9, wherein the inlet flow distribution mechanism is configured to generate, across the fixed-bed configuration of the cell culture substrate, at least one of (i) a uniform velocity profile, (ii) a uniform mass flow, and (iii) a uniform cell distribution.

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

12. The bioreactor system of claim 11, wherein the plurality of holes comprises a plurality of rings of holes concentrically arranged about a center of the inlet flow distribution mechanism.

13. The bioreactor system of claim 12, wherein a width of each of the plurality of rings of holes is about equal to a spacing between holes within that ring of the plurality of rings of holes.

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

15. The bioreactor system of any of claims 6-10, wherein the inlet flow distribution comprises a distributor plate, the distributor plate comprises a first face, a second face, and a plurality of holes extended through a thickness of the distributor plate from the first face to the second face, wherein the first face comprises a plurality of annular regions covering a surface of the first face, each of the plurality of annular regions comprises one ring of holes 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 (SAar) defined by the following equation:SAar = 71 ( R2- r2) wherein R is an outer radius of the annular region and r is an inner radius of the annular region, wherein each of the plurality of annular regions comprises a total hole area comprising a sum of the areas of all of the plurality of holes within the annular region, and wherein a ratio of SAarto the total hole area one of the plurality of annular regions is equal to the ratio of any of the others of the plurality of annular regions.

17. The bioreactor system of any of the preceding claims, further comprising a bubble trap disposed in the vessel.

18. The bioreactor system of claim 17, further comprising at least one of an inlet plenum and an outlet plenum, at least one of the inlet plenum and the outlet plenum comprising a perforated distributor plate, the bubble trap being disposed on the perforated distributor plate of at least one of the inlet plenum and the outlet plenum.

19. The bioreactor system of any of the preceding claims, wherein the perforated distributor plate comprises a tapered surface on an upstream-side of the perforated distributor plate, the tapered surface rising toward a perimeter of the perforated distributor plate.

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

21. The bioreactor system of claim 19 or claim 20, wherein the tapered surface comprises a low point at or near a center of the perforated distributor plate.

22. The bioreactor system of any of claims 18-21, wherein the perforated distributor plate comprises a groove at or near the perimeter of the perforated distributor plate, the groove being configured to at least temporarily confine bubbles.

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

24. The bioreactor system of any of the preceding claims, wherein the surface area is from about 1 m2to about 3000 m2.

25. The bioreactor system of any of the preceding claims, wherein the surface area is from about 10 m2to about 200 m2, about 20 m2to about 100 m2, about 100 m2to about 500 m2, or about 500 m2to about 1000 m2.

26. The bioreactor system of any of the preceding claims, wherein the surface area is scalable from about 10 m2to about 200 m2, from about 100 m2to about 500 m2, from about 500 m2to about 1000 m2, or from about 10 m2to about 1000 m2.

27. The bioreactor system of claim 25, wherein the surface area is scalable from about 20 m2, to about 50 m2, to about 100 m2, to about 500 m2, and to about 1000 m2.

28. The bioreactor system of any of the preceding claims, wherein the surface area is scalable 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 of any of the preceding claims, wherein the cell culture substrate comprises a plurality of porous disks in a stacked arrangement, wherein each of the plurality of porous disks comprises a surface configured to culture cells thereon.

30. A flow distribution plate comprising: a first side, a second side opposite the first side, a thickness separating the first side from the second side, and a plurality of holes extending through the thickness from the first side to the second side, and a bubble trap disposed on the first side, the bubble trap comprising a tapered surface on the first side, the tapered surface rising toward a perimeter of the flow distribution plate.

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

32. The flow distribution plate of claim 30 or claim 31, wherein the tapered surface comprises a low point near a center of the flow distribution plate.

33. The flow distribution plate of claim 30, wherein the flow distribution plate comprises a groove at or near the perimeter of the flow distribution plate, the groove being configured to at least temporarily confine bubbles.

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

35. The flow distribution plate of any of claims 30-34, wherein the plurality of holes comprises a plurality of rings of holes concentrically arranged about a center of the flow distribution plate.

36. The flow distribution plate of claim 35, wherein a width of each of the plurality of rings of holes is about equal to a spacing between holes within that ring of the plurality of rings of holes.

37. The flow distribution plate of any of claims 30-36, wherein the first side comprises a plurality of annular regions covering a surface of the first side, each of the plurality of annular regions comprises one ring of holes of the plurality of holes.

38. The flow distribution plate of claim 37, wherein each of the plurality of annular regions comprises an annular region surface area (SAar) defined by the following equation:SAar = 71 ( R2- r2) wherein R is an outer radius of the annular region and r is an inner radius of the annular region, wherein each of the plurality of annular regions comprises a total hole area comprising a sum of the areas of all of the plurality of holes within the annular region, and wherein a ratio of SAarto the total hole area one of the plurality of annular regions is equal to the ratio of any of the others of the plurality of annular regions.

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