Fixed bed bioreactor and methods of using the same

Abstract

A fixed bed bioreactor system is provided, the system including a vessel having a media inlet, a media outlet, and an interior chamber disposed between the media inlet and the media outlet and in fluid communication with the media inlet and the media outlet. The vessel further includes a cell culture media positioned in the interior chamber between the media inlet and the media outlet in a packed bed configuration, the cell culture media including a plurality of porous disks in a stacked arrangement. The interior chamber includes a cell culture zone and a spacer zone, the cell culture media defining the cell culture zone, and the spacer zone disposed between the cell culture zone and the media outlet, and each of the plurality of porous disks having a surface configured to culture cells thereon.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62,930,935, filed November 5, 2019, pursuant to 35 U.S.SC §120, 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. Specifically, this disclosure relates to cell culture substrates, fixed-bed bioreactor containers and systems comprising said substrates, and methods for culturing cells using said substrates and bioreactors. Background Technology

[0004] In the bioprocessing industry, large-scale cell culture is conducted for the production of hormones, enzymes, antibodies, vaccines, and cell therapies. Most cells used in bioprocessing are anchor-dependent, meaning they require a surface to adhere to in order to grow and function. Traditionally, adherent cell culture takes place on two-dimensional (2D) surfaces that adhere to the cells, contained in one of a variety of container forms, such as T-flasks, Piper dishes, cell factories, cell stacking containers, roller flasks, and... Containers. These methods may have significant drawbacks, including difficulty in achieving sufficiently high cell densities to make them suitable for large-scale cell or therapy production.

[0005] Alternative methods have been proposed to increase the volumetric density of cultured cells. These methods include microcarrier culture in stirred tanks. In this approach, cells adhering to the surface of the microcarriers are subjected to constant shear stress, resulting in a significant impact on proliferation and culture performance. Another example of a high-density cell culture system is the hollow fiber bioreactor, in which cells can form large three-dimensional aggregates as they proliferate within the interfiber spaces. However, cell growth and performance are significantly inhibited due to a lack of nutrients. To mitigate this problem, these bioreactors are made smaller, making them unsuitable for large-scale manufacturing.

[0006] Another example of a high-density culture system for anchorage-dependent cells is a packed bed bioreactor system. For example, packed bed bioreactor systems comprising a packed bed of a carrier or matrix system for capturing cells have been previously disclosed in U.S. Patents Nos. 4,833,083; 5,501,971; and 5,510,262. The packed bed matrix is often made of porous particles used as a substrate or non-woven microfibers of a polymer. Such bioreactors function as a recirculating flow-through bioreactor. One of the significant problems with such bioreactors is the non-uniform distribution of cells in the packed bed. For example, the packed bed functions as a depth filter and cells are primarily captured at the inlet region, resulting in a cell distribution gradient during the inoculation step. In addition, due to random fiber packing, the flow resistance and cell capture efficiency is non-uniform across the packed bed cross-section. For example, media flows rapidly through regions of low cell packing density, while it flows slowly through regions of high resistance due to a higher number of cells being captured. This creates a channeling effect where nutrients and oxygen are more efficiently delivered to regions of lower cell volume density, while regions of higher cell density remain under sub-optimal culture conditions. Another significant drawback of the packed bed systems disclosed in the prior art is the inability to efficiently harvest 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 recovering cells from a packed bed during the cell harvest step. The bioreactor design is based on loosening the packed bed matrix and agitating or stirring the packed bed particles to cause the porous matrix to collide and thus dislodge the cells. However, this method is laborious and can cause significant cell damage, thus reducing overall cell viability.

[0007] Roll bottles have several advantages, such as ease of handling and the ability to monitor cells on an adherent surface. However, the main disadvantage from a production perspective is the low surface area to volume ratio, while the roll bottle configuration occupies a large area of manufacturing floor space. Various methods have been used to increase the available surface area for adherent cells in roll bottle formats. Some solutions have been implemented in commercially available products, but there is still room for improvement to further increase roll bottle productivity. Traditionally, roll bottles are produced as a single structure by a blow molding process. This manufacturing simplicity makes roll bottles economically viable in the bioprocessing industry. Some roll bottle improvements to increase the available surface area for cell culture can be achieved without changing the manufacturing process, but the improved roll bottle surface area only achieves a marginal increase. Other changes to the roll bottle design significantly increase the complexity of the manufacturing process, making it economically unviable in the bioprocessing industry. There is therefore a need to provide a roll bottle with increased surface area and bioprocessing productivity, while its manufacture uses the same blow molding process.

[0008] While it is possible to produce viral vectors for early stage clinical trials using existing platforms, there is a need for platforms that are capable of producing more high quality product to reach later stage commercial production scales.

[0009] There is a need for cell culture substrates, packed bed bioreactor vessels and bioreactor systems and methods that are capable of culturing cells in high density form, and with uniform cell distribution, as well as increased ease of access and harvest yield, and that harvest viable cells. Further, there is a need for these substrates, vessels, systems and methods to enable a scalable solution that is capable of achieving adjustable production levels to accommodate the needs of various use cases at different scales, for example, from research to process development to manufacturing scales. SUMMARY

[0010] According to one embodiment of the disclosure, a fixed bed bioreactor system is provided. The bioreactor system includes a vessel having a media inlet, a media outlet, and an interior chamber disposed between the media inlet and the media outlet and in fluid communication with the media inlet and the media outlet. The bioreactor system further includes a cell culture substrate positioned in the interior chamber between the media inlet and the media outlet in a packed bed configuration, wherein the cell culture substrate includes a plurality of porous disks in a stacked arrangement. Each of the porous disks has a surface for culturing cells on the surface. The interior chamber includes a cell culture zone and a spacer zone, wherein the cell culture substrate defines the cell culture zone and the spacer zone is disposed between the cell culture zone and the media outlet.

[0011] Aspects of one or more embodiments further include a spacer disposed in the interior chamber between the cell culture substrate and the media outlet and defining a spacer zone between the cell culture substrate and the media outlet. The spacer spaces the cell culture substrate a distance from the media outlet and confines the cell culture substrate to the cell culture zone of the interior chamber. The spacer can include a plurality of spacer members extending in a direction parallel to a length of the spacer zone. As a further aspect of some embodiments, a packed bed retainer can be disposed between the cell culture substrate and the spacer, and the packed bed retainer provides structural support to a top of the cell culture substrate. The packed bed retainer can be porous, substantially rigid, and can extend over a majority of a width of the interior chamber.

[0012] In further aspects of one or more embodiments, each of the plurality of perforated disks has a first side, a second side opposite the first side, a disk thickness separating the first side and the second side, and a plurality of openings formed in the disk and passing through the disk thickness. The plurality of openings are arranged to allow at least one of a cell culture medium, cells, or cell byproducts to flow through the cell culture substrate.

[0013] The system can also include an inlet distribution plate disposed between the culture medium inlet and the cell culture region. The inlet distribution plate can distribute fluid entering the interior chamber from the culture medium inlet over the area of the cell culture substrate. The system can also include an outlet distribution plate disposed between the spacer region and the culture medium outlet.

[0014] As an aspect of one or more embodiments, the above-described bioreactor system further includes a packed bed retainer disposed between the cell culture substrate and the spacer region, the packed bed retainer configured to provide structural support to the top of the cell culture substrate. The packed bed retainer can be porous, substantially rigid, and can extend over a majority of the width of the interior chamber. The system can also include a spacer disposed in the interior chamber between the packed bed retainer and the culture medium outlet and defining a spacer region between the packed bed retainer and the culture medium outlet. The spacer spaces the cell culture substrate a distance from the culture medium outlet and confines the cell culture substrate to the cell culture region of the interior chamber. The packed bed retainer can be, for example, a rigid lattice structure.

[0015] As a further aspect of one or more embodiments, the spacer has an adjustable length and is adjustable to maintain various predetermined distances between the cell culture region and the culture medium outlet, whereby the number of perforated disks in the cell culture substrate that can be accommodated in the cell culture region can vary.

[0016] In further aspects of one or more embodiments, the system includes a plurality of detachable spacers of different lengths, each of the plurality of detachable spacers being positionable in the spacer region to maintain a predetermined distance between the cell culture region and the culture medium outlet that is different from the distance maintained by each of the other spacers, whereby the number of perforated disks in the cell culture substrate that can be accommodated in the cell culture region can be varied based on the length of the spacer disposed in the spacer region.

[0017] In one aspect of some embodiments, the system further comprises at least one porous spacer disk disposed between the cell culture substrate and the media inlet. The at least one porous spacer disk can comprise a spacer disk pore diameter, and each of the plurality of porous disks can have a disk pore diameter, such that the spacer disk pore diameter is greater than the disk pore diameter. The at least one porous spacer disk can comprise a first disk having a first spacer disk pore diameter and a second disk having a second spacer disk pore diameter, wherein the first spacer disk pore diameter is different than the second spacer disk pore diameter.

[0018] As an aspect of some embodiments, the plurality of porous disks comprises a plurality of woven mesh layers. Each of the plurality of woven mesh layers has a defined, substantially uniform array of pores. Each layer of the plurality of woven mesh layers comprises a plurality of interwoven fibers, the plurality of interwoven fibers comprising a first set of fibers running parallel to each other in a first direction, and a second set of fibers running parallel to each other in a second direction. The first direction can be substantially perpendicular to the second direction. In some embodiments, the plurality of interwoven fibers of the mesh consists of the first set of fibers and the second set of fibers.

[0019] As an aspect of one or more embodiments, the media inlet is configured to supply at least one of cells and cell media to the interior chamber prior to or during cell culturing, and the media outlet is configured to withdraw at least one of cells, cell media, and cell byproducts from the interior chamber during or after cell culturing. The media outlet is configured to supply pressurized fluid to the spacer region during a harvesting operation, and the media inlet is configured to withdraw at least one of cells, cell media, and cell byproducts from the interior chamber during the harvesting operation. The bioreactor system is configured to fill the interior chamber with pressurized fluid via the media outlet to expel at least one of cells, cell media, and cell byproducts through the media inlet.

[0020] Aspects of one or more embodiments include the plurality of porous disks comprising from 50 to 1000 porous disks, or from 100 to 500 porous disks. The plurality of porous disks can culture cells to an average density of from about 1.00 x 10 7 to about 2.00 x 10 7 cells per disk. Each disk of the plurality of porous disks can have fibers defining pores between the fibers, wherein the fibers have a diameter of from about 50 pm to about 1000 pm, and the pores have a diameter of from about 100 pm to about 1000 pm. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1AAccording to one or more embodiments of the present disclosure, a perspective view of a three-dimensional model of a cell culture substrate is shown.

[0022] Figure 1B is a two-dimensional plan view of the substrate of Figure 1A

[0023] Figure 1C is a cross-sectional view of the substrate in Figure 1B

[0024] Figure 2A is a photograph of an exemplary cell culture substrate having a first geometry according to some embodiments.

[0025] Figure 2B is a photograph of an exemplary cell culture substrate having a second geometry according to some embodiments.

[0026] Figure 2C is a photograph of an exemplary cell culture substrate having a third geometry according to some embodiments.

[0027] Figure 3A is a perspective view of a multi-layer cell culture substrate according to one or more embodiments.

[0028] Figure 3B is a plan view of the multi-layer cell culture substrate of Figure 3A

[0029] Figure 4 is a cross-sectional view of the multi-layer cell culture substrate of Figure 3B along line B-B according to one or more embodiments.

[0030] Figure 5 is a cross-sectional view of the multi-layer cell culture substrate of Figure 4 along line C-C according to one or more embodiments.

[0031] Figure 6 is a schematic view of a packed-bed cell culture system having a multi-layer cell culture substrate according to one or more embodiments.

[0032] Figure 7A is a cross-sectional view of a packed-bed cell culture system according to one or more embodiments.

[0033] Figure 7B is a cross-sectional view of a packed-bed cell culture system according to one or more other embodiments.

[0034] Figure 8 is a photograph of an exemplary component of a bioreactor vessel according to one or more embodiments.

[0035] Figure 9A ​​​is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments, Figures 7A to 8

[0036] Figure 9B is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments, Figures 7A to 8

[0037] Figure 10 is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments, Figures 7A to 8

[0038] Figure 11A is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments,

[0039] Figure 11B is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments,

[0040] Figure 11C is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments,

[0041] Figure 12 is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments,

[0042] Figure 13 is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments,

[0043] Figure 14A is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments,

[0044] Figure 14B is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments,

[0045] Figure 15A is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments,

[0046] Figure 15B is a plan view of a flow distribution plate of a cell culture system according to one or more embodiments, ​​​

[0047] Figure 15C is a micrograph of HEK293T cells stained according to one or more embodiments, wherein the cells were seeded at a third cell seeding density.

[0048] Figure 16A is a photograph of a disc of cell culture substrate with stained cells after cell culture in a packed bed bioreactor, but after a cell harvesting operation was performed on the cell culture substrate, according to one or more embodiments.

[0049] Figure 16B is a photograph of a disc of cell culture substrate with stained cells after cell culture in a packed bed bioreactor, and after a cell harvesting operation was performed on the cell culture substrate, according to one or more embodiments.

[0050] Figure 17A is a bar graph showing experimental results of total cells harvested according to two embodiments of the present disclosure compared to cells cultured using

[0051] Figure 17B is a bar graph showing experimental results of total genome copies per vessel according to two embodiments of the present disclosure compared to cells cultured using

[0052] Figure 17C is a bar graph showing experimental results of genome copies per unit area according to two embodiments of the present disclosure compared to cells cultured using

[0053] Figure 18 is a detailed schematic of a cell culture system according to one or more embodiments. DETAILED DESCRIPTION

[0054] Various embodiments of the present disclosure are described in detail below with reference to the attached drawing figures, if any. The embodiments described are not limiting and do not exhaust the scope of the present disclosure. The scope of the present disclosure is limited only by the claims. Furthermore, any embodiments listed in this specification are not exhaustive and are merely some of the many possible embodiments of the claimed invention.

[0055] ​​​Embodiments of the present disclosure relate to cell culture substrates, and cell culture or bioreactor systems comprising the substrates, and to methods of using the substrates to culture cells. Embodiments also include bioreactor vessels that are capable of cell seeding, culturing, transfection, and / or harvesting with cell culture substrates in the vessel, and are capable of being operated at different production scales.

[0056] In conventional large-scale cell culture bioreactors, different types of packed bed bioreactors have been used. Typically, these packed beds comprise porous matrices to retain adherent or suspended cells, and to support growth and proliferation. The packed bed matrix provides a high surface area to volume ratio, and thus cell densities can be higher than in other systems. The packed bed often acts as a deep filter, where cells are physically trapped or entangled in the fibers of the matrix. However, since the cell inoculum flows linearly through the packed bed, cells experience a non-uniform distribution in the packed bed. Thus, for example, there is a higher cell density in the inlet region of the bioreactor, and a significantly lower cell density in the outlet section of the bioreactor. This non-uniform distribution of cells within the packed bed greatly hinders the scalability of such bioreactors in bioprocess manufacturing.

[0057] Another problem encountered in prior art disclosed packed bed bioreactors is the channeling effect. Due to the random nature of the packed non-woven fibers, the local fiber density at any given cross-section of the packed bed is non-uniform. The media flows rapidly in areas of low fiber density (high bed permeability), and significantly slower in areas of high fiber density (lower bed permeability). The non-uniform media perfusion through the packed bed creates a channeling effect. This itself manifests in the formation of pronounced gradients of nutrients and metabolic products, which adversely affect the overall cell culture and bioreactor performance. Cells located in areas of low media perfusion starve and often die due to lack of nutrients or metabolic poisoning. Cell harvesting is another problem encountered when using bioreactors packed with non-woven fiber scaffolds. Since the packed bed acts as a deep filter, the cells released at the end of the cell culture process are retained within the packed bed, and cell recovery is extremely low. This greatly limits the use of such bioreactors in bioprocesses where viable cells are the product.

[0058] The present disclosure includes embodiments of cell growth substrates and / or packed bed systems for anchorage-dependent cells that can be easily and effectively scaled up to any practical production scale for cells or cell-derived products (e.g., proteins, antibodies, viral particles). In one embodiment, the substrate has a structurally defined surface area for adherent cell attachment and proliferation that, when assembled in a packed bed or other bioreactor, has good mechanical strength and forms a highly uniform, multiply interconnected fluid network. In particular embodiments, mechanically stable, non-degradable woven mesh can be used to support adherent cell production. Such substrates can enable uniform cell seeding, as well as efficient harvesting of cells or other products from the bioreactor. In addition, embodiments of the present disclosure support such cell culture to achieve confluent monolayer or multilayer adherent cells on the disclosed substrates and can avoid the formation of 3D cell aggregates with limited nutrient diffusion and increased metabolite concentrations. The structurally defined substrates of one or more embodiments enable complete cell recovery from the packed bed of the bioreactor and consistent cell harvesting. In another embodiment of the present disclosure, methods of cell culture using a bioreactor with the substrate are provided for the bioprocess production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0059] "Structurally defined" as used herein means a component having a non-random, ordered structure according to a defined structural design.

[0060] In one or more embodiments, a cell culture substrate is provided that supports anchorage-dependent cells to attach and proliferate in high volumetric density. The substrate can be assembled and used in a bioreactor system, e.g., a perfusion packed bed bioreactor, and provides uniform cell distribution during the seeding step while preventing the formation of large and / or uncontrolled cell aggregates within the substrate or packed bed. Thus, the substrate eliminates diffusion limitations during bioreactor operation. In addition, the substrate enables easy and efficient cell harvesting from the bioreactor.

[0061] The substrate can be formed from a base material having a thin or sheet-like structure and having a first side and a second side separated by a relatively small thickness. In other words, the thickness of the sheet-like base material is small relative to the width and / or length of the first and second sides of the base material. Further, a plurality of holes or openings through the thickness of the base material are formed. The size and geometry of the base material between the openings allows cells to adhere to the surface of the base material as if it were a two-dimensional (2D) surface while also allowing sufficient fluid flow around and through the openings of the base material. In some embodiments, the base material is a polymeric base material and can be formed as a molded polymeric sheet; a polymeric sheet having openings through the thickness; a plurality of filaments fused into a mesh layer; or a plurality of filaments woven into a mesh layer. The physical structure of the substrate has a high surface-to-volume ratio for culturing anchorage-dependent cells. According to various embodiments, the substrate can be arranged or populated in a bioreactor in certain ways to achieve uniform cell seeding, uniform media perfusion, and efficient cell harvesting.

[0062] Embodiments of the present disclosure can enable a viral vector platform with a practical size that can produce viral genomes at a scale of about 10 15 to about 10 18 viral genomes per batch. For example, in some embodiments, viral genome yield can be about 10 15 to about 10 16 viral genomes per batch, or about 10 16 to about 10 19 viral genomes per batch, or about 10 16 to about 10 18 viral genomes per batch, or about 10 17 to about 10 19 viral genomes per batch, or about 10 18 to about 10 19 viral genomes per batch, or about 10 18 or more viral genomes per batch. A “batch” can mean a single cell culture run of a single bioreactor vessel. Due to the scalability of the embodiments herein, the bioreactor vessel and contained cell culture substrate can be appropriately scaled to achieve these yields. This scalability is facilitated by the structurally defined nature of the cell culture substrate, which provides uniform cell seeding and culture, uniform media flow, and / or uniform harvesting. In some embodiments, a batch can include multiple bioreactor vessels used in concert for cell culture operations.

[0063] In addition, the embodiments disclosed herein are capable of not only cell attachment to the cell culture substrate and growth, but also harvesting of the cultured viable cells. The inability to harvest viable cells is a significant drawback of existing platforms and results in difficulty in building and maintaining a sufficient number of cells to form a production capacity. According to one aspect of the embodiments of the present disclosure, viable cells, including 80% to 100% viable cells, or about 85% to about 99% viable cells, or about 90% to about 99% viable cells, can be harvested from the cell culture substrate. For example, at least 80% of the harvested cells 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 can be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0064] Figure 1A and 1B According to an example of one or more embodiments of the present disclosure, a three-dimensional (3D) perspective view and a two-dimensional (2D) plan view of a cell culture substrate 100 are shown. The cell culture substrate 100 is a woven mesh layer made of a first plurality of fibers 102 running in a first direction and a second plurality of fibers 104 running in a second direction. The woven fibers of the substrate 100 form a plurality of openings 106. The size and shape of the openings can vary based on the type of weave (e.g., number, shape, and size of filaments; angle between intersecting filaments, etc.). The openings can be defined by a certain width or diameter, as shown by a first diameter D1 and a second diameter D2 in Figure 1B The woven mesh can be considered a two-dimensional sheet or layer on a macro scale. However, upon closer inspection of the woven mesh, it is found to have a three-dimensional structure due to the rise and fall of the intersecting fibers of the mesh. Thus, as shown in Figure 1C The thickness T of the woven mesh 100 can be thicker than the gauge of a single fiber, as shown in

[0065] In Figure 1B , the openings 106 have a diameter D1 defined as the distance between opposing fibers 102 and a diameter D2 defined as the distance between opposing fibers 104. Depending on the weave geometry, D1 and D2 can be equal or unequal. In the case where D1 and D2 are unequal, the larger one can be referred to as the major diameter and the smaller one can be referred to as the minor diameter. In some embodiments, the diameter of an opening can refer to the widest portion of the opening. Unless otherwise specified, the diameter of an opening as used herein shall refer to the distance between parallel fibers on opposite sides of the opening.

[0066] A given fiber of the plurality of fibers 102 has a thickness t1, and a given fiber of the plurality of fibers 104 has a thickness t2. In the case of circular cross-section fibers, as Figure 1A or other three-dimensional cross-sectional views, the thicknesses t1and t2are the largest diameter or thickness of the fiber cross-section. According to some embodiments, the plurality of fibers 102 all have the same thickness t1, and the plurality of fibers 104 all have the same thickness t2. Further, t1and t2may be equal. However, in one or more embodiments, t1and t2may not be equal. Further, each of the plurality of fibers 102 and the plurality of fibers 104 can include fibers of two or more different thicknesses (e.g., t 1a , t 1b , etc., and t 2a , t 2b , etc.). Due to the three-dimensional nature of the woven mesh, as Figures 1A-1C shown, the effective surface area of the fibers available for cell attachment and proliferation exceeds the surface area available for attachment on a comparable planar 2D surface having the same substrate dimensions at the macro-scale.

[0067] The woven mesh can include monofilament or multifilament polymeric fibers. In one or more embodiments, the monofilament fibers can have a diameter in the range of about 50 μιη to about 1000 μιη. At the micro-scale level, the surface of the monofilament fibers exists as a conventional 2D surface for adherent cell attachment and proliferation due to the scale of the fibers compared to the cells (e.g., fiber diameter is greater than cell). Such fibers are woven into a mesh having a defined pattern and amount of structural rigidity. The fibers can be woven into a mesh and the openings are in the range of about 100 μιη x 100 μιη to about 1000 μιη x 1000 μιη. These ranges of filament diameter and opening diameter are examples of some embodiments, but are not intended to limit the possible feature sizes of the mesh of all embodiments.

[0068] The substrate mesh can be fabricated from monofilament or multifilament fibers of polymeric materials that are compatible in 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 weave forms, including, for example, knit, warp knit, or woven (e.g., plain weave, twill weave, Dutch weave, five-needle weave).

[0069] Surface chemistry of the mesh filaments can need to be modified to provide desired cell adhesion properties. These modifications can be achieved through chemical treatment of the polymer material of the mesh, or by grafting cell adhesion molecules to the filament surface. Alternatively, biocompatible hydrogels that exhibit cell adhesion properties (e.g., including collagen or Alternatively, the filament fiber surface of the mesh can be made to have cell adhesion properties through a treatment process using various types of plasma, process gases, and / or chemicals known in the industry.

[0070] Figures 2A-2C Different examples of woven meshes are shown in accordance with some contemplated embodiments of the present disclosure. The fiber diameters and opening sizes of these meshes, as well as the approximate magnitudes of the increases in cell culture surface area provided by a single layer of the respective meshes relative to an equivalent 2D surface, are summarized in Table 1 below. In Table 1, Mesh A refers to a mesh having a fiber diameter of 0.2 pm and an opening size of 0.2 pm, Mesh B refers to a mesh having a fiber diameter of 0.4 pm and an opening size of 0.4 pm, and Mesh C refers to a mesh having a fiber diameter of 0.6 pm and an opening size of 0.6 pm. The three mesh geometries of Table 1 are merely examples, and embodiments of the present disclosure are not limited to these particular examples. Since Mesh C provides the highest surface area, it can be advantageous to achieve high density of cell adhesion and proliferation, and thus provide the most efficient substrate for cell culture. However, in some embodiments, it can be advantageous for the cell culture substrate to include a mesh with a lower surface area, such as Mesh A or Mesh B, or a combination of meshes with different surface areas, to achieve desired cell distribution or flow characteristics within the culture chamber, for example. Figure 2A Figure 2B Figure 2C The three mesh geometries of Table 1 are merely examples, and embodiments of the present disclosure are not limited to these particular examples. Since Mesh C provides the highest surface area, it can be advantageous to achieve high density of cell adhesion and proliferation, and thus provide the most efficient substrate for cell culture. However, in some embodiments, it can be advantageous for the cell culture substrate to include a mesh with a lower surface area, such as Mesh A or Mesh B, or a combination of meshes with different surface areas, to achieve desired cell distribution or flow characteristics within the culture chamber, for example.

[0071]

[0072] Table 1 Figures 2A-2C

[0073] As shown in the table above, the three-dimensional nature of the mesh provides an increased surface area for cell attachment and proliferation compared to a planar 2D surface of equivalent size. This increased surface area contributes to the scalability of embodiments of the present disclosure. For process development and process validation studies, small-scale bioreactors are often desired in order to conserve reagent costs and increase experimental throughput. Embodiments of the present disclosure can be applied to such small-scale studies, but can also be scaled up to industrial scale. For example, if a 100-layer Mesh C with a 2.2 cm diameter circular shape is packed into a cylindrical packed bed with an inner diameter of 2.2 cm, the total surface area available for cell attachment and proliferation is equal to approximately 935 cm2. In order to scale this bioreactor by a factor of ten, a similar setup using a cylindrical packed bed with an inner diameter of 7 cm and 100 layers of the same mesh can be used. In this case, the total surface area would be equal to 9,350 cm2. In some embodiments, the total surface area available is equal to or greater than approximately 99,000 cm2. 2 2 2 ​​​​​​ / L. In some embodiments, the bioreactor vessel may have a cell culture medium having more than 100 layers of cell culture medium, for example, comprising 100-1000 layers or more, or about 300 layers of cell culture medium. In some examples, the 300 layers of cell culture medium may have about 3 × 10⁻⁶ layers. 9 Up to 5×10 9 cm 2 The surface area. In some embodiments, the packed bed bioreactor has a surface area of ​​1m². 2 Up to 10m 2 The equivalent surface area, for example, including 1m² 2 2m 2 2.5m 2 3m 2 4m 2 5m 2 and 10m 2 And the range therein. Due to the plugged perfusion flow in packed beds, the same flow rate can be used in both smaller and larger bioreactor formats, in ml / min / cm². 2 (cm 2 (This is expressed as the cross-sectional surface area of ​​the packed bed).

[0074] High uniformity of flow resistance is achieved in the matrix or packed bed by using a culture substrate with a sufficiently rigid structure. Depending on the implementation, the substrate can be deployed in single or multiple layers. This flexibility eliminates diffusion limitations and provides uniform nutrient and oxygen delivery to cells attached to the substrate. Furthermore, the substrate has no cell retention areas in the packed bed construction, allowing for high-viability cell harvesting at the end of culture. The substrate also provides uniform filling of the packed bed and enables direct scalability from process development units to large-scale industrial bioprocessing units. The ability to harvest cells directly from the packed bed eliminates the need to resuspend the substrate in stirred or mechanically agitated containers. Furthermore, the high packing density of the cell culture substrate results in high bioprocess productivity in industrially manageable volumes.

[0075] Figure 3A An embodiment of a substrate having a multilayer substrate 200 is shown, and Figure 3B This is a plan view of the same multilayer substrate 200. The multilayer substrate 200 includes a first mesh substrate layer 202 and a second mesh substrate layer 204. Although the first substrate layer 202 and the second substrate layer 204 overlap, the geometry of the mesh (e.g., the ratio of the opening diameter to the fiber diameter) causes the openings of the first substrate layer 202 and the second substrate layer 204 to overlap and provide a path for fluid flow through the entire thickness of the multilayer substrate 200, such as... Figure 3B The filamentless opening 206 is shown in the diagram. AlthoughFigure 3A and 3B Only two substrate layers are shown, but it is understood that embodiments of the present disclosure include cell culture substrates comprising many layers of cell culture substrate arranged, for example, in a stacked arrangement as shown in Figure 3A and 3B While the substrate layers 202 and 204 are shown as not being perfectly aligned, the layers can also be aligned such that the openings 206 are aligned. Figure 3B

[0076] Figure 4 A cross-sectional view of the multi-layer substrate 200 at line B-B in Figure 3B is shown. The arrow 208 shows a possible fluid flow path through the opening in the second substrate layer 204 and then around the filament in the first substrate layer 202. The geometry of the mesh substrate layers is designed to allow efficient and generally uniform flow through the one or more substrate layers. The structure of the substrate 200 can allow fluid to flow through the substrate in multiple orientations. For example, as shown in Figure 4 , the overall fluid flow direction (as shown by arrow 208) is perpendicular to the major side surfaces of the first substrate layer 202 and the second substrate layer 204. However, the substrate can also be oriented with respect to the flow such that the side faces of the substrate layers are parallel to the overall flow direction. For example, Figure 5 A cross-sectional view of the multi-layer substrate 200 along line C-C of Figure 4 is shown. The structure of the substrate 200 allows fluid flow (arrow 210) through the fluid path in the multi-layer substrate 200. In addition to fluid flow perpendicular or parallel to the first and second side faces of the mesh layers, the substrate can also be arranged such that the multiple substrate sheets are at an intermediate angle, or even randomly arranged with respect to the fluid flow. The flexibility of such a substrate allows it to be used in a variety of applications and bioreactor or vessel designs.

[0077] As discussed herein, according to one or more embodiments, the cell culture substrate can be used within a bioreactor vessel. For example, the substrate can be used in a packed bed bioreactor configuration, or other configurations within a three-dimensional culture chamber. However, embodiments are not limited to three-dimensional culture spaces, and it is contemplated that the substrate can be used in configurations that can be considered two-dimensional culture surface configurations, where one or more layers of the substrate are laid flat, for example, in a flat bottom petri dish, to provide a culture substrate for cells. Due to contamination concerns, the vessel can be a single-use vessel, and can be discarded after use.

[0078] According to one or more embodiments, a packed bed bioreactor system for culturing cells is provided, wherein a cell culture substrate is used within a culture chamber of a bioreactor vessel. Figure 6 ​One example of a cell culture system 300 is shown, which includes a bioreactor vessel 302 having a cell culture chamber 304 in an interior chamber of the bioreactor vessel 302. Within the cell culture chamber 304 is a cell culture matrix 306 made of a plurality of substrate layers 308. The plurality of substrate layers 308 can include a plurality of separable and distinct substrate layers arranged in a stack, but can also include an integrated cell culture matrix in which the plurality of layers are fixed together. The substrate layers 308 are stacked such that a first side and a second side of a substrate layer face a first side or a second side of an adjacent substrate layer. The bioreactor vessel 300 has an inlet 310 at one end for inputting culture media, cells, and / or nutrients into the culture chamber 304, and an outlet 312 at an opposite end for removing culture media, cells, or cell products from the culture chamber 304. By allowing the substrate layers to be stacked in this manner, the system can be easily scaled up, and has no adverse effects on cell attachment and proliferation due to the defined structure and efficient flow of fluids through the stacked substrates.

[0079] In one or more embodiments, the flow resistance and bulk density of the packed bed can be controlled by interleaving substrate layers having different geometries. Specifically, the mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the flow resistance of the fluid in the form of a packed bed. By interleaving meshes having different sizes and geometries, the flow resistance in specific portions of the bioreactor can be controlled. This will enable better uniformity of liquid perfusion in a packed bed bioreactor. For example, 10 layers of mesh A (Table 1) can be stacked, followed by 10 layers of mesh B (Table 1), followed by 10 layers of mesh C (Table 1) to achieve the desired packed bed characteristics. As another example, the packed bed can start with 10 layers of mesh B, followed by 50 layers of mesh C, followed by 10 layers of mesh B. This repeating pattern can continue until the entire bioreactor is packed with meshes. These are examples only and are for illustrative purposes and are not intended to limit the possible combinations. In fact, a variety of combinations of meshes having different sizes are possible to achieve different distributions of bulk density and flow resistance of the cell growth surface. For example, a packed bed column having zones of varying cell bulk density (e.g., a series of zones producing a low / high / low / high, etc. density pattern) can be assembled by interleaving meshes of different sizes.

[0080] In Figure 6In this example, the overall flow direction is in the direction from the inlet 310 to the outlet 312 (as indicated by the direction of the arrows shown), and in this example, the first and second major sides of the substrate layer 308 are perpendicular to the overall flow direction. However, embodiments are not limited to this configuration. For example, the substrate can be arranged within the culture space such that the first and second sides are parallel to the overall flow direction, or at some intermediate angle relative to the overall flow direction. Thus, the matrices of embodiments of the present disclosure can be used in any one of these configurations. In any one of the configurations, the dimensions and shape of the substrate can be adjusted to properly fill the interior space defined by the culture chamber. Further, the dimensions of the individual layers of the substrate or the stack of substrate layers can be adjusted, as needed, to fill the interior space, or can fill less than the entire interior space. For example, in some embodiments, it can be desirable for the stack of substrate layers to occupy less than the full interior space.

[0081] As described above, embodiments of the present disclosure include bioreactor vessels that are capable of cell seeding, culturing, transfection, and / or harvesting using cell culture substrates in the vessel, and are capable of being operated at different production scales. Bioreactors according to embodiments of the present disclosure enable end users to run bioprocess experiments at scale from 1x to 10x using the same bioreactor unit. The simple scaling model of these embodiments enables the transition of bioprocesses from research to process development to production scale in one system. This flexibility in bioreactor vessel capacity configuration will save cost and time for process optimization and validation over a 1x to 10x scale range. Aspects of some embodiments will also allow end users to seed and harvest cells at the same predetermined flow rates without the need for re-optimization during scale-up of the bioreactor.

[0082] FIG. 7 illustrates a perfusion-type bioreactor 320, according to one or more embodiments. The bioreactor 320 includes a media inlet 321 through which media (including fluids, cells, and nutrients) can be fed to an interior chamber 327 of the bioreactor 320. The interior chamber 327 can be considered to include a cell culture zone 327a in which a cell culture substrate 323 is disposed and a spacer zone 327b in which a spacer 325 is disposed. The media inlet 321 leads to a fluid distribution plate 322. The fluid distribution plate 322 disperses and / or distributes the incoming media across the width of the interior space holding the cell culture substrate 323. Although not shown, a similar flow distribution plate can be disposed just prior to the outlet 326. The outlet distribution plate can help to concentrate media or components from across the width of the interior chamber 327 to the outlet. As will be explained in detail below, the bioreactor 320 can also be operated in a mode in which fluid is placed into the interior chamber 327 through the outlet 326, in which case the outlet distribution plate can help to distribute the fluid evenly across the width of the interior chamber 327. In this reverse flow mode, the inlet 321 can be used to remove fluid or components from the interior chamber 327.

[0083] The cell culture substrate 323 can correspond to embodiments disclosed herein, including, for example, a porous polymer substrate. The substrate 323 has a height h that extends from the flow distribution plate side of the substrate 323 to the opposite side or top of the substrate 323. Optionally, at the top of the substrate 323 opposite the flow distribution plate 322 is a packed bed retainer 324. The packed bed retainer 324 is designed to provide structural support to the substrate 323 to resist the flow of media from the media inlet 321. In this way, the packed bed retainer 324 can help to maintain the position and / or shape of the packed bed substrate 323. According to different embodiments, the packed bed retainer 324 can have many configurations, but in general has a structure sufficient to hold the substrate 323 in place while allowing media (including cells) to pass through the packed bed retainer 324. At the top of the packed bed retainer 324 is the spacer 325. The spacer 325 is sized to substantially fill the spacer zone 327b, or the interior space above the substrate 323 and / or packed bed retainer 324 and at the top of the interior chamber 327 prior to the media outlet 326. By filling this space, the spacer 325 abuts the top of the interior space or some support feature within the interior space and thus holds the packed bed retainer 324 and substrate 323 in place.

[0084] The spacer 325 also creates a headspace in a spacer region 327b above the cell culture region 327a. This headspace can have the effect of enhancing flow uniformity in the substrate 323, especially near the top of the substrate 323. For example, if the top of the substrate 323 is near the top of the interior chamber 327, flow restrictions at the top of the interior chamber 327 can create areas of increased pressure and affect flow uniformity in the packed bed. As will be discussed below, this headspace also provides a space for pressurized fluid to begin filling the interior chamber 327 during a harvest operation.

[0085] Figure 7B The display Figure 7A variations of the embodiments. In Figure 7B between the cell culture substrate 323 and the media inlet 321. The porous spacer disk 328 can take different forms, but generally extends across the width of the interior chamber 327 or the width of the substrate 323 and is porous to allow fluid flow through the porous spacer disk 328. According to embodiments, the porous spacer disk 328 has a spacer disk hole diameter that is larger than the hole diameter of the openings or holes in the substrate. In some embodiments, multiple porous spacer disks 328 can be stacked between the substrate 323 and the inlet 321. These multiple porous spacer disks 328 can each have the same configuration (e.g., thickness and spacer disk hole size) or different configurations (e.g., different thicknesses and spacer disk hole sizes). It is contemplated that the porous spacer disks 328 can further homogenize and distribute the fluid flow from the inlet 321 or distribution plate 322 before the fluid reaches the cell culture substrate 323.

[0086] Figure 7A The substrate 323 in 7B may be non-woven or woven, for example, a woven PET substrate. However, in some embodiments, the substrate can be non-woven. The substrate can include multiple layers of substrate material in a stacked arrangement, or a substrate material in a roll or spiral.

[0087] Figure 8 are Figure 7A and 7B are photographs of components of an example of a bioreactor 320. As shown, the bioreactor 320 can include two or more separable housing components 350a and 350b. In the lower housing component 350a, a flow distribution plate 352 is shown, which is located between the media inlet (in Figure 8Above (not shown in the image). An example of a packed bed retainer 354 is shown. The packed bed retainer 354 is generally grid-shaped and has a defined thickness. While the grid of the packed bed retainer 354 allows the culture medium and other components to flow through the grid, it still provides support to any substrate layer within the bioreactor. An example of a spacer 355 is also shown. The shapes and dimensions of the shell components 350a, 350b, distribution plate 352, packed bed retainer 354, and spacer 355 are shown by way of example only, and embodiments of this disclosure are not limited to the configurations shown. A first porous spacer disc 356, a second porous spacer disc 357, and a third porous spacer disc 358 are also shown near the inlet. In this example, the second porous spacer disc 357 and the third porous spacer disc 358 have the same construction, both made of a sieve or mesh material having a spacer disc pore diameter larger than the pore or opening diameter of the cell culture medium 360. The first porous spacer disc 356 is also made of a screen, mesh, or grid material, but has a larger spacer disc hole diameter than the second porous spacer disc 357 and the third porous spacer disc 358.

[0088] Figure 9A A plan view of the distribution plate 2 is shown, which is similar to... Figure 8 The distribution plate 352. The holes 7 in the distribution plate 2 are arranged to uniformly distribute the flow rate across the width of the distribution plate and within the internal space of the bioreactor. Figure 9B A plan view of a packed bed retainer 4 is shown according to some embodiments. The packed bed retainer 4 has a grid shape that defines a plurality of large openings 8 to minimize flow resistance, but still effectively retains the cell culture medium within the packed bed area.

[0089] Figure 10 It shows Figure 8 A three-dimensional model of spacer 355. The spacer can compress the substrate and / or retain the substrate in the packed bed region, while simultaneously establishing spacer region 327b in the internal chamber 327 of the bioreactor (see...). Figure 7A The spacers 355 are designed to define a top space above the packed bed of cell culture medium. Removable spacers 355 of varying heights allow users to culture cells in bioreactors with different volumes of cell culture medium and allow unrestricted flow of culture medium from the packed bed to the culture medium outlet port. That is, a spacer 355 with a greater height can correspondingly reduce the height of the packed bed, while a spacer 355 with a smaller height can correspondingly allow more space for a higher packed bed. In some embodiments, the bioreactor system may include height-adjustable spacers instead of using individual spacers of different heights.

[0090] FIG. 11 shows an example of an assembled packed bed bioreactor with spacers inserts of different heights, and correspondingly packed bed substrates of different heights. In this example, from left to right, the same bioreactor can be configured to scale up production by a factor of 3. However, it is expected that by correspondingly adjusting the component dimensions, embodiments of the present disclosure will allow scaling up production of a given bioreactor by an even greater range.

[0091] Figure 12 According to one or more embodiments, a bioreactor 402 is shown incorporated into a bioprocessing system 400. The system 400 includes a media conditioning vessel 411, for example, to properly maintain cell culture media parameters, e.g., pH, temperature, and oxygenation levels. An automated control pump 409 is used to perfuse media through the bioreactor 402. The bioreactor inlet 413 is equipped with an additional three-way port to facilitate cell seeding or harvesting of harvested cells. The system 400 can include inline sensors, as well as sensors 412 in the media conditioning vessel 411.

[0092] Figure 13 The bioreactor according to the above-described embodiments is shown in multiple stages during the process of harvesting cells from the bioreactor. The cell harvesting process involves pre-filling the bioreactor with a cell dissociation solution 422, and incubating the packed bed for a predetermined amount of time to allow the cells to detach from the substrate. The resulting cell suspension is withdrawn from the media inlet 421 by reversing the flow of media / cells through the inlet 421 and applying pressurized fluid (e.g., air) through the media outlet 426, as shown in the progression of stages from left to right in Figure 13

[0093] Table 3 shows AAV production run results in a 60 mm bioreactor according to the embodiments of FIGS. 7-13. This 60 mm bioreactor corresponds to a total substrate surface area of 6780 cm 2 . Transfected cell yield, transfection efficiency, and viral genome yield per cm 2 are shown.

[0094] ​The above described scalable container embodiments of the present disclosure are targeted packed bed bioreactor systems for anchorage dependent cells that allow process optimization or production at several different scales using the same platform and the same bioreactor container. In one embodiment, for example, the bioreactor container 320 holds a packed bed 323 for adherent cell attachment, proliferation, transfection, and production of a product. Cell culture media can be continuously perfused through the packed bed and bioreactor container to supply the cells with oxygen and nutrients and to remove harmful metabolic products. The media is introduced through the container inlet 321 at a calculated flow rate and exits the bioreactor through outlet 326. To achieve uniform flow distribution over the packed bed of substrate material, a flow distribution plate 322 is positioned in front of the packed bed area. This flow distribution plate can have a branched and radially interconnected design to allow uniform flow distribution over the packed bed as a whole.

[0095] In another embodiment, the layers of the packed bed are held in a close packed state by a rigid retention grid 4. This retention grid has large openings that minimize flow resistance during bioreactor perfusion, but it is structurally still rigid enough to uniformly exert pressure on the packed bed surface to hold the layers of PET mesh in a close packed state. The volume of the packed bed in the bioreactor of the present disclosure can be varied by fixing the retention grid 4 in place with spacers 5 of different sizes. The size of the bioreactor container and the diameter of the packed bed can vary from ~1 cm (laboratory scale) to ~10 cm (process development scale), to 50 cm (pilot scale), to 200 cm (manufacturing scale).

[0096] Figure 8 Parts of the bioreactor tested in the biological production of viral particles by HEK293T adherent cells are shown. The inner diameter of the bioreactor packed bed area is 60 mm and the packed bed of the assembled state of the bioreactor can consist of 10 to 300 layers of rigid PET substrate. This corresponds to a surface area of 678-20300 cm 2 for adherent cell attachment, growth, and production of a compound of interest. Figure 12A schematic of the bioreactor vessel 402 in an assembled state and connected to the main external components is shown, including media conditioning vessel, a pump allowing the pumping of media into the bioreactor, and external dissolved oxygen sensors supporting the process conditions required for a successful bioprocess. The cell media is conditioned in the media conditioning vessel 411 where the appropriate pH, temperature, and dissolved oxygen levels are maintained. Subsequently, the media is perfused through the bioreactor by the pump 409. The flow rate of the pump 409 is integrated into a feedback loop that automatically adjusts to maintain a minimum predetermined level of dissolved oxygen in the media exiting the bioreactor. Via the media conditioning vessel 411, all transfection reagents, nutrients, and additional media supplements required for a given bioprocess can be introduced into the total media, and waste media can be removed. At the end of the process, the media can be drained from the bioreactor and refilled with a cell harvest solution 422 Figure 13 ). The packed bed is incubated in the harvest solution for a predetermined time, and the predetermined time is sufficient for the cells to detach from the substrate, after which the cells are harvested by applying air pressure at the bioreactor outlet 6 to achieve a reverse flow with a flow rate of 70 ml / cm 2 (cross-sectional area of the packed bed) / minute. The cells are harvested at the three-way port 413 of the bioreactor. The cells can also be lysed directly in the bioreactor, and the lysate solution containing the AAV particles can be collected through the three-way port 413.

[0097] The media conditioning vessel 404 can include sensors and control components found in typical bioreactors used in the bioprocessing industry for suspension batch, fed batch, or perfusion cultures. These include, but are not limited to, DO oxygen sensors, pH sensors, oxygenators / gas sparging units, temperature probes, and nutrient addition and base addition ports. The gas mixture supplied to the sparging units can be controlled by gas flow controllers for N2, O2, and CO2 gases. The media conditioning vessel 404 also includes an impeller for media mixing. All media parameters measured by the sensors listed above can be controlled by the media conditioning control unit 418, which is in communication with the media conditioning vessel 404 and is capable of measuring and / or adjusting the conditions of the cell media 406 to achieve the desired levels.

[0098] Culture medium from the media conditioning vessel 404 is delivered to the bioreactor 402 through an inlet, which can also include an injection port for cell seeding, to seed cells and begin cell culture. The bioreactor vessel 402 can also include one or more outlets through which cell culture medium exits the vessel 402. In addition, cells or cell products can be output through the outlet. To analyze the contents of the effluent from the bioreactor 402, one or more sensors 412 can be provided in the line. In some embodiments, the system 400 includes a flow control unit for controlling the flow into the bioreactor 402. For example, the flow control unit can receive signals from the one or more sensors 412 and, based on the signals, adjust the flow into the bioreactor 402 by sending signals to a pump (e.g., a peristaltic pump) upstream of the inlet 408 of the bioreactor 402. Thus, based on a factor or a combination of factors measured by the sensors 412, the pump can control the flow into the bioreactor 402 to achieve desired cell culture conditions.

[0099] The rate of medium perfusion is controlled by a signal processing unit that collects and compares sensor signals from the media conditioning vessel 404 and sensor signals from sensors located at the outlet of the packed bed bioreactor. Due to the packed flow nature of the perfusion of culture medium through the packed bed bioreactor 402, gradients of nutrients, pH, and oxygen develop along the packed bed. According to the flow chart of Figure 14B The perfusion flow rate of the bioreactor can be automatically controlled by a flow control unit operatively connected to the peristaltic pump.

[0100] One or more embodiments of the present disclosure provide a cell seeding step that is different from conventional methods. In conventional methods, a packed bed having a conventional matrix is packed with culture medium and a concentrated inoculum is injected into the culture medium circulation loop. The cell suspension is pumped through the bioreactor at an increased flow rate, thereby reducing the non-uniformity of cell seeding by capture on the conventional packed bed matrix. In such conventional methods, the cells are pumped into the circulation loop at an elevated flow rate and for perhaps several hours until a majority of the cells are captured in the packed bed bioreactor. However, due to the non-uniform deep bed filtration properties of conventional packed bed bioreactors, the cells are distributed non-uniformly within the packed bed, and there is a higher cell density at the inlet region of the bioreactor and a lower cell density at the outlet region of the bioreactor.

[0101] In contrast, according to embodiments of the present disclosure, a cell inoculum having a volume equal to the void volume of the culture chamber of the bioreactor is injected directly into the packed bed through a cell inoculum injection port at the inlet of the bioreactor 402 Figure 12Due to the uniform and continuous fluid channels present in the cell culture substrate described herein, the cell suspension is then uniformly distributed within the packed bed. To prevent cell sedimentation due to gravity during the initial inoculation phase, culture medium perfusion can be initiated immediately after inoculum injection. The perfusion flow rate is maintained below a preset threshold to counteract gravity and prevent cells from being washed away from the packed bed bioreactor. Thus, during the initial cell attachment phase, cells are gently tumbled within the packed bed, achieving a uniform cell distribution and attachment to the available substrate surface.

[0102] Figure 14A According to some embodiments, process steps used in the cell culture system disclosed herein are shown. Figure 14A As shown, these process steps may include process preparation (S1), inoculation and attachment of cells (S2a, S2b), cell expansion (S3), transfection (S4a, S4b), production of viral vectors (S5a, S5b), and harvesting (S6a, S6b).

[0103] Figure 14B A method for controlling a perfusion bioreactor system (e.g., Figure 12 This is an example of a method 450 for controlling the flow rate of a system 400. According to method 450, certain parameters of the system 400 are predetermined at step S1 through optimized operation of the bioreactor. Based on these optimized operations, values ​​for pH1, pO1, [glucose]1, pH2, pO2, [glucose]2, and the maximum flow rate are determined. At step S2, the values ​​of pH1, pO1, and [glucose]1 are measured in the cell culture chamber of the bioreactor 402, and at step S3, pH2, pO2, and [glucose]2 are measured in the culture medium conditioning container 404 by sensor 412. Based on these values ​​at S2 and S3, the perfusion pump control unit makes a decision at S4 to maintain or adjust the perfusion flow rate. For example, if at least one of pH2 ≥ pH2 minimum, pO2 ≥ pO2 minimum, and [glucose]2 ≥ [glucose]2 minimum is satisfied, the perfusion flow rate of the cell culture medium reaching the cell culture chamber can continue at the current rate (S5). If the current flow rate is less than or equal to the predetermined maximum flow rate of the cell culture system, the perfusion flow rate is increased (S7). Further, if the current flow rate is not less than or equal to the predetermined maximum flow rate of the cell culture system, the controller of the cell culture system may reassess at least one of the following: (1) minimum pH2, minimum pO2, and minimum [glucose]2; (2) pH1, pO1, and [glucose]1; and (3) the height of the bioreactor vessel (S6).

[0104] Depending on the desired system, the cell culture substrate can be arranged in a variety of configurations within the culture chamber. For example, in one or more embodiments, the system includes one or more substrate layers and its width extends over the entire width of the defined cell culture space in the culture chamber. Multiple substrate layers can be stacked in this manner to a predetermined height. The substrate layers can be arranged such that the first and second sides of one or more layers are perpendicular to the general flow direction of the culture media through the defined culture space in the culture chamber. In some embodiments, the first and second sides of one or more layers can be parallel to the general flow direction. In one or more embodiments, the cell culture substrate includes one or more substrate layers in a first orientation relative to the general flow, and one or more other layers in a second orientation different from the first orientation. For example, the individual layers can have a first and second side that are parallel or perpendicular to the general flow direction, or at some angle between parallel and perpendicular.

[0105] In one or more embodiments, the cell culture system includes a plurality of discrete pieces of cell culture substrate in a packed bed configuration, wherein the length and / or width of the substrate pieces is relatively small with respect to the culture chamber. As used herein, a piece of substrate is considered to have a length and / or width that is relatively small with respect to the culture chamber when the length and / or width of the piece of substrate is equal to or less than about 50% of the length and / or width of the culture space. Thus, the cell culture system can include a plurality of pieces of substrate packed into the culture space in a desired arrangement. The arrangement of the pieces of substrate can be random or semi-random, or can have a predetermined order or array, for example, the pieces of substrate are oriented in a substantially similar orientation (e.g., horizontal, vertical, or at an angle between 0° and 90° with respect to the general flow direction).

[0106] A "defined culture space" as used herein refers to the space in the culture chamber that is occupied by the cell culture substrate and in which cell seeding and / or culturing will occur. The defined culture space can fill approximately the entire culture chamber, or can occupy a portion of the space within the culture chamber. A "general flow direction" as used herein is defined as the direction of the overall mass flow of fluid or media through or over the cell culture substrate during cell culturing, and / or during the flow of media into or out of the culture chamber.

[0107] In one or more embodiments, the cell culture substrate is secured within the culture chamber by a securing mechanism. The securing mechanism can secure a portion of the cell culture substrate to the walls of the culture chamber that surround the substrate, or to the walls of the chamber at one end of the culture chamber. In some embodiments, the securing mechanism adheres a portion of the cell culture substrate to a member that travels through the culture chamber, for example, a member that travels parallel to the longitudinal axis of the culture chamber, or to a member that travels perpendicular to the longitudinal axis. In one or more other embodiments, however, the cell culture substrate can be contained within the culture chamber and not fixedly attached to the walls of the chamber or bioreactor vessel. For example, the substrate can be contained within the chamber by the boundaries of the culture chamber or other structural members to keep the substrate within a predetermined area of the bioreactor vessel and not fixedly secure the substrate to these boundaries or structural members.

[0108] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. According to one or more embodiments of the present disclosure, the culture chamber is capable of containing a cell culture substrate and a substrate.

[0109] In a roller bottle configuration, the bioreactor vessel can be operatively attached to a device for moving the bioreactor vessel about its central longitudinal axis. For example, the bioreactor vessel can be rotated about the central longitudinal axis. The rotation can be continuous (e.g., continuously in one direction), or discontinuous (e.g., rotated intermittently in a single direction or alternating directions, or oscillated in a back-and-forth rotational direction). In operation, the rotation of the bioreactor vessel causes movement of cells and / or fluids within the chamber. This movement can be considered movement relative to the walls of the chamber. For example, when the bioreactor vessel is rotated about its central longitudinal axis, gravity can cause fluids, media, and / or unattached cells to be maintained toward the lower portion of the chamber. In one or more embodiments, however, the cell culture substrate is substantially fixed relative to the vessel, and thus, rotates with the vessel. In one or more other embodiments, the cell culture substrate can be unattached, and when the vessel is rotated, it moves freely relative to the vessel to a desired extent. Cells can be attached to the cell culture substrate, while movement of the vessel causes the cells to be exposed to cell culture media or liquid, and oxygen or other gases in the culture chamber.

[0110] By using the cell culture substrates of the present disclosure, e.g., substrates including a woven or mesh base material, the roller bottle vessel has an increased surface area available for adherent cell attachment, proliferation, and functionalization. Specifically, using a substrate of a woven mesh of monofilament polymeric material within a roller bottle, the surface area can be increased by about 2.4-fold to about 4.8-fold, or up to about 10-fold of a standard roller bottle. As described herein, each monofilament strand of the mesh substrate is able to present a 2D surface for adherent cell adhesion. Further, multiple layers of mesh can be arranged within the roller bottle such that the total surface area available is increased by about 2-fold to 20-fold compared to a standard roller bottle. Thus, by adding the improved cell culture substrates disclosed herein, existing roller bottle facilities and processes, including cell seeding, media exchange, and cell harvesting, can be altered with minimal impact on existing operational infrastructure and process steps.

[0111] The bioreactor vessel optionally includes one or more outlets that can be attached to inlet and / or outlet devices. Through the one or more outlets, liquid, media, or cells can be supplied to the chamber or removed from the chamber. A single port in the vessel can function as both an inlet and an outlet, or multiple ports can be provided for dedicated inlets and outlets.

[0112] The embodiments are not limited to rotation of the vessel about a central longitudinal axis. For example, the vessel can be rotated about an axis that is not centered with respect to the vessel. Further, the axis of rotation can be a horizontal or vertical axis.

[0113] Embodiments

[0114] To demonstrate the efficacy of the cell culture substrates, cell culture systems, and related methods of the present disclosure, studies were conducted with respect to cell seeding and culture according to the following embodiments.

[0115] In Example 1, a cell culture substrate having a polyethylene terephthalate (PET) woven mesh substrate (see Figures 15A-15C ) was tested in static cell culture conditions. The PET mesh was washed in ethanol and plasma treated in an oxygen RF plasma. Gelatin was adsorbed onto the surface of the mesh filaments to promote cell adhesion. Disc-shaped pieces of the mesh were placed into ultra-low attachment (ULA) six-well plates. HEK293T cells were seeded onto the mesh disc pieces at different seeding densities (50K / cm 2 , 75K / cm 2 , 100K / cm 2 , corresponding to Figure 15A , 15B and 15C, respectively, and cell culture was performed for three days. Cells on the surface of the filaments were stained with fluorescent green cell tracking dye. Figures 15A-15CThis visualization of cells on the surface of the filaments is shown. The size of the web filaments relative to the cell size allows the monofilament fibers to be effectively used as a two-dimensional surface for cell attachment and proliferation. Cells are harvested from the web and processed using materials from Beckman Coulter. Cell proliferation was measured using a cell counter. The results showed that, under static cell culture conditions, cells exhibited excellent adhesion and proliferation on the cell culture medium.

[0116] In Example 2, according to an embodiment of this disclosure, in a packed bed bioreactor system (e.g., Figure 6 Cells are cultured in the system shown. The packed bed has a cylindrical shape and is made of stacks of cell culture media, each of which is circular or disc-shaped. Specifically, in Example 2, the packed bed has a height of approximately 25 mm and comprises one hundred discs of PET mesh substrate, each disc having a diameter of approximately 20 mm. The mesh used corresponds to mesh C in Table 1. The estimated total two-dimensional surface area available for cell attachment is approximately 760 cm². 2 To inoculate the bioreactor, 8 ml of HEK293T cell suspension (two million cells / ml) was directly injected into the packed bed. Culture medium perfusion was initiated immediately after the introduction of the cell suspension, with a perfusion flow rate set to 3 ml / min. Perfusion was continued at this flow rate for 24 hours, then the flow rate was reduced to 1 ml / min. Thereafter, the perfusion flow rate was adjusted to maintain pO2 ≥ 50% saturation pressure and pH ≥ 7 at the bioreactor outlet. After two to three days, the cells in the bioreactor were stained with crystal violet, and the bioreactor was disassembled to demonstrate uniform cell attachment within the matrix. According to a preferred embodiment, the bioreactor can be inoculated using an inoculation method in which cells are continuously tumbled within the packed bed during the initial attachment phase. As a result, uniform cell distribution was achieved in all portions of the packed bed after two days of cell culture. This indicates that uniform cell distribution was achieved when the bioreactor was continuously perfused during the cell inoculation phase.

[0117] In Example 3, cells were cultured in a packed-bed bioreactor system, and HEK293T cells were transfected in the bioreactor for adeno-associated virus (AAV) production. Example 3 used the same bioreactor setup as Example 2 (see, e.g., Figure 6 The bed consists of discs containing 100 PET meshes (mesh C in Table 1). Each disc is approximately 20 mm in diameter, and the bed height is approximately 25 mm, resulting in a total two-dimensional surface area of ​​approximately 760 cm² available for cell attachment and proliferation. 2To seed the bioreactor, 8 ml of HEK293T cell suspension (2 million cells / ml) was directly injected into the packed bed. A culture medium storage container containing approximately 50 ml of culture medium was attached to the bioreactor vessel. Cells were cultured in Duchenne Modified E. E. MEM (DMEM). Incubate for 72 hours in medium (containing +10% FBS and +6 mM L-glutamine). When the pH of the medium in the storage container drops below 7, replace the medium with fresh medium. Accordingly, adjust the perfusion flow rate to maintain pO2 ≥ 50% saturation pressure and pH ≥ 7 at the bioreactor outlet. After 72 hours, use 50 ml of... The medium was modified with DMEM (15-018) (containing +10% FBS, +6mM L-glutamine), and transfection reagent was added to obtain a final concentration of 2ug / ml AAV2 and PEIpro in a 1:2 ratio. During the next 72 hours, if the pH in the reservoir dropped below 7, the medium was replaced with a fresh feed. Accordingly, the perfusion flow rate was adjusted to maintain pO2 ≥ 50% saturation pressure and pH ≥ 7 at the bioreactor outlet. Cells were harvested using a 5X TrypLE. Transfection efficiency was analyzed by fluorescence flow cytometry, and viral particle and viral genome titers were analyzed by ELISA and PCR assays. The cell culture results are shown in Table 2, where “VP” represents viral protein and “GC” represents genome copy.

[0118]

[0119]

[0120] Table 2 Results of HEK 293T cell transfection and AAV production in a packed bed bioreactor.

[0121] The embodiments disclosed herein offer several advantages over existing platforms used for cell culture and viral vector production. It should be noted that the embodiments disclosed herein can be used to produce a wide variety of cell types and cell byproducts, including, for example, adherent or semi-adherent cells, human embryonic kidney (HEK) cells (e.g., HEK23), including transfected cells, and viral vectors such as lentiviruses (stem cells, CAR-T) and adeno-associated viruses (AAV). These are examples of some common applications of the bioreactors or cell culture materials disclosed herein, but they are not intended to limit the use or application of the disclosed embodiments, as those skilled in the art will understand that the embodiments can be applied to other uses.

[0122] As noted above, one advantage of embodiments of the present disclosure is through the flow uniformity of the cell culture substrate. Without being bound by theory, it is believed that the regular and uniform structure of the cell culture substrate provides a consistent and uniform body through which the culture medium can flow. In contrast, existing platforms primarily rely on irregular or random substrates, e.g., felt-like or non-woven fibrous materials. The uniform nature of the substrate of the present disclosure can be illustrated by examining the uniform and consistent cell seeding achieved on the substrate. For example, Figure 16A Three disks (1801, 1802, 1803) of substrate material are shown in accordance with some embodiments of the present disclosure. Figure 16A The disks in were PET woven web material as described herein, and each disk was about 60 mm in diameter. The surface area of a bioreactor filled with 10 to 300 layers of similar disks would be about 678 to 20,300 cm 2 In this example, cell culture was performed using a stack of 100 disks. The first disk 1801 was the top disk in the stack of disks within the bioreactor, the second disk 1802 was the middle disk of the stack, and the third disk 1803 was the bottom disk of the stack.

[0123] Figure 16A The uniformity of cell distribution within the bioreactor is shown 72 hours after seeding. Substrate layers from three different regions of the packed bed were retrieved from the bioreactor and stained with crystal violet to visualize the attached HEK 293T cells. The uniformity of staining of the top, middle, and bottom layers of cells demonstrates that cells were uniformly distributed throughout the packed bed during the seeding and attachment steps. The packed bed substrate samples were also stained after harvest to demonstrate the efficiency of the cells harvested from the bioreactor of the present disclosure (see Figure 16B ). Figure 16B Stained substrate layers retrieved from three different regions of the packed bed after the harvest step are shown. As can be seen from Figure 16B , the harvest process recovered over 95% of the cells from the bioreactor. Cell culture results are shown in Table 3.

[0124] In generating Figure 16A and 16BThe images in FIG. 2 were taken after 72 hours of culture and before harvesting the cells from the substrate. 外 ≥ 45% saturation. At 72 hours post-seeding, the media was replaced with 500 ml of Corning DMEM (15-018) + 10% FBS + 6 mM L-glutamine and allowed to perfuse for 2 hours. The transfection mix (complex of plasmid DNA and PEI at a 1 :2 ratio; 0.8 ug total DNA / million cells) was added to achieve a final concentration of 2 ug total DNA / ml media at 24 hours post-transfection, and the media was replaced with 500 ml of fresh complete Corning DMEM (15-018) media to replenish depleted nutrients. The perfusion flow rate was automatically adjusted to maintain DO 外 ≥ 45% saturation. At 72 hours post-seeding, the media was replaced with 500 ml of Corning DMEM (15-018) + 10% FBS + 6 mM L-glutamine and allowed to perfuse for 2 hours. The transfection mix (complex of plasmid DNA and PEI at a 1 :2 ratio; 0.8 ug total DNA / million cells) was added to achieve a final concentration of 2 ug total DNA / ml media at 24 hours post-transfection, and the media was replaced with 500 ml of fresh complete Corning DMEM (15-018) media to replenish depleted nutrients. The perfusion flow rate was automatically adjusted to maintain DO

[0125] Crystal violet staining was used to highlight the uniform growth of cells across the surface of the disks in Figure 16A Although the first disk 1801, the second disk 1802, and the third disk 1803 are interspersed in the stack of cell culture matrix, the cell growth is uniform across all three disks. Figure 16A The images in FIG. 2 were taken after 72 hours of culture and before harvesting the cells from the substrate. Figure 16B shows the same three disks (1801', 1802', and 1803') after the cells have been harvested. As Figure 16BThe relative absence of crystal staining indicates that cells have been uniformly harvested on the surface of each disc and on the three discs of the cell culture substrate stack. Based on the analysis, over 95% of the cells were recovered from the bioreactor. Table 3 below shows the results for these cells on a total surface area of ​​6780 cm². 2 Cell culture results produced by AAV in a substrate stack / container with a diameter of 60 mm are shown, including transfected cell yield, transfection efficiency, and per cm⁻¹. 2 Viral genome yield. Furthermore, the uniform structure and flow properties of the substrate are believed to contribute to this efficient and uniform growth and harvesting capability.

[0126]

[0127]

[0128] Table 3 : Transfected cell yield, transfection efficiency, and per cm from a 60 mm bioreactor 2 Viral genome yield.

[0129] Table 4 below shows the above results under multiple experimental settings including bioreactor vessels of different diameters (29 mm and 60 mm). The data show smaller (e.g., 29 mm diameter, 1600 cm⁻¹) results. 2 Surface area) and larger (e.g., 60mm diameter, 6780cm²) 2 Good scalability between the surface area container and / or the packed bed matrix.

[0130]

[0131] Table 4 Consistent results across the entire size of the bioreactor.

[0132] Example AAV production via HEK 293T cells in a 60 mm bioreactor unit and AAV production in a packed bed bioreactor.

[0133] A packed bed bioreactor is assembled using α-units, as shown in Figure 7. Actual bioreactor components are shown below. Figure 8 A 60mm diameter packed bed containing 100 layers of structured and treated PET substrate was placed in a bioreactor. The bed height was 26mm, and the calculated total 2D surface area available for cell attachment and proliferation was 6780cm². 2The bioreactor was pre-filled with cell culture media and the system was pre-conditioned overnight to achieve steady state at pH 7.2, D.O. 100% and 37°C. The entire bioreactor system was filled with 400 ml of ATCC DMEM media + 10% FBS + 6 mM L-glutamine Figure 12 The suspended 30 ml of HEK293T cells (five million cells / ml) were injected directly into the packed bed through the three-way port 413 to form the inoculum.

[0134] The bioreactor was perfused with pre-conditioned media at a rate of 30 mL / min for the first 48 hours to allow for uniform cell distribution, attachment and initial growth in the packed bed. After 48 hours of culture, 200 ml of fresh complete ATCC DMEM media was added to the system to maintain glucose levels above 1 g / L. The perfusion flow rate was automatically adjusted to maintain DO 外 ≥ 45% saturation at the bioreactor outlet.

[0135] At 72 hours post-inoculation, the media was replaced with 500 ml of Corning DMEM (15-018) + 10% FBS + 6 mM L-glutamine and allowed to perfuse for 2 hours. The transfection mix (complex of plasmid DNA and PEI at a ratio of 1 :2; 0.8 ug total DNA / million cells) was added to achieve a final concentration of 2 ug total DNA / ml media at 24 hours post-transfection, and the media was replaced with 500 ml of fresh complete Corning DMEM (15-018) media to replenish depleted nutrients. The perfusion flow rate was automatically adjusted to maintain DO 外 ≥ 45% saturation at the bioreactor outlet.

[0136] Glucose levels were monitored during the subsequent 48 hours of culture and replenished as needed by media addition or replacement to maintain levels above 0.3 g / L. At 72 hours post-transfection, the cells were washed with DPBS and harvested using IX Accutase solution. Transfection efficiency was analyzed by fluorescent flow cytometry analysis, and viral particle and viral genome titers were analyzed by ELISA and qPCR assays. Figure 16A The uniformity of cell distribution within the bioreactor at 72 hours post-inoculation is shown. As can be seen from Figure 16B the harvest process recovered over 95% of the cells from the bioreactor. Cell culture results are shown in Table 3.

[0137] As described above, embodiments of the present disclosure can provide a packed bed cell culture substrate and / or bioreactor that is capable of culturing high density cells in a relatively small and practical footprint. For example, the 60 mm cell culture substrate in the example in Tables 3 and 4 above has an area of about 6870 cm2 surface area. For reference, Corning® has a surface area of about 1720 cm 2 The 60 mm diameter cell culture substrates of Tables 3 and 4 can be housed in smaller bioreactors than but still result in higher cell counts, higher total genomic copies (GC or viral genome (VG)) per vessel at harvest. Figure 17A , 17B and 17C show data from cell culture in two bioreactor vessels of the present disclosure having the 60 mm diameter substrates of Tables 3 and 4, compared to cell culture data from a 2D surface of 2 Figure 17C , although lower in this example than but compensated for by the higher surface area.

[0138] Figure 18 According to one or more embodiments, a more detailed schematic of a cell culture system 520 is shown. The basic configuration of system 520 is similar to system 400 of Figure 12 , with a packed bed bioreactor 522 having a vessel containing a packed bed of cell culture material (e.g., PET woven mesh), and a separate media conditioning vessel 524. However, in contrast to system 400, system 520 shows details of the system, including sensors, user interface and controls, and various inlets and outlets for media and cells. According to some embodiments, media conditioning vessel 524 is controlled by a controller 526 to provide appropriate temperature, pH, O2, and nutrients. While in some embodiments, bioreactor 522 can also be controlled by controller 526, in other embodiments, bioreactor 522 is provided in a separate perfusion loop 528, in which a pump is used to control the flow rate of media through perfusion loop 528 based on detection of O2 at or near the outlet of bioreactor 522.

[0139] Exemplary Embodiments

[0140] The following is a description of aspects of various embodiments of the disclosed subject matter. Each aspect can include one or more of the various features, characteristics or advantages of the disclosed subject matter. Embodiments are intended to be illustrative of several aspects of the disclosed subject matter and should not be considered to be exhaustive or complete.

[0141] Aspect 1 is directed to a cell culture system comprising: a bioreactor vessel; and

[0142] ​​A cell culture substrate disposed in a bioreactor vessel and configured for culturing cells; wherein the cell culture substrate comprises a substrate including a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and through the thickness of the substrate, and wherein the plurality of openings are configured to allow at least one of a cell culture media, a cell, or a cell product to flow through the thickness of the substrate.

[0143] Aspect 2 relates to the cell culture system of Aspect 1, wherein the substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0144] Aspect 3 relates to the cell culture system of Aspect 1 or Aspect 2, wherein the substrate comprises at least one of a molded polymeric lattice sheet, a 3D-printed lattice sheet, and a woven mesh.

[0145] Aspect 4 relates to the cell culture system of Aspect 3, wherein the substrate comprises a woven mesh comprising one or more fibers.

[0146] Aspect 5 relates to the cell culture system of Aspect 4, wherein the one or more fibers comprise a cross-sectional shape that is at least one of oblong, circular, rectangular, or polygonal.

[0147] Aspect 6 relates to the cell culture system of Aspect 4 or Aspect 5, wherein the one or more fibers comprise at least one of a monofilament fiber and a multifilament fiber.

[0148] Aspect 7 relates to the cell culture system of any one of Aspects 4-6, wherein the one or more fibers comprise a first fiber having a first fiber diameter of about 50 pm to about 1000 pm, about 50 pm to about 600 pm, about 50 pm to about 400 pm, about 100 pm to about 325 pm, or about 150 pm to about 275 pm.

[0149] Aspect 8 relates to the cell culture system of Aspect 7, wherein the one or more fibers further comprise a second fiber having a second fiber diameter of about 50 pm to about 1000 pm, about 50 pm to about 600 pm, about 50 pm to about 400 pm, about 100 pm to about 325 pm, or about 150 pm to about 275 pm.

[0150] Aspect 9 relates to the cell culture system of Aspect 8, wherein the second fiber diameter is different than the first fiber diameter.

[0151] Aspect 10 relates to the cell culture system of any one of aspects 1-7, wherein the plurality of openings comprises an opening diameter of about 100 pm to about 1000 pm, about 200 pm to about 900 pm, or about 225 pm to about 800 pm.

[0152] Aspect 11 relates to the cell culture system of aspect 10, wherein the fiber diameter is about 250 pm to about 300 pm and the opening diameter is about 750 pm to about 800 pm, or wherein the fiber diameter is about 270 pm to about 276 pm and the opening diameter is about 785 pm to about 795 pm.

[0153] Aspect 12 relates to the cell culture system of aspect 10, wherein the fiber diameter is about 200 pm to about 230 pm and the opening diameter is about 500 pm to about 550 pm, or wherein the fiber diameter is about 215 pm to about 225 pm and the opening diameter is about 515 pm to about 530 pm.

[0154] Aspect 13 relates to the cell culture system of aspect 10, wherein the fiber diameter is about 125 pm to about 175 pm and the opening diameter is about 225 pm to about 275 pm, or wherein the fiber diameter is about 150 pm to about 165 pm and the opening diameter is about 235 pm to about 255 pm.

[0155] Aspect 14 relates to the cell culture system of any one of aspects 10-13, wherein the ratio of the opening diameter to the fiber diameter is about 1.0 to about 3.5, about 1.25 to about 3.25, about 1.4 to about 3.0, about 1.5 to about 2.9, about 1.5 to about 2.4, or about 2.4 to about 2.9.

[0156] Aspect 15 relates to the cell culture system of any one of aspects 1-14, wherein the plurality of openings comprises openings that are square, rectangular, rhombus, long diagonal square, circular, or elliptical in shape.

[0157] Aspect 16 relates to the cell culture system of any one of aspects 1-15, wherein the plurality of openings are arranged in a regular pattern.

[0158] Aspect 17 relates to the cell culture system of any one of aspects 1-16, wherein the cell culture substrate comprises a monolayer of substrate.

[0159] Aspect 18 relates to the cell culture system of any one of aspects 1-17, wherein the cell culture substrate comprises a multilayer of substrate comprising at least a first substrate layer and a second substrate layer, wherein the first substrate layer comprises a first side and a second side opposite the first side, and the second substrate layer comprises a third side and a fourth side opposite the third side, the second side facing the third side.

[0160] Aspect 19 relates to the cell culture system of Aspect 18, wherein the multi-layered substrate is configured such that the first substrate layer has a predetermined arrangement relative to the second substrate layer.

[0161] Aspect 20 relates to the cell culture system of Aspect 19, wherein the multi-layered substrate is configured such that the fiber intersections on the first substrate layer face the openings in the second substrate layer.

[0162] Aspect 21 relates to the cell culture system of Aspect 19 or Aspect 20, wherein the openings in the first substrate layer at least partially overlap the openings in the second substrate layer.

[0163] Aspect 22 relates to the cell culture system of Aspect 21, wherein the openings in the first substrate layer and the openings in the second substrate layer are aligned.

[0164] Aspect 23 relates to the cell culture system of Aspect 18, wherein the multi-layered substrate is configured such that the first substrate layer has a random arrangement relative to the second substrate layer.

[0165] Aspect 24 relates to the cell culture system of any one of Aspects 1-23, wherein the cell culture matrix is disposed in a bioreactor vessel such that the overall flow direction of the culture media through the bioreactor vessel is parallel or perpendicular to the first side and the second side.

[0166] Aspect 25 relates to the cell culture system of any one of Aspects 1-24, wherein the cell culture matrix comprises a plurality of substrates randomly packed into the bioreactor vessel.

[0167] Aspect 26 relates to the cell culture system of any one of Aspects 1-25, wherein the bioreactor vessel is a packed bed bioreactor.

[0168] Aspect 27 relates to the cell culture system of any one of Aspects 1-26, wherein the bioreactor vessel comprises a culture space disposed within the bioreactor vessel and housing the cell culture matrix, one or more openings configured to provide fluid to or remove fluid from the culture space.

[0169] Aspect 28 relates to the cell culture system of Aspect 27, wherein the one or more openings comprise an inlet configured to provide fluid to an interior of the culture space and an outlet configured to remove fluid from the culture space of the bioreactor vessel.

[0170] Aspect 29 relates to the cell culture system of Aspect 28, wherein the bioreactor vessel comprises a first end comprising the inlet, a second end opposite the first end and comprising the outlet, the culture space disposed between the first end and the second end.

[0171] Aspect 30 relates to the cell culture system of Aspect 29, wherein the cell culture substrate has a shape that corresponds to a shape of the culture space.

[0172] Aspect 31 relates to the cell culture system of any one of Aspects 1-30, wherein the cell culture substrate comprises a polymeric mesh material in a cylindrical roll configuration.

[0173] Aspect 32 relates to the cell culture system of Aspect 31, wherein a central longitudinal axis of the cylindrical roll is parallel to a flow direction of the culture medium.

[0174] Aspect 33 relates to the cell culture system of Aspect 31 or Aspect 32, wherein the cylindrical roll is configured to expand into a shape of the culture space in the bioreactor vessel by unrolling of the cylindrical roll.

[0175] Aspect 34 relates to the cell culture system of any one of Aspects 31-33, wherein the cylindrical roll is configured to be inserted into the culture space when the cylindrical roll is in a collapsed state, and to expand within the culture space when disposed within the culture space.

[0176] Aspect 35 relates to the cell culture system of any one of Aspects 31-34, wherein the cylindrical roll and the culture space are configured such that a frictional force between the polymeric mesh material and a wall of the culture space holds the polymeric mesh material in place within the culture space.

[0177] Aspect 36 relates to the cell culture system of Aspect 34, wherein the cylindrical roll is configured to be inserted into the culture space through an opening in the bioreactor vessel.

[0178] Aspect 37 relates to the cell culture system of Aspect 36, wherein the opening is one of an inlet and an outlet of the bioreactor vessel.

[0179] Aspect 38 relates to the cell culture system of any one of Aspects 31-37, wherein the bioreactor vessel comprises a substrate support within the culture space, the substrate support configured to guide, align, or stabilize the cell culture substrate within the culture space.

[0180] Aspect 39 relates to the cell culture system of Aspect 38, wherein the substrate support comprises a support member extending from one of a first end or a second end toward the other of the first end or the second end, wherein the cylindrical roll is configured to enclose the support member such that the support member is parallel to a central longitudinal axis of the cylindrical roll.

[0181] Aspect 40 relates to the cell culture system of any one of Aspects 1-39, wherein the bioreactor vessel is configured to rotate about a central longitudinal axis of the bioreactor vessel during cell culture.

[0182] Aspect 41 is directed to the cell culture system of Aspect 40, wherein, during cell culturing, the central longitudinal axis is perpendicular to the direction of gravity.

[0183] Aspect 42 is directed to the cell culture system of Aspect 40 or Aspect 41, wherein the cell culture system is configured to move the substrates through the cell culture fluid during rotation of the bioreactor vessel.

[0184] Aspect 43 is directed to the cell culture system of any one of Aspects 40-42, wherein the cell culture system further comprises a rotation device operatively connected to the bioreactor vessel and configured to rotate the bioreactor vessel about the central longitudinal axis.

[0185] Aspect 44 is directed to the cell culture system of any one of Aspects 1-43, wherein the cell culture substrate comprises a plurality of substrates comprising woven meshes having different geometries, wherein the different geometries differ in at least one of fiber diameter, opening diameter, or opening geometry.

[0186] Aspect 45 is directed to the cell culture system of Aspect 44, wherein the woven meshes having different geometries are arranged in a predetermined arrangement in the bioreactor vessel based on desired flow characteristics within the bioreactor vessel.

[0187] Aspect 46 is directed to the cell culture system of Aspect 45, wherein the desired flow characteristics comprise at least one of: uniform perfusion of liquid culture media through the cell culture substrate, and distribution of cell growth across the cell culture substrate.

[0188] Aspect 47 is directed to the cell culture system of Aspect 45 or Aspect 46, wherein the woven meshes having different geometries comprise a first mesh having a first geometry and a second mesh having a second geometry, and wherein the predetermined arrangement comprises the first mesh being upstream of the second mesh relative to an overall flow direction.

[0189] Aspect 48 is directed to the cell culture system of Aspect 47, wherein the predetermined arrangement comprises a stack of the first mesh being arranged upstream of a stack of the second mesh.

[0190] Aspect 49 is directed to the cell culture system of Aspect 47 or Aspect 48, wherein the predetermined arrangement comprises stacks of the first mesh and stacks of the second mesh arranged in an alternating arrangement along the overall flow direction.

[0191] Aspect 50 is directed to the cell culture system of any one of Aspects 1-49, further comprising a device for harvesting adherent cells or cell byproducts.

[0192] Aspect 51 is directed to the cell culture system of Aspect 50, wherein the cell byproducts include at least one of a protein, an antibody, a virus, a viral vector, a virus-like particle (VLP), a microvesicle, an exosome, and a polysaccharide.

[0193] Aspect 52 is directed to the cell culture system of any one of Aspects 1-51, wherein the substrate includes a functionalized surface that is physically or chemically modified to improve adhesion of adherent cells to the polymeric mesh material.

[0194] Aspect 53 is directed to the cell culture system of any one of the preceding Aspects, wherein the cell culture matrix includes a surface configured to adsorb or absorb components in the culture medium onto the mesh surface.

[0195] Aspect 54 is directed to the cell culture system of any one of Aspects 1-53, wherein the cell culture matrix includes a coating on the surface of the polymeric mesh material configured to promote adhesion of adherent cells.

[0196] Aspect 55 is directed to the cell culture system of Aspect 54, wherein the cells adhere to the coating.

[0197] Aspect 56 is directed to the cell culture system of Aspect 54 or Aspect 55, wherein the coating is a biological or synthetic bioactive molecule configured to promote cell attachment to the cell culture matrix.

[0198] Aspect 57 is directed to the cell culture system of any one of Aspects 54-56, wherein the coating is at least one of a hydrogel, a collagen, a bioactive molecule or peptide, and a biological protein.

[0199] Aspect 58 is directed to the cell culture system of any one of Aspects 53-56, wherein the functionalized surface is plasma treated.

[0200] Aspect 59 is directed to the cell culture system of any one of the preceding Aspects, wherein the cells include at least one of adherent cells, suspended cells, and loosely adherent cells that adhere to the woven mesh.

[0201] Aspect 60 is directed to the cell culture system of any one of the preceding Aspects, further comprising a media conditioning vessel configured to supply culture medium to an inlet of the bioreactor vessel.

[0202] Aspect 61 is directed to a cell culture matrix comprising: a substrate including a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and through the thickness of the substrate, wherein the plurality of openings are configured to allow at least one of a cell culture medium, a cell, or a cell product to flow through the thickness of the substrate.

[0203] Aspect 62 relates to the cell culture substrate of Aspect 61, wherein the substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0204] Aspect 63 relates to the cell culture substrate of Aspect 61 or Aspect 62, wherein the substrate comprises at least one of a molded polymeric lattice sheet, a 3D-printed lattice sheet, and a woven mesh.

[0205] Aspect 64 relates to the cell culture substrate of Aspect 63, wherein the substrate comprises a woven mesh comprising one or more fibers.

[0206] Aspect 65 relates to the cell culture substrate of Aspect 64, wherein the one or more fibers comprise a cross-sectional shape that is at least one of oblong, circular, rectangular, or polygonal.

[0207] Aspect 66 relates to the cell culture substrate of Aspect 64 or Aspect 65, wherein the one or more fibers comprise at least one of a monofilament fiber and a multifilament fiber.

[0208] Aspect 67 relates to the cell culture substrate of any one of Aspects 64-66, wherein the one or more fibers comprise a first fiber having a first fiber diameter that is about 50 pm to about 1000 pm, about 50 pm to about 600 pm, about 50 pm to about 400 pm, about 100 pm to about 325 pm, or about 150 pm to about 275 pm.

[0209] Aspect 68 relates to the cell culture substrate of Aspect 67, wherein the one or more fibers further comprise a second fiber having a second fiber diameter that is about 50 pm to about 1000 pm, about 50 pm to about 600 pm, about 50 pm to about 400 pm, about 100 pm to about 325 pm, or about 150 pm to about 275 pm.

[0210] Aspect 69 relates to the cell culture substrate of Aspect 68, wherein the second fiber diameter is different than the first fiber diameter.

[0211] Aspect 70 relates to the cell culture substrate of any one of Aspects 61-69, wherein the plurality of openings comprises an opening diameter that is about 100 pm to about 1000 pm, about 200 pm to about 900 pm, or about 225 pm to about 800 pm.

[0212] Aspect 71 is directed to the cell culture substrate of Aspect 70, wherein the fiber diameter is about 250 pm to about 300 pm and the opening diameter is about 750 pm to about 800 pm, or wherein the fiber diameter is about 270 pm to about 276 pm and the opening diameter is about 785 pm to about 795 pm.

[0213] Aspect 72 is directed to the cell culture substrate of Aspect 70, wherein the fiber diameter is about 200 pm to about 230 pm and the opening diameter is about 500 pm to about 550 pm, or wherein the fiber diameter is about 215 pm to about 225 pm and the opening diameter is about 515 pm to about 530 pm.

[0214] Aspect 73 is directed to the cell culture substrate of Aspect 70, wherein the fiber diameter is about 125 pm to about 175 pm and the opening diameter is about 225 pm to about 275 pm, or wherein the fiber diameter is about 150 pm to about 165 pm and the opening diameter is about 235 pm to about 255 pm.

[0215] Aspect 74 is directed to the cell culture substrate of any one of Aspects 70-73, wherein the ratio of the opening diameter to the fiber diameter is about 1.0 to about 3.5, about 1.25 to about 3.25, about 1.4 to about 3.0, about 1.5 to about 2.9, about 1.5 to about 2.4, or about 2.4 to about 2.9.

[0216] Aspect 75 is directed to the cell culture substrate of any one of Aspects 1-74, wherein the plurality of openings comprises openings that are square, rectangular, rhombus, long diagonal, circular, or elliptical in shape.

[0217] Aspect 76 is directed to the cell culture substrate of any one of Aspects 1-75, wherein the plurality of openings are arranged in a regular pattern.

[0218] Aspect 77 is directed to the cell culture substrate of any one of Aspects 1-76, wherein the cell culture substrate comprises a single layer of substrate.

[0219] Aspect 78 is directed to the cell culture substrate of any one of Aspects 1-77, wherein the cell culture substrate comprises a multi-layer substrate comprising at least a first substrate layer and a second substrate layer, wherein the first substrate layer comprises a first side and a second side opposite the first side, and the second substrate layer comprises a third side and a fourth side opposite the third side, the second side facing the third side.

[0220] Aspect 79 is directed to the cell culture substrate of Aspect 78, wherein the multi-layer substrate is configured such that the first substrate layer has a predetermined arrangement relative to the second substrate layer.

[0221] Aspect 80 is directed to the cell culture substrate of Aspect 79, wherein the multi-layered substrate is configured such that the fiber intersections on the first substrate layer face the openings in the second substrate layer.

[0222] Aspect 81 is directed to the cell culture substrate of Aspect 79 or Aspect 80, wherein the openings in the first substrate layer at least partially overlap the openings in the second substrate layer.

[0223] Aspect 82 is directed to the cell culture substrate of Aspect 81, wherein the openings in the first substrate layer and the openings in the second substrate layer are aligned.

[0224] Aspect 83 is directed to the cell culture substrate of Aspect 78, wherein the multi-layered substrate is configured such that the first substrate layer has a random arrangement relative to the second substrate layer.

[0225] Aspect 84 is directed to the cell culture substrate of any of Aspects 61-83, wherein the cell culture substrate comprises a plurality of substrates, each substrate of the plurality of substrates being in a random orientation relative to other substrates of the plurality of substrates.

[0226] Aspect 85 is directed to the cell culture substrate of any of Aspects 61-83, wherein the cell culture substrate comprises a plurality of substrates in a stacked arrangement.

[0227] Aspect 86 is directed to the cell culture substrate of Aspect 85, wherein the first side and the second side of one substrate of the plurality of substrates are substantially parallel to the first side and the second side of other substrates in the stacked arrangement.

[0228] Aspect 87 is directed to the cell culture substrate of any of Aspects 61-83, wherein the substrate is in a cylindrical roll configuration.

[0229] Aspect 88 is directed to the cell culture substrate of Aspect 87, wherein the cylindrical roll is configured to expand within a bioreactor vessel into a shape of a culture chamber by uncoiling of portions of the cylindrical roll when disposed within the culture chamber.

[0230] Aspect 89 is directed to the cell culture substrate of Aspect 88, wherein the cylindrical roll is configured to be inserted into a culture space when the cylindrical roll is in a collapsed state, and to expand within the culture space when disposed within the culture space.

[0231] Aspect 90 is directed to the cell culture substrate of any of Aspects 61-89, wherein the cell culture substrate comprises a plurality of substrates, the plurality of substrates comprising woven meshes having different geometries, wherein the different geometries differ in at least one of fiber diameter, opening diameter, or opening geometry.

[0232] Aspect 91 relates to the cell culture substrate of Aspect 90, wherein the woven meshes having different geometries are arranged in a predetermined arrangement based on desired flow characteristics within the bioreactor vessel.

[0233] Aspect 92 relates to the cell culture substrate of Aspect 91, wherein the desired flow characteristics include at least one of: uniform perfusion of liquid media through the cell culture substrate, and distribution of cell growth across the cell culture substrate.

[0234] Aspect 93 relates to the cell culture substrate of Aspect 91 or Aspect 92, wherein the woven meshes having different geometries include a first mesh having a first geometry and a second mesh having a second geometry, and wherein the predetermined arrangement includes the first mesh being upstream of the second mesh relative to a desired overall flow direction of the cell culture media through the cell culture substrate.

[0235] Aspect 94 relates to the cell culture substrate of Aspect 93, wherein the predetermined arrangement includes a stack of the first mesh disposed upstream of a stack of the second mesh.

[0236] Aspect 95 relates to the cell culture substrate of Aspect 93 or Aspect 94, wherein the predetermined arrangement includes stacks of the first mesh and stacks of the second mesh in an alternating arrangement along the overall flow direction.

[0237] Aspect 96 relates to the cell culture substrate of any one of Aspects 61-95, wherein the cell culture substrate is configured for culturing and / or harvesting at least one of: cells, proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvessicles, exosomes, and polysaccharides.

[0238] Aspect 97 relates to the cell culture substrate of any one of Aspects 61-96, wherein the substrate includes a functionalized surface that is physically or chemically modified to improve adhesion of adherent cells to the polymer mesh material.

[0239] Aspect 98 relates to the cell culture substrate of any one of Aspects 61-97, wherein the cell culture substrate includes a surface configured to adsorb or absorb components in the culture media onto the mesh surface.

[0240] Aspect 99 relates to the cell culture substrate of any one of Aspects 61-98, wherein the cell culture substrate includes a coating on the surface of the polymer mesh material configured to promote adhesion of adherent cells.

[0241] Aspect 100 relates to the cell culture substrate of Aspect 99, wherein cells adhere to the coating.

[0242] Aspect 101 is directed to the cell culture substrate of Aspect 99 or Aspect 100, wherein the coating is a biological or synthetic bioactive molecule configured to promote cell attachment to the cell culture substrate.

[0243] Aspect 102 is directed to the cell culture substrate of any one of Aspects 99-101, wherein the coating is at least one of a hydrogel, collagen, a bioactive molecule or peptide, and a biological protein.

[0244] Aspect 103 is directed to the cell culture substrate of any one of Aspects 99-102, wherein the functionalized surface is plasma treated.

[0245] Aspect 104 is directed to the cell culture substrate of any one of Aspects 61-103, wherein the cells comprise at least one of adherent cells, suspension cells, and loosely adherent cells adhered to the woven mesh.

[0246] Aspect 105 is directed to a method of culturing cells in a bioreactor, the method comprising: providing a bioreactor vessel, the bioreactor vessel comprising: a cell culture chamber within the bioreactor vessel, and a cell culture substrate disposed in the cell culture chamber and configured for culturing cells thereon, the cell culture substrate comprising a substrate, the substrate comprising a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and through the thickness of the substrate; seeding cells on the cell culture substrate; culturing the cells on the cell culture substrate; and harvesting a product of the cell culture, wherein the plurality of openings in the substrate are configured to allow at least one of a cell culture medium, a cell, or a cell product to flow through the thickness of the substrate.

[0247] Aspect 106 is directed to the method of Aspect 105, wherein the substrate comprises at least one of a molded polymeric lattice sheet, a 3D printed lattice sheet, and a woven mesh sheet.

[0248] Aspect 107 is directed to the method of Aspect 105 or Aspect 106, wherein the substrate comprises a polymeric material.

[0249] Aspect 108 is directed to the method of Aspect 107, wherein the polymeric material is at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0250] Aspect 109 is directed to the method of any one of Aspects 105-108, wherein the seeding comprises allowing the cells to attach to the substrate.

[0251] Aspect 110 relates to the method of any one of aspects 105-109, wherein the seeding comprises injecting the cell inoculum directly into the cell culture matrix.

[0252] Aspect 111 relates to the method of aspect 110, wherein the cell inoculum is injected through a cell inoculum injection port in the bioreactor vessel.

[0253] Aspect 112 relates to the method of aspect 110 or aspect 111, wherein the volume of the cell inoculum is about equal to the void volume of the cell culture chamber.

[0254] Aspect 113 relates to the method of any one of aspects 110-112, further comprising: after injecting the cell inoculum, perfusing the cell culture media through the culture chamber.

[0255] Aspect 114 relates to the method of any one of aspects 105-113, further comprising: during the culturing, supplying the cells with at least one of cell culture media and oxygen.

[0256] Aspect 115 relates to the method of aspect 114, wherein the supplying the cell culture media comprises flowing the cell culture media through the cell culture chamber and over the substrate.

[0257] Aspect 116 relates to the method of aspect 114 or aspect 115, wherein the supplying the cell culture media comprises providing a media conditioning vessel that is fluidly connected to the bioreactor vessel and supplying the cell culture media from the media conditioning vessel to the bioreactor vessel.

[0258] Aspect 117 relates to the method of aspect 116, wherein during or after the culturing, at least a portion of the media is recovered from the bioreactor vessel and returned to the media conditioning vessel.

[0259] Aspect 118 relates to the method of any one of aspects 105-117, further comprising: controlling the flow of cell culture media to the cell culture chamber, wherein the cell culture media comprises at least one of cells, cell culture nutrients, or oxygen.

[0260] Aspect 119 relates to the method of any one of aspects 105-118, further comprising: analyzing the cell culture media, cells, and / or cell products within or output from the bioreactor vessel.

[0261] Aspect 120 relates to the method of Aspect 119, wherein the analyzing comprises measuring at least one of pH1, pO1, [glucose]1, pH2, pO2, [glucose]2, and flow rate, wherein pH1, pO1, and [glucose]1 are measured within the cell culture chamber, and wherein pH2, pO2, and [glucose]2 are measured at an outlet of the cell culture chamber or bioreactor vessel.

[0262] Aspect 121 relates to the method of Aspect 119 or Aspect 120, wherein the cell culture media flow to the cell culture chamber is controlled based at least in part on the results of analyzing the cell culture media, cells, and / or cell products.

[0263] Aspect 122 relates to the method of any one of Aspects 120-121, wherein if at least one of pH2≥ pH 2最小 , pO2≥ pO 2最小 , and [glucose]2≥ [glucose] 2最小 is satisfied, the perfusion flow rate of the cell culture media to the cell culture chamber continues at the current rate, wherein pH 2最小 , pO 2最小 , and [glucose] 2最小 are predetermined based on the design of the cell culture system.

[0264] Aspect 123 relates to the method of any one of Aspects 120-122, wherein the perfusion flow rate is increased if the current flow rate is less than or equal to a predetermined maximum flow rate of the cell culture system.

[0265] Aspect 124 relates to the method of any one of Aspects 120-123, wherein if the current flow rate is not less than or equal to a predetermined maximum flow rate of the cell culture system, a controller of the cell culture system reevaluates at least one of pH 2最小 , pO 2最小 , and [glucose] 2最小 ; pH1, pO1, and [glucose]1; and a height of the bioreactor vessel.

[0266] Aspect 125 relates to the method of any one of Aspects 105-124, wherein the cells have a viability of greater than about 90% or greater than about 95% after being cultured for at least about 24 hours, at least about 48 hours, or at least about 72 hours.

[0267] Aspect 126 relates to the method of any one of Aspects 105-125, wherein the cells comprise at least one of adherent cells, suspension cells, and loosely adherent cells adhered to a cell culture substrate.

[0268] Aspect 127 relates to the method of any one of aspects 105-126, wherein the product of the cell culture comprises at least one of cells, proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0269] Aspect 128 relates to the method of aspect 127, wherein the product of the cell culture comprises cells that are at least 80% viable, at least 85% viable, at least 90% viable, at least 91% viable, at least 92% viable, at least 93% viable, at least 94% viable, at least 95% viable, at least 96% viable, at least 97% viable, at least 98% viable, or at least 99% viable.

[0270] Aspect 129 relates to a bioreactor system comprising: a cell culture vessel comprising at least one reservoir; and a cell culture matrix disposed in the at least one reservoir, the cell culture matrix comprising a woven substrate, the woven substrate having a plurality of interwoven fibers, and a surface of the fibers configured to allow cells to adhere thereto.

[0271] Aspect 130 relates to the system of aspect 129, wherein the woven substrate comprises a uniform arrangement of the plurality of interwoven fibers.

[0272] Aspect 131 relates to the system of aspect 129 or aspect 130, wherein the woven substrate comprises a plurality of openings disposed between the plurality of fibers.

[0273] Aspect 132 relates to the system of any one of aspects 129-131, wherein the plurality of fibers comprises polymeric fibers.

[0274] Aspect 133 relates to the system of aspect 132, wherein the polymeric fibers comprise at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0275] Aspect 134 relates to the system of any one of aspects 129-133, wherein the cell culture matrix comprises a plurality of woven substrates.

[0276] Aspect 135 relates to the system of aspect 134, wherein each substrate of the plurality of substrates comprises a first side, a second side opposite the first side, a thickness separating the first side and the second side, wherein the plurality of openings pass through the thickness of the substrate.

[0277] Aspect 136 relates to the system of aspect 134 or aspect 135, wherein a substrate of the plurality of substrates is arranged adjacent to one another such that one of the first side and the second side of the substrate is adjacent to the other of the first side or the second side of an adjacent substrate.

[0278] Aspect 137 relates to the system of any one of aspects 134-136, wherein at least a portion of the plurality of substrates are not separated by a spacer material or barrier.

[0279] Aspect 138 relates to the system of any one of aspects 134-137, wherein at least a portion of the plurality of substrates are in physical contact with each other.

[0280] Aspect 139 relates to the system of any one of aspects 129-138, wherein the cell culture vessel comprises at least one port configured to supply material to or remove material from the at least one reservoir through the at least one port.

[0281] Aspect 140 relates to the system of aspect 139, wherein the at least one port comprises at least one inlet for supplying material to the at least one reservoir and at least one outlet for removing material from the at least one reservoir.

[0282] Aspect 141 relates to the system of aspect 140, wherein the material comprises at least one of a culture medium, cells, or cell products.

[0283] Aspect 142 relates to a bioreactor system comprising: a cell culture vessel comprising a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed in the at least one reservoir, the cell culture matrix comprising a plurality of woven substrates, each woven substrate comprising a plurality of interwoven fibers, and a surface of the fibers configured for cell adhesion, wherein the bioreactor system is configured to flow material through the at least one reservoir in a flow direction from the first end to the second end, wherein the substrates of the plurality of woven substrates are stacked such that each woven substrate is substantially parallel to each other woven substrate and substantially perpendicular to the flow direction.

[0284] Aspect 143 relates to the system of aspect 142, wherein each of the substrates comprises a first side, a second side opposite the first side, a thickness separating the first side and the second side, wherein a plurality of openings pass through the thickness of the substrate.

[0285] Aspect 144 relates to a bioreactor system comprising: a cell culture vessel comprising a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed in the at least one reservoir, the cell culture matrix comprising a plurality of woven substrates, each woven substrate comprising a plurality of interwoven fibers, and a surface of the fibers configured for cell adhesion, wherein the bioreactor system is configured to flow material through the at least one reservoir in a flow direction from the first end to the second end, wherein the substrates of the plurality of woven substrates are stacked such that each woven substrate is substantially parallel to each other woven substrate and substantially parallel to the flow direction.

[0286] Aspect 145 relates to the system of Aspect 144, wherein each of the substrates comprises a first side, a second side opposite the first side, a thickness separating the first side and the second side, wherein a plurality of openings pass through the thickness of the substrate.

[0287] Aspect 146 relates to a bioreactor system comprising: a cell culture vessel comprising a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed in the at least one reservoir, the cell culture matrix comprising a woven substrate, the woven substrate comprising a plurality of interwoven fibers, and a surface of the fibers configured for cell adhesion, and wherein at least one of the at least one reservoir and the cell culture matrix is configured to rotate about a central longitudinal axis of the bioreactor vessel during cell culture.

[0288] Aspect 147 relates to the system of Aspect 146, wherein the woven substrate is disposed in the at least one reservoir as a cylindrical substrate, the cylindrical substrate at least partially encircling the central longitudinal axis of the bioreactor vessel.

[0289] Aspect 148 relates to the system of Aspect 146 or Aspect 147, wherein the bioreactor system is configured to flow material through the at least one reservoir in a flow direction from the first end to the second end.

[0290] Aspect 149 relates to the system of Aspect 148, wherein the central longitudinal axis of the cylindrical substrate is parallel to the flow direction of the culture media.

[0291] Aspect 150 relates to the system of any of Aspects 146-149, wherein the cylindrical substrate comprises a roll of woven substrate configured to expand into contact with a wall of the at least one reservoir by unrolling the roll of woven substrate.

[0292] Aspect 151 relates to the system of any of Aspects 146-150, wherein the roll of woven substrate is configured to expand in the cell culture vessel into a shape of an interior of the at least one reservoir.

[0293] Aspect 152 relates to the system of Aspect 151, wherein the rolled woven substrate is configured to be inserted into the culture space when the rolled woven substrate is in a collapsed roll state, and to expand within the reservoir when disposed within the reservoir.

[0294] Aspect 153 relates to the system of Aspect 151 or Aspect 152, wherein the rolled woven substrate and the reservoir are configured such that a frictional force between the woven substrate and the reservoir wall substantially holds the woven substrate in place within the reservoir.

[0295] Aspect 154 relates to the system of any one of Aspects 151-153, wherein the rolled woven substrate is configured to be inserted into the reservoir through an opening in the cell culture vessel.

[0296] Aspect 155 relates to the system of Aspect 154, wherein the opening is one of an inlet and an outlet of the cell culture vessel.

[0297] Aspect 156 relates to the system of any one of Aspects 146-155, wherein the cell culture vessel comprises a substrate support positioned within the reservoir, the substrate support configured to guide, align, or stabilize the woven substrate within the culture space.

[0298] Aspect 157 relates to the system of Aspect 156, wherein the substrate support comprises a support member extending from one of the first end or the second end to the other of the first end or the second end, wherein the rolled woven substrate is configured to surround at least a portion of a perimeter of the support member such that the support member is parallel to a central longitudinal axis of the rolled woven substrate.

[0299] Aspect 158 relates to the system of any one of Aspects 146-157, wherein the central longitudinal axis is perpendicular to a direction of gravity during cell culturing.

[0300] Aspect 159 relates to the system of any one of Aspects 146-158, wherein the bioreactor system is configured to move the substrate through the cell culture fluid during rotation of the cell culture vessel.

[0301] Aspect 160 relates to the system of any one of Aspects 146-159, wherein the bioreactor system further comprises a rotation device operatively connected to the cell culture vessel and configured to rotate the cell culture vessel about the central longitudinal axis.

[0302] Aspect 161 relates to a cell culture substrate, comprising: a woven substrate comprising a plurality of fibers interwoven and a plurality of openings disposed between the plurality of fibers, wherein the fibers each comprise a surface configured to support cell adhesion.

[0303] Aspect 162 relates to the substrate of Aspect 161, wherein the surface of the fiber is configured for cells to releasably adhere thereto.

[0304] Aspect 163 relates to the substrate of Aspect 161 or Aspect 162, wherein the plurality of fibers comprises polymeric fibers.

[0305] Aspect 164 relates to the substrate of Aspect 163, wherein the polymeric fibers comprise at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0306] Aspect 165 relates to the substrate of any one of Aspects 161-164, wherein the cell culture substrate further comprises a plurality of woven substrates.

[0307] Aspect 166 relates to the substrate of Aspect 165, wherein each substrate of the plurality of substrates comprises a first side, a second side opposite the first side, a thickness separating the first side and the second side, wherein the plurality of openings pass through the thickness of the substrate.

[0308] Aspect 167 relates to the substrate of Aspect 165 or Aspect 166, wherein the substrates of the plurality of substrates are arranged adjacent to one another such that one of the first side and the second side of a substrate is adjacent to the other of the first side or the second side of an adjacent substrate.

[0309] Aspect 168 relates to the substrate of any one of Aspects 165-167, wherein at least a portion of the plurality of substrates is not separated by a spacer material or a barrier.

[0310] Aspect 169 relates to the substrate of any one of Aspects 165-168, wherein at least a portion of the plurality of substrates are in physical contact with one another.

[0311] Aspect 170 relates to a bioreactor system comprising: a vessel comprising a media inlet, a media outlet, and a cell culture space disposed in an interior of the vessel and in fluid communication with and between the media inlet and the media outlet; wherein the cell culture space comprises a cell culture substrate region and a spacer region disposed between the cell culture substrate region and the media outlet, and wherein the cell culture space is configured to house a cell culture substrate in a packed bed configuration.

[0312] Aspect 171 relates to the bioreactor system of Aspect 170, wherein the cell culture substrate comprises the cell culture substrate of any one of Aspects 61-104.

[0313] Aspect 172 is directed to the bioreactor system of Aspect 170 or Aspect 171, further comprising a flow distribution plate disposed between the media inlet and the cell culture space.

[0314] Aspect 173 is directed to the bioreactor system of any one of Aspects 170-172, further comprising a packed bed retention layer disposed between the cell culture space and the spacer region.

[0315] Aspect 174 is directed to the bioreactor system of any one of Aspects 170-173, further comprising a spacer insert disposed between the media outlet and the cell culture space.

[0316] Aspect 175 is directed to the bioreactor system of Aspect 174, wherein the spacer insert is disposed between the media outlet and the packed bed retention layer.

[0317] Definitions

[0318] “Fully synthetic” or “total synthesis” means that the cell culture article (e.g., microcarriers or the surface of a culture vessel) is composed entirely of synthetic source materials and does not have any materials of animal origin or animal-derived materials. The fully synthetic cell culture articles disclosed eliminate the risk of xeno-contamination.

[0319] “Comprise,”“comprising,” and like terms mean to include, but not to be limited to.

[0320] According to embodiments herein,“user” refers to a person using the systems, methods, articles of manufacture, or kits disclosed herein, including a person culturing cells to harvest cells or cell products, or a person using the cultured and / or harvested cells or cell products.

[0321] “About” as used to describe a quantity, a dimension, a range of values, or the like, or a dimension of a component, refers to a variation that can occur in, for example: typical measuring and handling procedures used in the preparation of materials, compositions, compounds, concentrates, component parts, articles of manufacture, or application formulations; unintentional error; variations in the manufacturing, sourcing, or purity of the raw materials or components used to practice the described methods; and like considerations. The term“about” also includes amounts that vary due to differences in the amount of composition or formulation that has been applied, or due to differences in the initial concentration or mixture of the composition or formulation.

[0322] “Optional” or“optionally” means that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0323] As used herein, the indefinite article "a" or "an" and its corresponding plural forms mean "at least one" or one or more unless otherwise indicated.

[0324] Abbreviations familiar to those skilled in the art can be employed (e.g., "h" or "hrs" for hour(s), "g" or "gm" for gram(s), "mL" for milliliter(s), "rt" for room temperature, "nm" for nanometer(s), and the like).

[0325] The specific and preferred values and ranges of values disclosed herein for components, ingredients, additives, dimensions, conditions and like aspects are for illustration only and do not exclude other defined values or other values within defined ranges. The systems, kits and methods of the disclosure can include any of the values described herein or any combination of values, including intermediate values and ranges, not expressly stated herein.

[0326] Unless otherwise stated, no aspect of any method described herein is intended to require its steps to be performed in a particular order. Hence, when a method claim recites steps in a particular order, that order is not intended to be the only order in which the steps can be performed, unless the claim states otherwise. No language in the specification should be construed as implying any particular order to the steps of any method.

[0327] It will be apparent to those skilled in the art that various modifications and variations can be made in the disclosed embodiments 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 disclosure can occur to those skilled in the art, it is intended that the disclosure shall not be limited to the particular embodiments described in the specification but that the disclosure will include all embodiments falling within the scope of the appended claims and their equivalents.

Claims

1. A fixed-bed bioreactor system, comprising: A container, the container including a culture medium inlet, a culture medium outlet, and an internal chamber disposed between the culture medium inlet and the culture medium outlet and in fluid communication with the culture medium inlet and the culture medium outlet; and A cell culture medium, wherein the cell culture medium is located in an internal chamber between a culture medium inlet and a culture medium outlet in a filled bed configuration, the cell culture medium comprising a plurality of porous discs arranged in a stacked manner, wherein the plurality of porous discs are in direct physical contact with each other and are stacked together. Each of the plurality of porous discs includes a surface configured to culture cells on the surface. The fixed-bed bioreactor system also includes a spacer disposed in an internal chamber between the cell culture medium and the culture medium outlet. The internal chamber includes a cell culture zone and a spacer zone. The cell culture medium defines the cell culture zone, and the spacer zone is located between the cell culture zone and the culture medium outlet. The spacer is configured to separate the cell culture medium from the culture medium outlet and confine the cell culture medium to the cell culture area of ​​the internal chamber. The spacer includes a plurality of spacer members extending in a direction parallel to the length of the spacer region. The plurality of porous discs include a plurality of woven mesh layers. Each of the plurality of porous disks contains fibers with a diameter of 50 μm to 1000 μm. Each of the plurality of porous disks includes a plurality of openings formed in the disk and extending through the thickness of the disk. The plurality of openings have an opening diameter of 100 μm to 1000 μm, and The plurality of openings are arranged in a regular pattern.

2. The fixed-bed bioreactor system of claim 1, further comprising a packed-bed retainer disposed between the cell culture medium and the spacer, the packed-bed retainer being configured to provide structural support to the top of the cell culture medium.

3. The fixed-bed bioreactor system as described in claim 2, wherein, The filled bed retainer is porous, essentially rigid, and extends over most of the width of the internal chamber.

4. The fixed-bed bioreactor system as described in claim 1, wherein, Each of the plurality of porous disks includes a first side, a second side opposite to the first side, and a disk thickness separating the first side and the second side. The plurality of openings are arranged to allow at least one of cell culture medium, cells, or cell byproducts to flow through the cell culture medium.

5. The fixed-bed bioreactor system of claim 1, further comprising an inlet distribution plate disposed between the culture medium inlet and the cell culture medium substrate, the inlet distribution plate being configured to distribute fluid entering the internal chamber from the culture medium inlet over the area of ​​the cell culture medium substrate.

6. The fixed-bed bioreactor system of claim 1, further comprising an outlet distribution plate disposed between the cell culture medium and the culture medium outlet.

7. The fixed-bed bioreactor system of claim 1, further comprising a packed-bed retainer disposed between the cell culture medium and the spacer area, the packed-bed retainer being configured to provide structural support to the top of the cell culture medium.

8. The fixed-bed bioreactor system as described in claim 7, wherein, The filled bed retainer is porous, essentially rigid, and extends over most of the width of the internal chamber.

9. The fixed-bed bioreactor system of claim 7, further comprising a spacer disposed in an internal chamber between the packed bed retainer and the culture medium outlet, and defining a spacer region between the packed bed retainer and the culture medium outlet, the spacer being configured to space the cell culture medium from the culture medium outlet and to confine the cell culture medium to a cell culture area within the internal chamber.

10. The fixed-bed bioreactor system of claim 7, wherein, The filled bed retainer is a rigid grid structure.

11. The fixed-bed bioreactor system as described in claim 1, wherein, The spacer has an adjustable length and is configured to be adjustable to maintain various predetermined distances between the cell culture zone and the culture medium outlet, thereby allowing the number of porous discs in the cell culture medium that can be contained in the cell culture zone to vary.

12. The fixed-bed bioreactor system of claim 1, further comprising a plurality of removable spacers of different lengths, each of the plurality of removable spacers being placed in a spacer area to maintain a predetermined distance between the cell culture zone and the culture medium outlet, the predetermined distance being different from the distance maintained by the other spacers, thereby allowing the number of porous discs in the cell culture medium that can be accommodated in the cell culture zone to be varied based on the length of the spacers disposed in the spacer area.

13. The fixed-bed bioreactor system of claim 1, further comprising at least one porous spacer disc disposed between the cell culture medium and the culture medium inlet.

14. The fixed-bed bioreactor system of claim 13, wherein, The at least one porous spacer disk includes a spacer disk hole diameter, and each of the plurality of porous disks has a disk hole diameter such that the spacer disk hole diameter is larger than the disk hole diameter.

15. The fixed-bed bioreactor system of claim 13, wherein, The at least one porous spacer disk includes a first disk having a first spacer disk hole diameter and a second disk having a second spacer disk hole diameter, wherein the first spacer disk hole diameter is different from the second spacer disk hole diameter.

16. The fixed-bed bioreactor system of claim 1, wherein, Each of the plurality of woven web layers has a defined, substantially uniform array of holes.

17. The fixed-bed bioreactor system of claim 1, wherein, Each of the plurality of woven web layers includes a plurality of interwoven fibers, the plurality of interwoven fibers including a first group of fibers traveling parallel to each other in a first direction and a second group of fibers traveling parallel to each other in a second direction.

18. The fixed-bed bioreactor system of claim 17, wherein, The first direction is basically perpendicular to the second direction.

19. The fixed-bed bioreactor system of claim 17, wherein, The web consists of multiple interwoven fibers, which are composed of a first group of fibers and a second group of fibers.

20. The fixed-bed bioreactor system of claim 1, wherein, The culture medium inlet is configured to supply at least one of cells and cell culture medium to the internal chamber before or during cell culture, and the culture medium outlet is configured to recover at least one of cells, cell culture medium, and cell byproducts from the internal chamber during or after cell culture.

21. The fixed-bed bioreactor system of claim 20, wherein, The culture medium outlet is configured to supply pressurized fluid to the spacer area during harvesting operations, and the culture medium inlet is configured to recover at least one of cells, cell culture medium, and cell byproducts from the internal chamber during harvesting operations.

22. The fixed-bed bioreactor system of claim 21, wherein, The bioreactor system is configured to fill an internal chamber with pressurized fluid via a culture medium outlet to expel at least one of cells, cell culture medium, and cell byproducts via a culture medium inlet.

23. The fixed-bed bioreactor system as described in claim 1, wherein, The plurality of porous disks includes 50 to 1000 porous disks.

24. The fixed-bed bioreactor system of claim 23, wherein, The plurality of porous disks includes 100 to 500 porous disks.

25. The fixed-bed bioreactor system as described in claim 1, wherein, The plurality of porous discs are configured to culture cells to 1.00 × 10⁻⁶. 7 Cells / disc up to 2.00 × 10⁻⁶ 7 Average density per cell / disc.

26. The fixed-bed bioreactor system of claim 1, wherein, The fibers define pores between the fibers, and the pores have a diameter of 100 μm to 1000 μm.

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