Fixed bed bioreactor for cell culture with reduced bypass

Abstract

A bioreactor system for culturing cells is provided that include a cell culture vessel having an inlet, outlet, and interior reservoir. The cell culture matrix includes a plurality of substrates for adhering cells thereto, which are stacked in a stacking direction parallel to a flow direction through the reservoir. The cell culture matrix extends across the width of the reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted. The substrates can be under compression with a compression force in a direction that is substantially parallel to the stacking direction and substantially perpendicular to the reservoir width to reduce bypass fluid flow between the cell culture matrix and the reservoir wall. Edges of some of the substrates can also be joined to increase rigidity against fluid flow and thus reduce bypass.

Description

FIXED BED BIOREACTOR FOR CELL CULTURE WITH REDUCED BYPASSCROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 654,623 filed on May 31, 2024, the content of which is relied upon and incorporated herein by reference in its entirety their entireties.FIELD OF THE DISCLOSURE

[0002] This disclosure general relates to cell culture substrates and perfusion-based, fixed-bed bioreactors incorporating such substrates, as well as systems and methods for culturing cells. In particular, the present disclosure relates to fixed bed cell culture substrates and bioreactors with such substrates with reduced bypass around the fixed bed under perfusion cell culture conditionsBACKGROUND

[0003] In the bioprocessing industry, large-scale cultivation of cells is performed for purposes of the production of hormones, enzymes, antibodies, vaccines, and cell therapies. Cell and gene therapy markets are growing rapidly, with promising treatments moving into clinical trials and quickly toward commercialization. However, one cell therapy dose can require billions of cells or trillions of viruses. As such, being able to provide a large quantity of cell products in a short amount of time is critical for clinical success.

[0004] A significant portion of the cells used in bioprocessing are anchorage dependent, meaning the cells need a surface to adhere to for growth and functioning. Traditionally, the culturing of adherent cells is performed on two-dimensional (2D) cell-adherent surfaces incorporated in one of a number of vessel formats, such as T-flasks, petri dishes, cell factories, cell stack vessels, roller bottles, and HYPERStack® vessels. These approaches can havesignificant drawbacks, including the difficulty in achieving cellular density high enough to make it feasible for large scale production of therapies or cells.

[0005] Alternative methods have been suggested to increase volumetric density of cultured cells. These include microcarrier cultures performed in stir tanks. In this approach, cells that are attached to the surface of microcarriers are subject to constant shear stress, resulting in a significant impact on proliferation and culture performance. Another example of a high-density cell culture system is a hollow fiber bioreactor, in which cells may form large three-dimensional aggregates as they proliferate in the interspatial fiber space. However, the cells growth and performance are significantly inhibited by the lack nutrients. To mitigate this problem, these bioreactors are made small and are not suitable for large scale manufacturing.

[0006] Another example of a high-density culture system for anchorage dependent cells is a packed-bed bioreactor system. In this this type of bioreactor, a cell substrate is used to provide a surface for the attachment of adherent cells. Medium is perfused along the surface or through the semi-porous substrate to provide nutrients and oxygen needed for the cell growth. For example, packed bed bioreactor systems that contain a packed bed of support or matrix systems to entrap the cells have been previously disclosed U.S. Patent Nos. 4,833,083; 5,501,971; and 5,510,262. Packed bed matrices usually are made of porous particles as substrates or non-woven microfibers of polymer. Such bioreactors can function as recirculation flow-through bioreactors. One of the significant issues with such bioreactors is the non-uniformity of cell distribution inside the packed bed. The packed bed often functions as depth filter with cells predominantly trapped at the inlet regions, resulting in a gradient of cell distribution during the inoculation step. In addition, due to random fiber packaging, flow resistance and cell trapping efficiency of cross sections of the packed bed are not uniform. For example, medium flows fast though the regions with low cell packing density and flows slowly through the regions where resistance is higher due to higher number of entrapped cells. This creates a channeling effect where nutrients and oxygen are delivered more efficiently to regions with lower volumetric cells densities and regions with higher cell densities are being maintained in suboptimal culture conditions.

[0007] Another significant drawback of packed bed systems disclosed in a prior art is the inability to efficiently harvest intact viable cells at the end of culture process. Harvesting of cellsis important if the end product is cells, or if the bioreactor is being used as part of a “seed train,” where a cell population is grown in one vessel and then transferred to another vessel for further population growth. U.S. Patent No. 9,273,278 discloses a bioreactor design to improve the efficiency of cell recovery from the packed bed during cells harvesting step. It is based on loosening the packed bed matrix and agitation or stirring of packed bed particles to allow porous matrices to collide and thus detach the cells. However, this approach is laborious and may cause significant cells damage, thus reducing overall cell viability.

[0008] Certain examples of packed-bed bioreactors currently on the market use small strips of cell substrate material consisting of randomly oriented fibers in a non-woven arrangement. These strips are randomly packed into a vessel to create a packed bed. However, the non-uniform packing of the substrate strips creates visible channels within the packed bed, leading to preferential and non-uniform media flow and nutrient distribution through the packed bed. Studies of such commercially available bioreactors have noted a “systemic inhomogeneous distribution of cells, with their number increasing from top to bottom of fixed bed,” as well as a “nutrient gradient... leading to restricted cell growth and production,” all of which lead to the “unequal distribution of cells [that] may impair transfection efficiency.” (Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells. BiotechnoL J. 2016, 11, 290-297). Studies have noted that agitation of the packed bed may improve dispersion, but would have other drawbacks (i.e., “necessary agitation for better dispersion during inoculation and transfection would induce increased shear stress, in turn leading to reduced cell viability.” Id.). Another study noted that the uneven distribution of cells makes monitoring of the cell population using biomass sensors difficult (“... if the cells are unevenly distributed, the biomass signal from the cells on the top carriers may not show the general view of the entire bioreactor.” Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale.Human Gene Therapy, Vol. 26, No. 8, 2015).

[0009] In addition, because of the random arrangement of fibers in the substrate strips and the variation in packing of strips between one packed bed and another of a given packed bed reactor system, it can be difficult for users to predict cell culture performance, since the substrate variesbetween cultures. Furthermore, the packed substrate of many existing bioreactors makes efficiently harvesting cells very difficult or impossible, as it is believed that cells are entrapped by the packed bed.

[0010] Existing fixed bed or packed bed bioreactor systems are designed around the concept of a loosely -packed bed, which has been suggested to improve fluid flow. This loose packing results in a low-occupancy bed region (i.e., open spaces are left in the reactor volume due to the reactor not being filled with a substrate to maximum occupancy, let alone having the substrate being compressed into a tightly packed bed). In addition, the loosely-packed beds can result in fluid channeling through the packed bed which reduces flow uniformity, and thus also nutrient and cell uniformity in the bed. Even attempts at higher-density packed or fixed bed can suffer from channeling effects, either through (i.e., “channeling”) or around (i.e., “bypass”) the bed.

[0011] There is a need for cell culture substrates and / or matrices, bioreactors, systems, and methods that enable culturing of cells in a high-density format, with uniform cell distribution, and easily attainable and increased harvesting yields. In particular, there is a need for cell culture substates for fixed bed bioreactors that have improved fluid flow characteristics with reduced fluid flow channeling and reduced fluid flow bypass around the cell culture substrate.Furthermore, there is a need for bioreactors that can deliver high-surface-area densities for the fixed bed using small-diameter fibers in the substrate layers without exacerbating fluid bypass and negatively impacting uniformity of the fluid flow and cell culture.SUMMARY

[0012] According to an embodiment of this disclosure, a bioreactor system for culturing cells is provided. The system comprises a cell culture vessel comprising at least one interior reservoir defined by an interior wall; and a cell culture matrix disposed in the reservoir, the cell culture matrix comprising a plurality of substrates configured for adhering cells thereto, the plurality of substrates being in a stacked arrangement, wherein the cell culture matrix comprises a matrix width extending across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted.

[0013] According to embodiments of this disclosure, a bioreactor system for culturing cells is provided. The bioreactor system includes a cell culture vessel comprising an inlet, an outlet, andat least one interior reservoir defined by an interior wall and disposed in a fluid path between the inlet and the outlet. The cell culture vessel is configured to allow fluid to flow in from the inlet, through the interior reservoir in a bulk flow direction, and out via the outlet. The system also includes a cell culture matrix disposed in the reservoir. The cell culture matrix includes a plurality of substrates for adhering cells thereto. The substrates of the plurality of substrates are stacked in a stacking direction parallel to the bulk flow direction of fluid through the reservoir. The cell culture matrix comprises a matrix width extending across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted. This reduces fluid bypass around the cell culture matrix to improve fluid flow uniformity through the fixed bed.

[0014] According to aspects of embodiments, the plurality of substrates is under compression with a compression force in a compression direction that is substantially parallel to the stacking direction and substantially perpendicular to the reservoir width. In embodiments, the compression force is at least 1 lb / in2, at least 1.5 lb / in2, greater than or equal to 1 lb / in2and less than or equal to 3 lb / in2, less than or equal to 2 lb / in2, less than or equal to 3 lb / in2, greater than or equal to 1.5 lb / in2and less than or equal to 3 lb / in2, greater than or equal to 1 lb / in2and less than or equal to 2 lb / in2, greater than or equal to 1.5 lb / in2and less than or equal to 2 lb / in2, greater than 0 lb / in2and less than or equal to 3 lb / in2, or greater than 0 lb / in2and less than or equal to 2 lb / in2. As an aspect of embodiments, the cell culture matrix comprises a height measured in the stacking direction, and the height is compressed by at least 10% compared to the plurality of substrates when not subject to the compression force. The height may be compressed by at least 20% compared to the plurality of substrates when not subject to the compression force. The compression force produces a radial force exerted by the plurality of substrates on the interior wall, the radial force being orthogonal to the compression force.

[0015] The bioreactor system can further include an O-ring disposed at a top or a bottom of the cell culture matrix in the stacking direction. The O-ring can be, for example, disposed along a circumference of the interior wall. The O-ring comprises an elastomeric material, in embodiments. The O-ring can be disposed between the cell culture matrix and a sealing surface, the O-ring being compressed between the cell culture matrix and the sealing surface. The sealingsurface can be a fluid flow distribution plate, a cell culture matrix support member, a top wall of the interior reservoir, or a bottom wall of the interior reservoir.

[0016] According to additional aspects of embodiments, at least one substrate of the plurality of substates comprises a peripheral edge surrounding an interior of the at least one substrate. The peripheral edge has an edge thickness that is greater than a thickness of the interior of the at least one substrate. The thickness of the interior and the edge thickness are measured in a direction parallel to the stacking direction. In aspects of embodiments, the peripheral edge is compressible in the edge thickness direction. In embodiments, two or more consecutive layers of the plurality of substrates are joined at peripheral edges of the two or more consecutive layers forming a joined layer. The plurality of substrates can include a plurality of joined layers. The peripheral edges are joined by at least one of an adhesive material, a molded material, and a melted edge of one or more of the peripheral edges. In additional aspects, the joined layer comprises layers of differing properties. The differing properties can be at least one of a physical dimension, a material composition, a stiffness, and a porosity of the two or more consecutive layers forming the joined layer. In aspects of embodiments, the plurality of substates includes fibers and pores separated by the fibers, and the physical dimension can be at least one of a fiber diameter and a pore diameter.

[0017] In aspects of embodiments, the plurality of substrates include a substrate layer comprising: a first major surface; a second major surface separated from the first major surface by a thickness; a plurality of fibers comprising a fiber diameter; and a plurality of pores comprising a pore diameter, each separated from others of the plurality of pores by the plurality of fibers and passing through the substrate layer from the first major surface to the second major surface. The fiber diameter is greater than 0 pm and can be less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, or less than or equal to 30 pm. The opening diameter is greater than 0 pm and can be less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, less than or equal to 30 pm, or less than or equal to 10 pm.

[0018] According to aspects, the cell culture vessel is configured to flow fluid into the interior reservoir via the inlet, through the cell culture matrix, and out of the interior reservoir via the outlet in the bulk flow direction such that the fluid flows substantially in a linear path between the inlet and the outlet. The cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix, such that the uniform flow satisfies the following expressions:A0 = 02 - 0i, andA0 <N, wherein P is greater than or equal to at least one of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.95, and wherein A is less than or equal to at least one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0.

[0019] The uniform flow is maintained through a bed height of the fixed bed, the bed height being greater than or equal to 10 cm; 20 cm; 30 cm; 40 cm; 50 cm; 60 cm; 70 cm; 80 cm; 90 cm; or 100 cm. The cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix, such that E(0) is less than or equal to 0.2 or less than or equal to 0.1, when 0 is less than or equal to 0.8, 0.75, 0.7, 0.6, or 0.5.

[0020] According to additional embodiments of this disclosure, a bioreactor system for culturing cells is provided. The system includes a cell culture vessel comprising an inlet, an outlet, and at least one interior reservoir defined by an interior wall and disposed in a fluid path between the inlet and the outlet, the cell culture vessel being configured allow fluid to flow in from the inlet, through the interior reservoir in a bulk flow direction, and out via the outlet. The system also includes a cell culture matrix disposed in the reservoir, the cell culture matrix comprising a plurality of substrates configured for adhering cells thereto. The plurality of substrates comprising a substrate layer comprising: a first major surface; a second major surface separated from the first major surface by a thickness of the substrate layer; and a peripheral edge at the boundary for the first major surface and the second major surface. The plurality of substrates are stacked in a stacking direction parallel to the bulk flow direction. The plurality of substrates comprises two or more of the substrate layers that are attached to each other at the peripheral edges of the two or more substrate layers forming a joined layer.

[0021] According to aspects, the two or more substrate layers of the joined layer are not attached to each other at the first major surface and / or the second major surface within the peripheral edges. The cell culture matrix comprises a matrix width extending across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted. The plurality of substrates can include a plurality of joined layers. In aspects, the peripheral edges are joined by at least one of an adhesive material, a molded material, and a melted edge of one or more of the peripheral edges.

[0022] In aspects of embodiments, the joined layer comprises two or more substrate layers of differing properties. The differing properties can include at least one of a physical dimension, a material composition, a stiffness, and a porosity of the two or more consecutive layers forming the jointed layer. The plurality of substates comprises fibers and pores separated by the fibers, and the physical dimension comprises at least one of a fiber diameter and a pore diameter.

[0023] In embodiments, a thickness of the peripheral edge of the substrate layer is greater than a thickness of an interior of the substrate layer located radially within the peripheral edge. The cell culture matrix is under compression with a compression force in a compression direction that is substantially parallel to the stacking direction and substantially perpendicular to the reservoir width. The joined layer has a rigidity that is greater than a rigidity of a same number of substrate layers that are not joined, the rigidity being measured with respect to a force applied in a direction of perpendicular to the first major surface.

[0024] Individual substrate layers of the joined layer are oriented with respect to each other to achieve maximum packing density in the joined layers. The individual substrate layers of the joined layer are oriented with respect to each other by rotating one or more of the substrate layers about a rotational axis that is perpendicular to the first major surface. The degree of rotation of the one or more substrate layers achieves an interlocking of fibers of the one or more substrate layers. In an aspect, the joined layer includes two or more different types of substrate layers arranged in an alternating order in the stack of the joined layer. The two or more different types of substrate layers can include substrates having different fiber diameters, different pore diameters, or different weave patterns. In an aspect, the joined layer comprises an even number of substrate layers.

[0025] According to embodiments, the plurality of substrates include one or more substrate layers comprising a fiber diameter that is greater than 0 pm and can be less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, or less than or equal to 30 pm. The plurality of substrates can include one or more substrate layers comprising an opening diameter that is greater than 0 pm and can be less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, less than or equal to 30 pm, or less than or equal to 10 pm.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A shows a perspective view of a three-dimensional model of a cell culture substrate, according to one or more embodiments of this disclosure.

[0027] Figure IB is a two-dimensional plan view of the substrate of Figure 1A.

[0028] Figure 1C is a cross-section along line A-A of the substrate in Figure IB.

[0029] Figure 2 A shows an example of a cell culture substrate, according to some embodiments.

[0030] Figure 2B shows an example of a cell culture substrate, according to some embodiments.

[0031] Figure 2C shows an example of a cell culture substrate, according to some embodiments.

[0032] Figure 2D shows an example of a cell culture substrate, according to some embodiments.

[0033] Figure 3 shows a schematic view of a cell culture system, according to one or more embodiments.

[0034] Figure 4A shows a plan view of a modeled multi-layer woven mesh cell culture substrate in a tightly packed arrangement, according to one or more embodiments of this disclosure.

[0035] Figure 4B shows a side cross-section view of the multi-layer woven mesh cell culture substrate of Figure 4A, according to one or more embodiments of this disclosure.

[0036] Figure 5A shows a plan view of a modeled multi-layer woven mesh cell culture substrate in a loosely packed arrangement, according to one or more embodiments of this disclosure.

[0037] Figure 5B shows a side cross-section view of the multi-layer woven mesh cell culture substrate of Figure 5 A, according to one or more embodiments of this disclosure.

[0038] Figure 6 A shows the modeled empty space in the dotted-line volume shown in Figures4 A and 4B.

[0039] Figure 6B shows the modeled empty space in the dotted-line volume shown in Figures 5 A and 5B.

[0040] Figure 7 is a schematic drawing of an experimental setup for measuring residence time distribution of different cell culture substrate samples.

[0041] Figure 8 is a graph showing the change in dye concentration vs. time during residence time distribution measurement for a woven and non-woven cell culture substrate.

[0042] Figure 9 is a graph showing the change in dye concentration vs. normalized volume of flow through a bioreactor having a woven substrate according to embodiments of this disclosure versus other commercially available non-woven cell culture substrates.

[0043] Figure 10A is a photograph of a stack of substrate disks, according to embodiments of this disclosure, at 100% occupancy for an experimental bioreactor,

[0044] Figure 10B is a photograph of a stack of substrate disks, according to embodiments of this disclosure, at 95% occupancy for an experimental bioreactor.

[0045] Figure 11 A is a photograph of a bioreactor with a cell culture matrix of stacked layers of substate packed at 95% occupancy with loose packing, according to one or more embodiments.

[0046] Figure 1 IB is a photograph of a bioreactor with a cell culture matrix of stacked layers of substate packed at 95% occupancy with compressed packing, according to one or more embodiments.

[0047] Figure 12A is a graph of the residence time distribution data from three different packing densities in compress packing configurations, according to one or more embodiments.

[0048] Figure 12B is a graph of the residence time distribution data from three different packing densities in loose packing configurations, according to one or more embodiments.

[0049] Figure 13A is a graph of the residence time distribution data from a fixed bed at 102.5% occupancy at different flow rates, according to one or more embodiments.

[0050] Figure 13B is a graph of the residence time distribution data from a fixed bed at 95% occupancy with compressed packing at different flow rates, according to one or more embodiments.

[0051] Figure 13C is a graph of the residence time distribution data from a fixed bed at 95% occupancy with loose packing at different flow rates, according to one or more embodiments.

[0052] Figure 14A is a graph of the residence time distribution data from fixed beds in various occupancy percentages with compressed packing and the presence of bubbles, according to one or more embodiments.

[0053] Figure 14B is a graph of the residence time distribution data from fixed beds in various occupancy percentages with loose packing and the presence of bubbles, according to one or more embodiments.

[0054] Figure 15A is a cross-section view of a bioreactor vessel with a plurality of high- density substrate layers, according to embodiments.

[0055] Figure 15B is cross-section view of a bioreactor vessel with a plurality of high-density substrate layers subject to a compression force, according to embodiments.

[0056] Figure 16 is a graph of the residence time distribution data for the bioreactors shown in Figures 15A and 15B, according to embodiments.

[0057] Figure 17 is an illustration of the formation of a joined layer of substrate material, according to embodiments.DETAILED DESCRIPTION

[0058] Various embodiments of the disclosure will be described in detail with reference to drawings, if any. Reference to various embodiments does not limit the scope of the invention,which is limited only by the scope of the claims attached hereto. Additionally, any examples set forth in this specification are not limiting and merely set forth some of the many possible embodiments of the claimed invention.

[0059] Embodiments of this disclosure include a cell culture substrate, as well as cell culture or bioreactor systems incorporating such a substrate, and methods of culturing cells using such a substrate and bioreactor systems. The substrate and bioreactor systems incorporating the same exhibit improved flow characteristics through the substrate. For example, more uniform flow is achieved through the substrate, and non-uniform flow resulting from channeling or turbulent flow is reduced or eliminated. Flow “dead zones” in the substrate or fixed bed of the bioreactor are greatly reduced or eliminated compared to existing solutions. The result is a substrate or fixed bed that allows for uniform perfusion throughout the substrate or fixed bed, which promotes cell health during cell culture and an efficient cell culture process in terms of not only the culturing of cells, but also cell seeding, and harvesting of cells or cell by-products.

[0060] In conventional large-scale cell culture bioreactors, different types of fixed bed or packed bed bioreactors have been used. Usually these packed beds contain porous matrices to retain adherent or suspension cells, and to support growth and proliferation. Packed-bed matrices provide high surface area to volume ratios, so cell density can be higher than in the other systems. However, the packed bed often functions as a depth filter, where cells are physically trapped in the entangled fibers of the matrix. Thus, because of linear flow of the cell inoculum through the packed bed, cells are subject to heterogeneous distribution inside the packed bed, leading to variations in cell density through the depth or width of the packed bed. For example, cell density may be higher at the inlet region of a bioreactor and significantly lower nearer to the outlet of the bioreactor. This non-uniform distribution of the cells inside of the packed bed significantly hinders scalability and predictability of such bioreactors in bioprocess manufacturing, and can even lead to reduced efficiency in terms of growth of cells or viral vector production per unit surface area or volume of the packed bed.

[0061] Another problem encountered in packed bed bioreactors disclosed in prior art is the channeling effect. Due to random nature of packed nonwoven fibers, the local fiber density at any given cross section of the packed bed is not uniform. Liquid media flows quickly in theregions with low fiber density (high bed permeability) and much slower in the regions of high fiber density (lower bed permeability). The resulting non-uniform media perfusion across the packed bed creates the channeling effect, which manifests itself as significant nutrient and metabolite gradients that negatively impact overall cell culture and bioreactor performance. Cells located in the regions of low media perfusion will starve and very often die from the lack of nutrients or metabolite poisoning. The flow can be so non-uniform that there are effectively flow “dead zones” in the packed bed, where perfusion does not occur and the delivery of any nutrients to cells in those areas is limited to diffusion mechanics in the media.

[0062] Cell harvesting is yet another problem encountered when bioreactors packed with nonwoven fibrous scaffolds are used. Due to packed-beds often functioning as a depth filter, cells that are released at the end of cell culture process are entrapped inside the packed bed, and cell recovery is very low. This significantly limits utilization of such bioreactors in bioprocesses where live cells are the products or where live cells need to be harvested for further processing to capture cell by-products. Thus, the non-uniformity leads to areas with different exposure to flow and shear, effectively reducing the usable cell culture area, causing non-uniform culture, and interfering with transfection efficiency and cell release.

[0063] Channeling can also occur at the periphery of a packed bed of fixed bed. This effect is referred to herein as “bypass” — that is, the fluid flows around or bypasses the cell culture substrate, preferentially flowing in a space between the substrate and the cell culture vessel wall.

[0064] In addition, while existing solutions suggest that loosely packed beds are preferable, the current disclosure will illustrate the converse: densely packed beds, in accordance with embodiments of the present disclosure, can actually improve performance of a fixed bed for cell culture. For example, embodiments of this disclosure show superior flow uniformity at high packed densities of substrate material in the bioreactor. Embodiments also show a reduction in the presence of bubbles in the fixed bed and the bioreactor system, in general. Nonetheless, even if bubbles are present in the system, the uniform fluid flow characteristics described herein can be maintained. Bubbles are commonly observed during cell cultures and can easily be trapped in fixed bed bioreactors, including the substrates. Once trapped, they disturb flow direction and reduce flow uniformity, therefore interfering with cell seeding and nutrient supply. For cellculture in a fixed bed reactor, higher flow rates are consistently used to maintain oxygen and nutrient supply to support high density cell growth. High flow rates can potentially lift mesh disks in the packed bed if the disks are not restricted. This can change local permeability and disturb flow direction. By using high compression and high occupancy, the disks are restricted in place and the negative impacts from bubbles and high flow rates can be mitigated. The loose packing or shifting of the packed bed can also mean inconsistent packing of packed beds in conventional systems, which can result in variations in permeability and channeling. However, when the bed is compacted according to embodiments of this disclosure, these negative flow characteristics can be avoided.

[0065] The above advantages of embodiments of this disclosure are quite unexpected in light of the preference for loosely-packed beds in the marketplace. These improvements also exist in embodiments regardless of fluid flow rate (including, e.g., channeling) or size of the fixed bed or bioreactor. Thus, the advantages are present across a range of bioreactor scales. A further advantage includes reducing or eliminating shifting of the fixed bed or a change in compaction of the fixed bed due to being subjected to various flow rates or due to mechanical forces during handling and / or shipping. In addition, the higher compaction of the fixed bed results in more surface area for cells.

[0066] As used herein, “occupancy” is defined as percentage of substrate packed into the bed volume of the reactor, relative to 100% occupancy. To determine 100% occupancy, the space height of the reactor bed space is divided by the thickness of each substrate layer to obtain the number of substrate layers for 100% occupancy. Occupancies of less than 100% (also referred to as loose packing) is obtained by removing the requisite percentage of mesh layers. For example, removing 5% of the layers from the 100% occupancy condition results in 95% occupancy. It is also possible to achieve greater than 100% occupancy. For example, due to some elasticity in the material of a substrate, the bed could be compressed to some extent and thus create additional space for most substate layers. If additional layers are added to that space above the 100% occupancy number of layers, then the resulting occupancy would be greater than 100%. For example, if 2.5% extra layers are added, the occupancy would be 102.5%.

[0067] To address these and other problems of existing cell culture solutions, embodiments of the present disclosure provide cell growth substrates, matrices of such substrates, and / or packed- bed systems using such substrates that enable efficient and high-yield cell culturing for anchorage-dependent cells and production of cell products (e.g., proteins, antibodies, viral particles). Embodiments include a porous cell-culture matrix made from an ordered and regular array of porous substrate material that enables uniform cell seeding and media / nutrient perfusion, as well as efficient cell harvesting. Embodiments also enable scalable cell-culture solutions with substrates and bioreactors capable of seeding and growing cells and / or harvesting cell products from a process development scale to a full production size scale, without sacrificing the uniform performance of the embodiments. For example, in some embodiments, a bioreactor can be easily scaled from process development scale to product scale with comparable viral genome per unit surface area of substrate (VG / cm2) across the production scale. The harvestability and scalability of the embodiments herein enable their use in efficient seed trains for growing cell populations at multiple scales on the same cell substrate. In addition, the embodiments herein provide a cell culture matrix having a high surface area that, in combination with the other features described, enables a high yield cell culture solution.

[0068] In one embodiment, a matrix is provided with a structurally defined surface area for adherent cells to attach and proliferate that has good mechanical strength and forms a highly uniform multiplicity of interconnected fluidic networks when assembled in a packed bed or other bioreactor. In particular embodiments, a mechanically stable, non-degradable woven mesh can be used as the substrate to support adherent cell production. The cell culture matrix disclosed herein supports attachment and proliferation of anchorage dependent cells in a high volumetric density format. Uniform cell seeding of such a matrix is achievable, as well as efficient harvesting of cells or other products of the bioreactor. In addition, the embodiments of this disclosure support cell culturing to provide uniform cell distribution during the inoculation step and achieve a confluent monolayer or multilayer of adherent cells on the disclosed matrix, and can avoid formation of large and / or uncontrollable 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. Thus, the matrix eliminates diffusional limitations during operation of the bioreactor. In addition, the matrix enables easy and efficientcell harvest from the bioreactor. The structurally defined matrix of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of the bioreactor.

[0069] According to some embodiments, a method of cell culturing is also provided using bioreactors with the matrix for bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0070] In contrast to existing cell culture substrates used in cell culture bioreactors (i.e., nonwoven substrates of randomly ordered fibers), embodiments of this disclosure include a cell culture substrate having a defined and ordered structure. The defined and order structure allows for consistent and predictable cell culture results. In addition, the substrate has an open porous structure that prevents cell entrapment and enables uniform flow through the packed bed. This construction enables improved cell seeding, nutrient delivery, cell growth, and cell harvesting. According to one or more particular embodiments, the matrix is formed with a substrate material having a thin, sheet-like construction having first and second sides separated by a relatively small thickness, such that the thickness of the sheet is small relative to the width and / or length of the first and second sides of the substrate. In addition, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is of a size and geometry that allows cells to adhere to the surface of the substrate material as if it were approximately a two-dimensional (2D) surface, while also allowing adequate fluid flow around the substrate material and through the openings. In some embodiments, the substrate is a polymer-based material, and can be formed as a molded polymer sheet; a polymer sheet with openings punched through the thickness; a number of filaments that are fused into a mesh-like layer; a 3D-printed substrate; or a plurality of filaments that are woven into a mesh layer. The physical structure of the matrix has a high surface-to-volume ratio for culturing anchorage dependent cells. According to various embodiments, the matrix can be arranged or packed in a bioreactor in certain ways discussed here for uniform cell seeding and growth, uniform media perfusion, and efficient cell harvest.

[0071] In addition, the embodiments disclosed herein enable not only cell attachment and growth to a cell culture substrate, but also the viable harvest of cultured cells. The inability toharvest viable cells is a significant drawback in current platforms, and it leads to difficulty in building and sustaining a sufficient number of cells for production capacity. According to an aspect of embodiments of this disclosure, it is possible to harvest viable cells from the cell culture substrate, including between 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, of the cells that are harvested, at least 80% are viable, at least 85% are viable, at least 90% are viable, at least 91% are viable, at least 92% are viable, at least 93% are viable, at least 94% are viable, at least 95% are viable, at least 96% are viable, at least 97% are viable, at least 98% are viable, or at least 99% are viable. Cells may be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0072] Figures 1 A and IB show a three-dimensional (3D) perspective view and a two- dimensional (2D) plan view, respectively, of a cell culture substrate 100, according to an example of one or more embodiments of this disclosure. 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, which can be defined by one or more widths or diameters (e.g., Di, D2). 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.). A woven mesh may be characterized as, on a macro-scale, a two-dimensional sheet or layer. However, a close inspection of a woven mesh reveals a three-dimensional structure due to the rising and falling of intersecting fibers of the mesh. Thus, as shown in Figure 1C, a thickness T of the woven mesh 100 may be thicker than the thickness of a single fiber (e.g., ti). As used herein, the thickness T is the maximum thickness between a first side 108 and a second side 110 of the woven mesh. Without wishing to be bound by theory, it is believed that the three-dimensional structure of the substrate 100 is advantageous as it provides a large surface area for culturing adherent cells, and the structural rigidity of the mesh can provide a consistent and predictable cell culture matrix structure that enables uniform fluid flow.

[0073] In Figure IB, the openings 106 have a diameter Di, defined as a distance between opposite fibers 102, and a diameter D2, defined as a distance between opposite fibers 104. Di and D2 can be equal or unequal, depending on the weave geometry. Where Di and D2 are unequal,the larger can be referred to as the major diameter, and the smaller as the minor diameter. In some embodiments, the diameter of an opening may refer to the widest part of the opening. Unless otherwise specified, the opening diameter, as used herein, will refer to a distance between parallel fibers on opposite sides of an opening. In this disclosure, the spaces between the fibers may be referred to as “openings” or “pores.”

[0074] A given fiber of the plurality of fibers 102 has a thickness ti, and a given fiber of the plurality of fibers 104 has a thickness t2. In the case of fibers of round cross-section, as shown in Figure 1 A, or other three-dimensional cross-sections, the thicknesses ti and t2 are the maximum diameters or thicknesses of the fiber cross-section. According to some embodiments, the plurality of fibers 102 all have the same thickness ti, and the plurality of fiber 104 all have the same thickness t2. In addition, ti and t2 may be equal. However, in one or more embodiments, ti and t2 are not equal such as when the plurality of fibers 102 are different from the plurality of fiber 104. In addition, each of the plurality of fibers 102 and plurality of fibers 104 may contain fibers of two or more different thicknesses (e.g., tia, tib, etc., and t2a, t2b, etc.). According to embodiments, the thicknesses ti and t2 are large relative to the size of the cells cultured thereon, so that the fibers provide an approximation of a flat surface from the perspective of the cell, which can enable better cell attachment and growth as compared to some other solutions in which the fiber size is small (e.g., on the scale of the cell diameter). Due to three-dimensional nature of woven mesh, as shown in Figures 1A-1C, the 2D surface area of the fibers available for cell attachment and proliferation exceeds the surface area for attachment on an equivalent planar 2D surface. In this disclosure, with respect to fibers, the terms “thickness” and “diameter” may be used interchangeably and both refer to the same physical dimension of the fiber.

[0075] In one or more embodiments, a fiber may have a diameter in a range of about 10 pm to about 1000 pm, about 20 pm to about 1000 pm, about 30 pm to about 1000 pm, about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm; about 150 pm to about 300 pm, about 10 pm to about 200 pm, about 10 pm to about 100 pm, about 10 pm to about 75 pm, about 10 pm to about 50 pm, or about 10 pm to about 30 pm. On a microscale level, due to the scale of the fiber compared to the cells (e.g., the fiber diameters being largerthan the cells), the surface of monofilament fiber is presented as an approximation of a 2D surface for adherent cells to attach and proliferate. Fibers can be woven into a mesh with openings ranging from about 100 pm x 100 pm to about 1000 pm x 1000 pm. In some embodiments, the opening may have a diameter of about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm, about 10 pm to about 100 pm, or about 10 pm to about 50 pm. These ranges of the filament diameters and opening diameters are examples of some embodiments, but are not intended to limit the possible feature sizes of the mesh according to all embodiments. The combination of fiber diameter and opening diameter is chosen to provide efficient and uniform fluid flow through the substrate when, for example, the cell culture matrix is comprises a number of adjacent mesh layers (e.g., a stack of individual layers or a rolled mesh layer).

[0076] Factors such as the fiber diameter, opening diameter, and weave type / pattern will determine the surface area available for cell attachment and growth. In addition, when the cell culture matrix includes a stack, roll, or other arrangement of overlapping substrate, the packing density of the cell culture matrix will impact the surface area of the packed bed matrix. Packing density can vary with the packing thickness of the substrate material (e.g., the space needed for a layer of the substrate). For example, if a stack of cell culture matrix has a certain height, each layer of the stack can be said to have a packing thickness determined by dividing the total height of the stack by the number of layers in the stack. The packing thickness will vary based on fiber diameter and weave, but can also vary based the alignment of adjacent layers in the stack. For instance, due to the three-dimensional nature of a woven layer, there is a certain amount of interlocking or overlapping of fibers that adjacent layers can accommodate based on their alignment with one another. In a first alignment, the adjacent layers can be tightly nestled together, but in a second alignment, the adjacent layers can have zero or almost zero overlap, such as when the lower-most point of the upper layer is in direct contact with the upper-most point of the lower layer. It may be desirable for certain applications to provide a cell culture matrix with a lower density packing of layers (e.g., when higher permeability is a priority) or a higher density of packing (e.g., when maximizing substrate surface area is a priority). Accordingto one or more embodiments, the packing thickness can be from about 10 pm to about 1000 pm; about 10 pm to about 100 pm; about 10 pm to about 50 pm; about 50 pm to about 1000 pm; about 100 pm to about 750 pm; about 125 pm to about 600 pm; about 150 pm to about 500 pm; about 200 pm to about 400 pm; about 200 pm to about 300 pm.

[0077] The above structural factors can determine the surface area of a cell culture matrix, whether of a single layer of cell culture substrate or of a cell culture matrix having multiple layers of substrate. For example, in a particular embodiment, a single layer of woven mesh substrate having a circular shape and diameter of 6 cm can have an effective surface area of about 68 cm2. The “effective surface area,” as used herein, is the total surface area of fibers in a portion of substrate material that is available for cell attachment and growth. Unless stated otherwise, references to “surface area” refer to this effective surface area. According to one or more embodiments, a single woven mesh substrate layer with a diameter of 6 cm may have an effective surface area of about 50 cm2to about 90 cm2; about 53 cm2to about 81 cm2; about 68 cm2; about 75 cm2; or about 81 cm2. These ranges of effective surface area are provided for example only, and some embodiments may have different effective surface areas. The cell culture matrix can also be characterized in terms of porosity, as discussed in the Examples herein.

[0078] The substrate mesh can be fabricated from monofilament or multifilament fibers of polymeric materials compatible in cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide. Mesh substrates may have a different patterns or weaves, including, for example knitted, warp-knitted, or woven (e.g., plain weave, twilled weave, dutch weave, five needle weave).

[0079] The surface chemistry of the mesh filaments may need to be modified to provide desired cell adhesion properties. Such modifications can be made through the chemical treatment of the polymer material of the mesh or by grafting cell adhesion molecules to the filament surface. Alternatively, meshes can be coated with thin layer of biocompatible hydrogels that demonstrate cell adherence properties, including, for example, collagen or Matrigel®. Alternatively, surfaces of filament fibers of the mesh can be rendered with cell adhesiveproperties through the treatment processes with various types of plasmas, process gases, and / or chemicals known in the industry. In one or more embodiments, however, the mesh is capable of providing an efficient cell growth surface without surface treatment.

[0080] Figures 2A-2D show different examples of woven mesh according to some contemplated embodiments of this disclosure. The fiber diameter of the meshes are approximately 250 pm (Figure 2A), approximately 200 pm (Figure 2B), approximately 105 pm (Figure 2C), and approximately 53 pm (Figure 2D). The four mesh geometries of Figures 2A-2D are examples only, and embodiments of this disclosure are not limited to these specific examples. The mesh of Figure 2D offers the highest surface area of the four, which may be advantageous in achieving a high density in cell adhesion and proliferation, and thus provide the most efficient substrate for cell culturing. However, in some embodiments, it may be advantageous for the cell culture matrix to include a mesh with lower surface area, such as those in Figures 2A-2C, or a combination of meshes of different surface areas, to achieve a desired cell distribution or flow characteristics within the culture chamber, for example.

[0081] Another advantage of the higher surface areas and high cell seeding or growing densities is that the cost of the embodiments disclosed herein can be the same or less than competing solution. Specifically, the cost per cellular product (e.g., per cell or per viral genome) can be equal to or less than other packed bed bioreactors.

[0082] By using a structurally defined culture matrix of sufficient rigidity, high-flow- resistance uniformity across the matrix or packed bed is achieved. According to various embodiments, the matrix can be deployed in monolayer or multilayer formats. This flexibility eliminates diffusional limitations and provides uniform delivery of nutrients and oxygen to cells attached to the matrix. In addition, the open matrix lacks any cell entrapment regions in the packed bed configuration, allowing for complete cell harvest with high viability at the end of culturing. The matrix also delivers packaging uniformity for the packed bed, and enables direct scalability from process development units to large-scale industrial bioprocessing unit. The ability to directly harvest cells from the packed bed eliminates the need of resuspending a matrix in a stirred or mechanically shaken vessel, which would add complexity and can inflict harmfulshear stresses on the cells. Further, the high packing density of the cell culture matrix yields high bioprocess productivity in volumes manageable at the industrial scale.

[0083] The geometry of the mesh substrate layers is designed to allow efficient and uniform flow through one or multiple substrate layers. In addition, the structure of the matrix can accommodate fluid flow through the matrix in multiple orientations. For example, the direction of bulk fluid flow can be perpendicular to the major side surfaces of the first and second substrate layers. However, the matrix can also be oriented with respect to the flow such that the sides of the substrate layers are parallel to the bulk flow direction. In addition to fluid flow being perpendicular or parallel to the first and second sides of the mesh layers, the matrix can be arranged with multiple pieces of substrate at intermediate angles, or even in random arrangements with respect to fluid flow. This flexibility in orientation is enabled by the essentially isotropic flow behavior of the woven substrate. In contrast, substrates for adherent cells in existing bioreactors do not exhibit this behavior and instead their packed beds tend to create preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the matrix of the current disclosure allows for its use in various applications and bioreactor or container designs while enabling better and more uniform permeability throughout the bioreactor vessel.

[0084] As discussed herein, the cell culture substrate can be used within a bioreactor vessel, according to one or more embodiments. For example, the substrate can be used in a packed bed bioreactor configuration, or in other configurations within a three-dimensional culture chamber. However, embodiments are not limited to a three-dimensional culture space, and it is contemplated that the substrate can be used in what may be considered a two-dimensional culture surface configuration, where the one or more layers of the substrate lay flat, such as within a flat- bottomed culture dish, to provide a culture substrate for cells. Due to contamination concerns, the vessel can be a single-use vessel that can be disposed of after use.

[0085] A cell culture system is provided, according to one or more embodiments, in which the cell culture matrix is used within an interior reservoir or a culture chamber of a bioreactor vessel. Figure 3 shows an example of a cell culture system 300 that includes a bioreactor vessel 302 having a cell culture chamber 304 in the interior reservoir of the bioreactor vessel 302. Withinthe cell culture chamber 304 is a cell culture matrix 306 that is made from a stack of substrate layers 308. The substrate layers 308 are stacked with the first or second side of a substrate layer facing a first or second side of an adjacent substrate layer. The bioreactor vessel 300 has an inlet 310 at one end for the input of media, cells, and / or nutrients into the culture chamber 304, and an outlet 312 at the opposite end for removing media, cells, or cell products from the culture chamber 304. By allowing stacking of substrate layers in this way, the system can be easily scaled up without negative impacts on cell attachment and proliferation, due to the defined structure and efficient fluid flow through the stacked substrates. While the vessel 300 may generally be described as having an inlet 310 and an outlet 312, some embodiments may use one or both of the inlet 310 and outlet 312 for flowing media, cells, or other contents both into and out of the culture chamber 304. For example, inlet 310 may be used for flowing media or cells into the culture chamber 304 during cell seeding, perfusion, or culturing phases, but may also be used for removing one or more of media, cells, or cell products through the inlet 310 in a harvesting phase. Thus, the terms “inlet” and “outlet” are not intended to restrict the function of those openings.

[0086] In one or more embodiments, flow resistance and volumetric density of the packed bed can be controlled by interleaving substrate layers of different geometries. In particular, mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the fluid flow resistance in packed bed format. By interlaying meshes of different sizes and geometries (e.g., the different meshes shown in Figures 2A-2D), flow resistance can be controlled or varied in one or more specific portions of the bioreactor. This will enable better uniformity of liquid perfusion in the packed bed. Various combinations of meshes of different sizes are possible to obtain different profiles of volumetric density of cells growth surface and flow resistance. For example, a packed bed column with zones of varying volumetric cells densities (e.g., a series of zones creating a pattern of low / high / low / high, etc. densities) can be assembled by interleaving meshes of different sizes.

[0087] In Figure 3, the bulk flow direction is in a direction from the inlet 310 to the outlet 312, and, in this example, the first and second major sides of the substrate layers 308 are perpendicular to the bulk flow direction. However, embodiments are not limited to thisorientation and can include embodiments in which the system includes a bioreactor vessel and stack of substrates within the culture space that have first and second sides that are parallel to a bulk flow direction. Thus, the matrices of embodiments of this disclosure can be employed in either configuration. In system 300, the substrates 308 are sized and shaped to fill the interior space defined by the culture chamber 304 so that the culture space in the vessel is filled for cell growth surfaces to maximize efficiency in terms of cells per unit volume.

[0088] The cell culture matrix can be arranged in multiple configurations within the culture chamber depending on the desired system. For example, in one or more embodiments, the system includes one or more layers of the substrate with a width extending across the width of a defined cell culture space in the culture chamber. Multiple layers of the substrate may be stacked in this way to a predetermined height. As discussed above, the substrate layers may be arranged such that the first and second sides of one or more layers are perpendicular to a bulk flow direction of culture media through the defined culture space within the culture chamber, or the first and second sides of one or more layers may be parallel to the bulk flow direction. In one or more embodiments, the cell culture matrix includes one or more substrate layers at a first orientation with respect to the bulk flow, and one or more other layers at a second orientation that is different from the first orientation. For example, various layers may have first and second sides that are parallel or perpendicular to the bulk flow direction, or at some angle in between.

[0089] The “defined culture space,” as used herein, refers to a space or an interior reservoir within the culture chamber occupied by the cell culture matrix and in which cell seeding and / or culturing is to occur. The defined culture space can fill approximately the entirety of the culture chamber, or may occupy a portion of the space within the culture chamber. As used herein, the “bulk flow direction” is defined as a direction of bulk mass flow of fluid or culture media through or over the cell culture matrix during the culturing of cells, and / or during the inflow or outflow of culture media to the culture chamber.

[0090] The bioreactor vessel optionally includes one or more outlets capable of being attached to inlet and / or outlet means. Through the one or more outlets, liquid, media, or cells can be supplied to or removed from the chamber. A single port in the vessel may act as both the inlet and outlet, or multiple ports may be provided for dedicated inlets and outlets.

[0091] The packed bed cell culture matrix of one or more embodiments can consist of the woven cell culture mesh substrate without any other form of cell culture substrate disposed in or interspersed with the cell culture matrix. That is, the woven cell culture mesh substrate of embodiments of this disclosure are effective cell culture substrates without requiring the type of irregular, non-woven substrates used in existing solution. This enables cell culture systems of simplified design and construction, while providing a high-density cell culture substrate with the other advantages discussed herein related to flow uniformity, harvestability, etc.

[0092] As discussed herein, the cell culture substrates and bioreactor systems provided offer numerous advantages. For example, the embodiments of this disclosure can support the production of any of a number of viral vectors, such as AAV (all serotypes) and lentivirus, and can be applied toward in vivo and ex vivo gene therapy applications. The uniform cell seeding and distribution maximizes viral vector yield per vessel, and the designs enable harvesting of viable cells, which can be useful for seed trains consisting of multiple expansion periods using the same platform. In addition, the embodiments herein are scalable from process development scale to production scale, which ultimately saves development time and cost. The methods and systems disclosed herein also allow for automation and control of the cell culture process to maximize vector yield and improve reproducibility. Finally, the number of vessels needed to reach production-level scales of viral vectors (e.g., 1016to 1018AAV VG per batch) can be greatly reduced compared to other cell culture solutions.

[0093] As discussed herein, the embodiments of this disclosure provide cell culture substrates, bioreactor systems, and methods of culturing cells or cell by-products that are scalable and can be used to provide a cell seed train to gradually increase a cell population. One problem in existing cell culture solutions is the inability for a given bioreactor system technology to be part of a seed train. Instead, cell populations are usually scaled up on various cell culture substrates. This can negatively impact the cell population, as it is believed that cells become acclimated to certain surfaces and being transferred to a different type of surface can lead to inefficiencies. Thus, it would be desirable to minimize such transitions between cell culture substrates or technologies. By using the same cell culture substrate across the seed train, as enabled by embodiments of this disclosure, efficiency of scaling up a cell population is increased. Forexample, the seed train can begin with a vial of starter cells which are seeded into a first vessel (such as a T175 flask from Corning), then into a second vessel (such as a HyperFlask® from Corning), then into a process-development scale bioreactor system according to embodiments of this invention (effective surface area of substrate of about 20,000 cm2), and then into a larger bioreactor pilot system according to embodiments of this invention (effective surface area of substrate is about 300,000 cm2). At the end of this seed train, the cells can be seeded into a production-scale bioreactor vessel according to embodiments of this disclosure, with a surface area of about 5,000,000 cm2, for example. Harvest and purification steps can then be performed when the cell culture is complete. Harvest can be accomplished via in situ cell lysis with a detergent (such as Triton X-100), or via mechanical lysis; and further downstream processing can be performed, as needed.

[0094] The benefits of using the same cell culture substrate within the seed train (e.g., from process development level to pilot level, or even to production level) include efficiencies gained from the cells being accustomed to the same surface during the seed train and production stages; a reduced number of manual, open manipulations during seed train phases; more efficient use of the packed bed due to uniform cell distribution and fluid flow, as described herein; and the flexibility of using mechanical or chemical lysis during viral vector harvest.

[0095] Examples

[0096] To demonstrate the efficacy of the cell culture matrix, cell culture systems, and related methods of this disclosure, studies were conducted on the seeding and culturing of cells, according to the following examples.

[0097] Example 1

[0098] To further examine the flow uniformity or permeability of the substrates of this disclosure, modeling was used to understand the porosity of the three-dimensional cell culture matrix. Sheets of woven PET mesh substrate were modeled in a tight-packed configuration and a loose-packed configuration, which represent upper and lower boundaries of the packing densityof a substrate stack for the particular mesh sheet that was modeling. In particular, Figure 4A shows a plan view of the tight-packed configuration, and Figure 4B shows a cross-section side view of the same stack. Figures 5 A and 5B show plan and cross-section views, respectively, of the loose-packed configuration. For each modeled configuration, a sample cell 600, 602 was defined that encloses the same volume of mesh material to analyze the porosity per unit volume of the sample cell 600, 602. The modeled volume of open space within each cell is shown in Figures 6A (for tight-packed stack) and 6B (for loose-packed stack). The porosity in terms of percentage of open space was about 40.8% for the loose-packed cell, and 61.4% for the tight- packed cell. Because the modeled stacks in Figures 4A-5B represent the tightest- and loosest- packed configurations for the given mesh material, the porosities of 40.8% and 61.4% are the upper and lower bounds of porosity for this particular mesh material. Depending on the alignment and real-world packing density when using this mesh material, the porosity may fall in between these extremes. However, embodiments of this disclosure are not limited to this porosity range, as variations in the mesh dimensions and arrangement of the substrate within the cell culture vessel can lead to a different range of porosities.

[0099] In addition to the modeled porosity range, porosity was measured using real packed beds of PET woven mesh substrate. The measurements were made using one hundred disks, each with a diameter of 22.4 mm, stacked with random alignment. The total weight of the 100-disk stack was 5.65±0.2 g. Volume of the PET material of the stack was calculated, assuming a PET density of 1.38 g / cm3, using the following formula:VPET = (total weight of stack) / (density of PET) Equation 2Thus, the PET volume VPET of 5.65 g of PET (for 100 disks of 22.4 mm diameter) was calculated to be 4.1 ml. The total volume Vtotai of the stack, including the PET volume VPET and the volume of the open space within the stack, was then calculated using the following formula:Vtotai = itx(0.5 x disk diameter) x (stacked bed height) Equation 3The 100-disk stack had a stack height of 25±1 mm. Thus, with a disk diameter of 22.4 mm, Vtotai was found to be 9.85 ml. Accordingly, porosity of the stacked bed can be calculated using the following:Porosity = (Vtotai - VPET) / Vtotai Equation 4Using Equation 4 and the above values, the porosity was calculated to be 58.4%, which is within the range predicted by the model.

[0100] Example 2

[0101] To understand the potential increased viral production yield of substrates of the present disclosure, the performance of a PET woven mesh substrate was compared to that of a substrate material similar to the substrate used in the iCellis®. Table 1 summarizes the total viral particles produces using these substrates in a simplified bioreactor.Table 1. Viral particles produced using PET woven mesh substrate material and non-woven substrate.From the results in Table 1, it is possible to calculate the volume of substrate material needed to produce a certain number of viral particles. For example, if the goal for production-scale viral vector production is 3.00E+18 viral particles, as shown in Table 2, the volume of PET woven mesh needed is about one-seventh the amount of the non-woven substrate needed.Table 2. Comparison of PET woven mesh substrate and non-woven substrate

[0102] Example 3

[0103] Residence time distribution (RTD) is a useful tool to study the flow in a vessel. Its theory, measurement, and analysis can be found in the textbook: Levenspiel, O. Chemical Reaction Engineering. 3ed. 1999. Wiley. New York. Figure 7 shows the schematic drawing of the setup to measure RTD. The bioreactor vessel containing the fixed bed is cylindrical with diameter of 29 mm and the total fixed bed volume is 36 ml. The fixed bed consisted of 3.6 g of non-woven mesh or, according to embodiments of this disclosure, 200 layers of open woven mesh. 1 : 2000 diluted McCormick Green Food Color was used as a tracer for the RTD measurement. A UV-vis with a Flowcell was used to monitor the change of tracer concentration. A flow rate of 22.5 ml / ml was used for all experiment. The chamber was first filled with water. After switched to green dye, the change of the optical density (OD) was recorded. The results in terms of normalized dye concentration versus time are shown in Figure 8. As shown in Figure 8, the non-woven mesh resulted in slower increase in dye concentration over a longer period of time as compared to the open woven mesh. For example, the normalized dye concentration for the non-woven mesh has a lower slope than for the open woven mesh, and takes more time to reach a comparable normalized concentration level.

[0104] The following equations were used to calculate mean residence time t (Equation (6)) and variance c (Equation (7)): t = j0(1 — F)dt Equation (6)Equation (7) where F is normalized concentration in a step tracer response. Table 3 summarizes the calculated mean residence time and variance from the measurement. The open woven mesh shows shorter mean residence time, which was likely caused by the lower porosity and decreased dead zones. In a packed bed of open woven mesh, the porosity was about 60% while the porosity of the nonwoven mesh has higher porosity which was about 93%. The much higher normalized variance detected in packed bed of non-woven mesh suggests that the flow in non-woven mesh was less uniform or ideal.Table 3. Means residence time and variance of non-woven and open woven mesh from measurement.

[0105] Using the same setup as described above, the RTDs of other commercially available packed bed substrate materials were measured. The packing density was calculated based on the vendor recommendation and to avoid loose packing. The change of dye concentration with the volume of water flowed through the vessels are shown in Figure 9. The measured changes of dyes were used to calculate their normalized variance as described in previous section. The calculated variances are listed in Table 4.Table 4. Normalized variance of different commercially available packed bed substrates vs. a woven mesh substrate of the present disclosure.

[0106] From the above permeability and residence time experiments, it is shown that the type of non-woven, irregular cell culture substrate used in current bioreactors has lower permeability than the substrate of the present disclosure. These non-woven substrates also have different permeability or flow rates depending on the direction of flow relative to the non-woven substrate, whereas the substrates of the present disclosure exhibit essentially isotropic flow behavior. Due to the non-uniform flow and lower residence time of the non-woven substrates, nutrients and transfection reagents can take longer to reach to the cells on the substrate surface or the other side of a substrate layer, as compared to the uniform, woven mesh substrate of the present disclosure. Adding to this is the higher permeability of the randomly packed non-woven substrate, which suggest a strong channeling effect and thus non-uniform delivery of cells or nutrients.

[0107] Example 4

[0108] To further quantify the flow uniformity in substrates according to embodiments of this disclosure, the compaction or occupancy of the fixed bed was controlled in experiments using a setup similar to that shown in Figure 16. Specifically, to compare and quantify the flow uniformity through different fixed beds, the RTD was measured. In each experiment, while maintaining flow through the bioreactor fixed bed, a small volume of a green dye solution was injected at the inlet of the bioreactor and the change of dye concentration over time was measured at the outlet at the outlet. The dye was injected over a direction of within 2 seconds, which is considered as a pulse input. The change in dye concentration at the outlet was recorded using a UV-vis spectrometer. Different flow rates were used during the measurement depending on the experimental designs.

[0109] The reactor used in these experiments had an internal diameter of 63 mm and 201.7 mm bed space height to hold substrate material as a fixed bed or packed bed. Substrates included open woven mesh disks, according to embodiments of this disclosure. To pack the reactor, each disk was aligned relative to a disk according to a 45° rotation about a z-axis, where the z-axis is parallel to the longitudinal axis of the bioreactor bed space and is perpendicular to the first and second major surfaces of the disk. Thus, the fibers in each woven disk were rotated 45° relative to the fibers in the neighboring disk. This rotation prevented interlocking of the fibers between disks and provided more consistent permeability. To control the packing density or occupancy, the height of the reactor was divided by the thickness of each mesh disk to obtain the number of disks for 100% occupancy. In this situation, the mesh disks filled all the way to the top of reactor space. Figure 10A shows this stack of disks at 100% occupancy. By removing 5% of the disk count from the 100% occupancy condition, we obtained 95% occupancy as shown in Figure 10B.

[0110] At lower occupancy, either the disks were compressed down, in which case empty space was created above the packed bed (this is referred to as compressed packing), as shown in Figure 1 IB, or the extra space was distributed across the packed bed without clear space on the top as shown in Figure 11 A (this is referred to as loose packing). Even at 100% occupancy, due to some elasticity of the mesh layer, the bed can still be compressed to some extent and result in a little space left above the fixed bed. This is called compressed packing at 100% occupancy, whereas without the applied compression there would be loose packing. In compressedconfiguration, about 2.5% extra disks were added to create 102.5% occupancy which means that the mesh disks were always maintained at highly compressed condition.

[0111] The experiments showed that compressed packing, including different occupancies from 95% to 102.5%, surprisingly provided good and comparable RTD profiles, demonstrating flow uniformity, as shown in Figure 12A. However, loose packing generated comparably worse results, which indicated more bypass at 100 ml / min, as shown in Figure 12B, as represented by a larger earlier peak of the E(0) curve. With increased flow rate, such as 300ml / min, the bypass was increased further, and more significant for lower occupancies and loose packing conditions, as demonstrated in Figure 12B and Figures 13A, 13B, and 13C. This means that, when a higher flow rate is needed during higher density cell culture, the flow uniformity will suffer more.

[0112] Figures 13A, 13B, and 13C show the E(0) curves using substate disks according to embodiments of this disclosure under different packing conditions. In Figure 13 A, the bed is packed at 102.5% occupancy. In Figure 13B, the bed is packed at 95% occupancy with compressed packing. In Figure 13C, the bed is paced at 95% occupancy with loose packing. As shown, the loose packing condition in Figure 13C results in an early peak (indicating channeling), a wider base of the E(0) curve, and more variability between flow rates, with a higher flow rate creating worse flow. In comparison, compressed packing, at both 102.5% and 95% occupancy, creates more uniform flow conditions that do not vary significantly with flow rate.

[0113] It should be noted that the E(0) curve is normalized and dimensionless, and thus bioreactors of different sizes can be easily compared on the same curve. According to embodiments of this disclosure, the substate and fixed beds show remarkably consistent E(0) curves regardless of the size of the reactor. For example, a fixed bed having a substrate surface area of 5 m2and one having a surface area of 100 m2have similar overlapping E(0) curves. These sizes are given for example, only, as it is anticipated that fixed beds of any size according to embodiments herein will have similar flow characteristics.

[0114] As would be understood by a person of ordinary skill in the art, an “early” peak means that the peak of the E(0) comes earlier 0 = 1 (and, in the above experiment, earlier than those of the compressed packing). A distribution of the RTD curve closer to 0 = 1 indicates more uniformflow or flow closer to a true “plug flow” condition. For example, a single, sharp peak in the E(0) curve indicates that all (or nearly all) dye particles injected spend about the same amount of time in the reactor, which indicates uniform flow that is close to idealized “plug flow”. However, in some substrates or beds, channeling can occur meaning that the permeability of the bed will be variable and the particles of dye used in the RTD experiment will spend variable amounts of time in the reactor. This can be indicated by an “early” peak on the E(0) curve, indicating dye that reached the bioreactor outlet early, and / or by a wider E(0) curve, indicating a longer length of time to clear the dye from the reactor.

[0115] Without wishing to be bound by theory, it is believed that with loose packing, the substrate disks may not be able to keep flat in the fixed bed due to less restriction from neighboring disks. Lifted or curved disks can create more gaps between the disks and vessel wall, and lead to more bypass. At higher flow rate, the flow can further lift the disks and make greater gaps and more bypass flow.

[0116] Example 5

[0117] To simulate the effect of bubbles, the setup described above for Example 4 was used, but 8 ml of air was injected at the inlet before corresponding RTD measurements. The obtained RTD profiles were compared with the same reactor before bubble introduction. As shown in Figures 14A and 14B, at lower occupancies, the RTD profile, or E(0) curve, has a wider spread, suggesting poor uniformity compared to those at higher occupancies. This was similar for both compressed packing (Figure 14A) and loose packing (Figure 14B).

[0118] Without wishing to be bound by theory, it is believed that, at high occupancy such as 100% or 102.5%, there is not much extra space in the packed bed for bubbles and most of the bubbles were pushed out of the bed by perfusion flow through the reactor, which made the impact much less. While at lower occupancies, bubbles were easily trapped in the extra spaces in the bed and then disturbed the perfusion flow through the bed. Since bubbles are difficult to avoid during cell culture, using high occupancy can help minimize the negative impact of bubbles, and maintain uniform flow to support better cell growth.

[0119] As discussed above, different substrate geometries (e.g., fiber diameter and pore diameter) can result in different amounts of substrate surface area being available for cell culture. As a result, the surface area density (e.g., the amount of available substrate surface area in a given space) can also increase. For example, the mesh shown in Figure 2D has a very fine fiber diameter and small pore diameter, resulting in a high surface area density when arranged as a fixed bed in a bioreactor vessel. However, there are challenges when implementing such high- density mesh substrate, due at least in part to the fine fibers used to achieve such small fiber diameters. For example, the smaller-diameter fibers can result in a mesh substrate layer that is far less rigid that substrate layers made from fibers that are two- to five-times thicker in diameter. As a result, the high-density mesh substrates may more easily deform within in the bioreactor, even when stacked with many other substrate layers. It can thus be difficult to ensure that the substrate layers lay flat within the bioreactor’s culture space. For example, the flow of fluid through the reactor culture space in a perfusion bioreactor can be sufficient to deform the high- density substrates to a degree that fluid bypass around the substrate layers (i.e., between the substrate layers and the wall of the reactors interior reservoir) increases, to detrimental effect on the cell culture performance.

[0120] Another challenge discovered with this high-density mesh is that processes for manufacturing the substrate layers can lead to increased thickness at the peripheral edges of the substrate layers. For example, mechanical cutting or laser cutting of mesh sheets to form substrate layers of the desired shape can lead to this increased thickness at the peripheral edge. This increased edge thickness makes uniformly stacking the substrate layers difficult. In addition, due to these high-density meshes having smaller openings / pores and a tighter weave, they provide more resistance to fluid flow. Therefore, it is possible that fluid bypass around the substrate layers is further exacerbated.

[0121] The increase in bypass for finer-diameter meshes has been confirmed by RTD analysis in accordance with the above-discussed RTD protocols. Accordingly, there is a need for bioreactors that can deliver high- surface-area densities without exacerbating fluid bypass and negatively impacting uniformity of the fluid flow and cell culture.

[0122] According to embodiments of this disclosure, a cell culture bioreactor with a fixed bed comprising high-density mesh substrate layers can be subject to a compression force to alleviate bypass issues. Specifically, a compression force applied in a direction that is orthogonal to the first and / or second major faces of the substrate layers (and, for example, parallel to the direction of bulk fluid flow through the fixed bed) is effective in reducing bypass. The compression force can be applied by a spring pushing down on the top of the fixed bed or pushing up on the bottom of the fixed bed. Also, or alternatively, the compression force can be applied when assembling the bioreactor by pressing one or more sections or an end cap (e.g., lid) to close the reactor while also exerting a compression force on the fixed bed.

[0123] In embodiments, the rigid member, such as a screen, grid, flow distribution plate, or other rigid member is used to press the fixed bed. The rigid member may be pushed against the fixed bed by a spring-like mechanism or by the walls of the reactor itself. In aspects of embodiments, an O-ring can also be employed to help evenly distribute force around the edge of the fixed bed, which has the benefit of sealing off that area to any bypass flow. The O-ring can be made of an elastomeric material, for example. Figures 15A and 15B show illustrations of cross-section elevation views of bioreactors with high-density substrate layers. In Figure 15 A, the bioreactor 700 contains a loosely-packed fixed bed comprising high-density substrate layers 702. The increased thicknesses of the peripheral edges 704 of the high-density substrate layers 702 are also shown. The layers 702 are loosely packed in this case because the layers are not compressed. However, in Figure 15B, the bioreactor 710 contains a densely -packed fixed bed comprising high-density substrate layers 712 that are subject to a compression force F. In this example, bioreactor 710 also includes an O-ring 716 and a rigid member 718 (e.g., flow distribution plate or grid) to help distribute the compression force F to the fixed bed. It is also contemplated, according to embodiments, that the peripheral edges 714 of the substrate layers 712 may be slightly compressed in response to the compression force F. The compression of the thickened portion of the peripheral edge may act to press those thickened portions outward against the vessel wall, thereby helping to reduce bypass.

[0124] Figure 16 shows the results from an RTD experiment comparing the two bioreactors 700 and 710. The same mesh substrate material was used in both bioreactors: a 53 / 40 mesh,which has fiber diameters of 53 microns. Because of the different packing densities, the bioreactor from Figure 15A has fewer substrate layers (550 layers) due to loose packing, compared to the higher packing density of the bioreactor from Figure 15B, which resulted in more substrate layers (670). The results show that the loosely-packed substrate layers led to significant bypass, whereas the substrate layers subject to a compression force showed very little or no bypass. Specifically, about 90% of the flow had a residence time of around 1, which indicates good flow through the fixed bed with little to no bypass.

[0125] To achieve the compression force, the packing density of the fixed bed can be increased by adding additional layers to the stack, and then using pressure to compress and flatten the layers into the culture space. For example, in experiment used to generate Figure 16, 20% more disks were added and about 1.5 to 2.0 lb / inch2of pressure was used to compress the layers. The RTD measurement showed that about majority of flow can go through the packed bed instead of bypass through the gap or guide rod keyhole. Without wishing to be bound by theory, it is believed that this solution prevents bypass by a combination of three mechanisms: (1) the axial force compresses the disks and causes them to expand radially, thus filling the gap around the edge of the disks; (2) the extra pressure compresses the outer peripheral edge forming a seal against radial flow to the outer bypass channel from fluid internal to the bed; and (3) the O-ring used to apply pressure on peripheral edge can limit prevent flow of media to outer channel by sealing.

[0126] In embodiments of this disclosure, the increased thickness at the edge of the substrate layers is an artefact of a process of cutting the layers from a mesh sheet. In some cases, the mesh sheet is cut by a laser. The heat from the laser causes the mesh to melt, resulting in the laser melt edge with increased thickness. According to embodiments of this disclosure, this process can be taken advantage of to provide a solution for preventing bypass, particularly for high-density mesh substrates. In particular, when cutting the substrate layers, multiple layers of mesh can be stacked on top of each other and cut simultaneously with a laser. As a result, the substrate layers that are simultaneously cut can be melded together along the cut line (i.e., the peripheral edge of the substrate layers) due to the melting and solidification of the substrate material from the laser cutting process. The result is a joined layer comprising two or more substrate layers joined attheir peripheral edges. This melt edge of the jointed layer can help fix the multiple layers together and provide several benefits. First, the relative thickness increase at the melt edge of a joined layer is smaller than the total thickness of the individual layers if not joined. Therefore, the resulting substrate layers become much flatter and easier to be stacked closely together. This also allows tighter packing density of substrate layers and higher density of substrate surface area in the bioreactor. Second, the joined layers formed by multiple layers joined together are much more rigid than a single layer of substrate. This can prevent or minimize substrate layer curving or deforming during assembly of the fixed bed or under perfusion flow conditions. Third, cutting multiple layers together can also help reduce process time and improve cutting and assembling efficiency. The presence of joined layers reduces the number of individual layers per reactor that are required to be packed, and the handling of the joined layers is easier due to them being more rigid than the individual substate layers.

[0127] Figure 17 shows the process of forming a joined layer and using a plurality of joined layers in a bioreactor. Specifically, two or more substrate layers 800 are provided. The substrate layers are cut simultaneously while lying on top of one another so that the laser melt edge 804 is formed and fuses the two or more substrate layers together as a joined layer 802. One of more of these joined layers can then be placed in a bioreactor vessel 806. According to embodiments, the fixed bed may be comprised entirely of joined layers or a mixture of one or more joined layers with one or more individual substrate layers. Additionally, embodiments are not limited to forming the joined layer by simultaneous laser cutting. Any suitable means for joining the lasers may be used, including laser ablation after the substrate layers are cut, or another form of heating to partially melt the peripheral edges together. Additionally, the peripheral edges can be joined by the use of an adhesive or polymer, such as a molded polymer.

[0128] In aspects of embodiments, the individual substrate layers of the joined layer may all be the same type of substrate material or different materials; or may all have the same physical characteristics or may have different physical characteristics. These physical characteristics can include, but are not limited to, fiber diameter, opening diameter, weave type, and more.

[0129] Several combinations of mesh layers can be used to form the joined layers. For example, the multiple substrate layers can be made from a same type of mesh. When two layers 1of the same woven mesh are stacked next to each other, if the fibers are perfectly aligned, the mesh can interlock and form a much tighter packing. The permeability at the interlocked location can be lOx smaller than the overlayed location as illustrated by CFD simulation. Since interlock does not always happen during stacking, this can create significantly variation in the permeability of the packed bed and therefore cause nonuniform flow. However, the fine pattern in the high-density mesh substrates can make perfect alignment difficult to happen. This can reduce the chance of interlock or minimize variation in permeability.

[0130] Alternatively, the multiple layers can be made from two or more types of mesh. The two or more types of mesh can be slightly different in their spacing (e.g., fiber diameter and / or opening diameter), such as 10% or higher, to prevent interlock. In each joined layer, the different types of mesh can be stacked in alternating order. When the total layer count in each disks is an odd number, such as 3, 5, and 7 layers, both top and bottom layers of the joined layer are the same type of mesh. Two groups of multiple layer disks can be made. Group A has mesh 1 on the outside (both top and bottom) and Group B has mesh 2 on the outside. By alternating Group A and Group B during stacking, direct contact of the same mesh can be prevented and interlock can be avoided.

[0131] In a variation of using two or more different meshes together in a joined layer, when the total layer count in each joined layer is an even number, such as 2, 4, and 6 layers, the top and bottom layers are made of different meshes. If one type of mesh is always kept on the top during staking, direct contact of the same type of mesh in the adjacent joined layer in a stack of joined layers can be prevented, therefore interlock can be avoided.

[0132] In an aspect of embodiments, the joining of different layers can aid in assembly by ensuring the substrate layers of a joined layer are oriented relative to one another in a desirable way. This is particularly helpful for high-density mesh substrates, which may be difficult to handle individually. Thus, the layers of mesh can be rotated relative one another prior to cutting so that the fibers of the layers in the joined layer will be in predetermined, fixed orientations after the simultaneous cutting operation due to the peripheral edges being joined. The multiple laser cut disks maintain relative orientation eliminating the need for a guid rod. Further sets of disks can include an orientation mark (from laser or other marking method) to indicate orientation.Because of easier handling, orientation marks can be used to assess quality control for the desired orientation (or alternating orientation)

[0133] Embodiments of this disclosure includes aspects of embodiments disclosed in U. S. Patent Application Nos. 16 / 781,685; and 16 / 781,723; and U.S. Provisional Patent Application No. 63 / 227,693, the contents of which are hereby incorporated herein in their entireties.

[0134] Illustrative Implementations

[0135] The following is a description of various aspects of implementations of the disclosed subject matter. Each aspect may include one or more of the various features, characteristics, or advantages of the disclosed subject matter. The implementations are intended to illustrate a few aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible implementations.

[0136] Aspect 1 pertains to a bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising an inlet, an outlet, and at least one interior reservoir defined by an interior wall and disposed in a fluid path between the inlet and the outlet, the cell culture vessel being configured allow fluid to flow in from the inlet, through the interior reservoir in a bulk flow direction, and out via the outlet; and a cell culture matrix disposed in the reservoir, the cell culture matrix comprising a plurality of substrates configured for adhering cells thereto, the plurality of substrates being stacked in a stacking direction parallel to the bulk flow direction, wherein the cell culture matrix comprises a matrix width extending across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted, and_wherein the plurality of substrates is under compression with a compression force in a compression direction that is substantially parallel to the stacking direction and substantially perpendicular to the reservoir width.

[0137] Aspect 2 pertains to the bioreactor system of Aspect 1, wherein the compression force is at least 1 lb / in2, at least 1.5 lb / in2, greater than or equal to 1 lb / in2and less than or equal to 3 lb / in2, less than or equal to 2 lb / in2, less than or equal to 3 lb / in2, greater than or equal to 1.5 lb / in2and less than or equal to 3 lb / in2, greater than or equal to 1 lb / in2and less than or equal to 2lb / in2, greater than or equal to 1.5 lb / in2and less than or equal to 2 lb / in2, greater than 0 lb / in2and less than or equal to 3 lb / in2, or greater than 0 lb / in2and less than or equal to 2 lb / in2.

[0138] Aspect 3 pertains to the bioreactor system of Aspect 1 or Aspect 2, wherein the cell culture matrix comprises a height measured in the stacking direction, and the height is compressed by at least 10% compared to the plurality of substrates when not subject to the compression force.

[0139] Aspect 4 pertains to the bioreactor system of Aspect 3, wherein the height is compressed by at least 20% compared to the plurality of substrates when not subject to the compression force.

[0140] Aspect 5 pertains to the bioreactor system of any of the preceding Aspects, wherein the compression force produces a radial force exerted by the plurality of substrates on the interior wall, the radial force being orthogonal to the compression force.

[0141] Aspect 6 pertains to the bioreactor system of any of the preceding Aspects, further comprising an O-ring disposed at a top or a bottom of the cell culture matrix in the stacking direction, the O-ring being disposed along a circumference of the interior wall.

[0142] Aspect 7 pertains to the bioreactor system of Aspect 6, wherein the O-ring comprises an elastomeric material.

[0143] Aspect 8 pertains to the bioreactor system of Aspect 6 or Aspect 7, wherein the O-ring is disposed between the cell culture matrix and a sealing surface, the O-ring being compressed between the cell culture matrix and the sealing surface.

[0144] Aspect 9 pertains to the bioreactor system of Aspect 8, wherein the sealing surface is a fluid flow distribution plate, a cell culture matrix support member, a top wall of the interior reservoir, or a bottom wall of the interior reservoir.

[0145] Aspect 10 pertains to the bioreactor system of any of the preceding Aspects, wherein at least one substrate of the plurality of substates comprises a peripheral edge surrounding an interior of the at least one substrate, the peripheral edge comprising an edge thickness that is greater than a thickness of the interior of the at least one substrate, and wherein the thickness of the interior and the edge thickness are measured in a direction parallel to the stacking direction.

[0146] Aspect 11 pertains to the bioreactor system of Aspect 10, wherein the peripheral edge is compressible in the edge thickness direction.

[0147] Aspect 12 pertains to the bioreactor system of Aspects 1-11, wherein two or more consecutive layers of the plurality of substrates are joined at peripheral edges of the two or more consecutive layers forming a joined layer.

[0148] Aspect 13 pertains to the bioreactor system of Aspect 12, wherein the plurality of substrates comprises a plurality of joined layers.

[0149] Aspect 14 pertains to the bioreactor system of Aspect 12 or Aspect 13, wherein the peripheral edges are joined by at least one of an adhesive material, a molded material, and a melted edge of one or more of the peripheral edges.

[0150] Aspect 15 pertains to the bioreactor system of Aspects 12-14, wherein the joined layer comprises layers of differing properties.

[0151] Aspect 16 pertains to the bioreactor system of Aspect 15, wherein the differing properties comprises at least one of a physical dimension, a material composition, a stiffness, and a porosity of the two or more consecutive layers forming the joined layer.

[0152] Aspect 17 pertains to the bioreactor system of Aspect 16, wherein the plurality of substates comprises fibers and pores separated by the fibers, and the physical dimension comprises at least one of a fiber diameter and a pore diameter.

[0153] Aspect 18 pertains to the bioreactor system of any of the preceding Aspects, wherein the plurality of substrates comprises a substrate layer comprising: a first major surface; a second major surface separated from the first major surface by a thickness; a plurality of fibers comprising a fiber diameter; and a plurality of pores comprising a pore diameter, each separated from others of the plurality of pores by the plurality of fibers and passing through the substrate layer from the first major surface to the second major surface.

[0154] Aspect 19 pertains to the bioreactor system of Aspect 18, wherein the fiber diameter is greater than 0 pm and is less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, or less than or equal to 30 pm.

[0155] Aspect 20 pertains to the bioreactor system of Aspect 18 or Aspect 19, wherein the opening diameter is greater than 0 pm and is less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, less than or equal to 30 pm, or less than or equal to 10 pm.

[0156] Aspect 21 pertains to the bioreactor system of Aspects 1-20, wherein the cell culture vessel is configured to flow fluid into the interior reservoir via the inlet, through the cell culture matrix, and out of the interior reservoir via the outlet in the bulk flow direction such that the fluid flows substantially in a linear path between the inlet and the outlet.

[0157] Aspect 22 pertains to the bioreactor system of any of the preceding Aspects, wherein the cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix, such that the uniform flow satisfies the following expressions:^ E(0)d0 = P,A0 = 02 - 0i, andA0 <N, wherein P is greater than or equal to at least one of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.95, and wherein Ais less than or equal to at least one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0.

[0158] Aspect 23 pertains to the bioreactor system of Aspect 22, wherein the uniform flow is maintained through a bed height of the fixed bed, the bed height being greater than or equal to 10 cm; 20 cm; 30 cm; 40 cm; 50 cm; 60 cm; 70 cm; 80 cm; 90 cm; or 100 cm.

[0159] Aspect 24 pertains to the bioreactor system of any of the preceding Aspects, wherein the cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix, such that E(0) is less than or equal to 0.2 or less than or equal to 0.1, when 0 is less than or equal to 0.8, 0.75, 0.7, 0.6, or 0.5.

[0160] Aspect 25 pertains to a bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising an inlet, an outlet, and at least one interior reservoir defined by an interior wall and disposed in a fluid path between the inlet and the outlet, the cell culture vessel being configured allow fluid to flow in from the inlet, through the interior reservoir in a bulk flow direction, and out via the outlet; and a cell culture matrix disposed in the reservoir, the cellculture matrix comprising a plurality of substrates configured for adhering cells thereto, the plurality of substrates comprising a substrate layer comprising: a first major surface; a second major surface separated from the first major surface by a thickness of the substrate layer; and a peripheral edge at the boundary for the first major surface and the second major surface, wherein the plurality of substrates are stacked in a stacking direction parallel to the bulk flow direction, and wherein the plurality of substrates comprises two or more of the substrate layers that are attached to each other at the peripheral edges of the two or more substrate layers forming a joined layer.

[0161] Aspect 26 pertains to the bioreactor system of Aspect 25, wherein the two or more substrate layers of the joined layer are not attached to each other at the first major surface and / or the second major surface within the peripheral edges.

[0162] Aspect 27 pertains to the bioreactor system of Aspect 25 or Aspect 26, wherein the cell culture matrix comprises a matrix width extending across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted.

[0163] Aspect 28 pertains to the bioreactor system of any of Aspects 25-27, wherein the plurality of substrates comprises a plurality of joined layers.

[0164] Aspect 29 pertains to the bioreactor system of any of Aspects 25-28, wherein the peripheral edges are joined by at least one of an adhesive material, a molded material, and a melted edge of one or more of the peripheral edges.

[0165] Aspect 30 pertains to the bioreactor system of any of Aspects 25-29, wherein the joined layer comprises two or more substrate layers of differing properties.

[0166] Aspect 31 pertains to the bioreactor system of Aspect 30, wherein the differing properties comprises at least one of a physical dimension, a material composition, a stiffness, and a porosity of the two or more consecutive layers forming the jointed layer.

[0167] Aspect 32 pertains to the bioreactor system of Aspect 30 or Aspect 31, wherein the plurality of substates comprises fibers and pores separated by the fibers, and the physical dimension comprises at least one of a fiber diameter and a pore diameter.

[0168] Aspect 33 pertains to the bioreactor system of any of Aspects 25-32, wherein a thickness of the peripheral edge of the substrate layer is greater than a thickness of an interior of the substrate layer located radially within the peripheral edge.

[0169] Aspect 34 pertains to the bioreactor system of any of Aspects 25-33, wherein the cell culture matrix is under compression with a compression force in a compression direction that is substantially parallel to the stacking direction and substantially perpendicular to the reservoir width.

[0170] Aspect 35 pertains to the bioreactor system of any of Aspects 25-34, wherein the joined layer has a rigidity that is greater than a rigidity of a same number of substrate layers that are not joined, the rigidity being measured with respect to a force applied in a direction of perpendicular to the first major surface.

[0171] Aspect 36 pertains to the bioreactor system of Aspects 25-35, wherein individual substrate layers of the joined layer are oriented with respect to each other to achieve maximum packing density in the joined layers.

[0172] Aspect 37 pertains to the bioreactor system of Aspect 36, wherein the individual substrate layers of the joined layer are oriented with respect to each other by rotating one or more of the substrate layers about a rotational axis that is perpendicular to the first major surface.

[0173] Aspect 38 pertains to the bioreactor system of Aspect 37, wherein the degree of rotation of the one or more substrate layers achieves an interlocking of fibers of the one or more substrate layers.

[0174] Aspect 39 pertains to the bioreactor system of any of Aspects 25-38, wherein the joined layer comprises two or more different types of substrate layers arranged in an alternating order in the stack of the joined layer.

[0175] Aspect 40 pertains to the bioreactor system of Aspect 39, wherein the two or more different types of substrate layers comprise substrates having different fiber diameters, different pore diameters, or different weave patterns.

[0176] Aspect 41 pertains to the bioreactor system of any of Aspects 25-40, wherein the joined layer comprises an even number of substrate layers.

[0177] Aspect 42 pertains to the bioreactor system of any of Aspects 25-41, wherein the plurality of substrates comprise one or more substrate layers comprising a fiber diameter that is greater than 0 pm and is less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, or less than or equal to 30 pm.

[0178] Aspect 43 pertains to the bioreactor system of any of Aspects 25-42, wherein the plurality of substrates comprise one or more substrate layers comprising an opening diameter that is greater than 0 pm and is less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, less than or equal to 30 pm, or less than or equal to 10 pm.Definitions

[0179] “Wholly synthetic” or “fully synthetic” refers to a cell culture article, such as a microcarrier or surface of a culture vessel, that is composed entirely of synthetic source materials and is devoid of any animal derived or animal sourced materials. The disclosed wholly synthetic cell culture article eliminates the risk of xenogeneic contamination.

[0180] ‘ ‘Include,” “includes,” or like terms means encompassing but not limited to, that is, inclusive and not exclusive.

[0181] ‘ ‘Users” refers to those who use the systems, methods, articles, or kits disclosed herein, and include those who are culturing cells for harvesting of cells or cell products, or those who are using cells or cell products cultured and / or harvested according to embodiments herein.

[0182] ‘ ‘About” modifying, for example, the quantity of an ingredient in a composition, concentrations, volumes, process temperature, process time, yields, flow rates, pressures, viscosities, and like values, and ranges thereof, or a dimension of a component, and like values, and ranges thereof, employed in describing the embodiments of the disclosure, refers to variation in the numerical quantity that can occur, for example: through typical measuring and handling procedures used for preparing materials, compositions, composites, concentrates, componentparts, articles of manufacture, or use formulations; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of starting materials or ingredients used to carry out the methods; and like considerations. The term “about” also encompasses amounts that differ due to aging of a composition or formulation with a particular initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a particular initial concentration or mixture.

[0183] “Optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0184] The indefinite article “a” or “an” and its corresponding definite article “the” as used herein means at least one, or one or more, unless specified otherwise.

[0185] Abbreviations, which are well known to one of ordinary skill in the art, may be used (e.g., “h” or “hrs” for hour or hours, “g” or “gm” for gram(s), “mL” for milliliters, and “rt” for room temperature, “nm” for nanometers, and like abbreviations).

[0186] Specific and preferred values disclosed for components, ingredients, additives, dimensions, conditions, and like aspects, and ranges thereof, are for illustration only; they do not exclude other defined values or other values within defined ranges. The systems, kits, and methods of the disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values described herein, including explicit or implicit intermediate values and ranges.

[0187] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that any particular order be inferred.

[0188] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the disclosed embodiments. Since modifications, combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and substance of the embodiments may occur to persons skilled in the art,the disclosed embodiments should be construed to include everything within the scope of the appended claims and their equivalents.

Claims

What is claimed:

1. A bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising an inlet, an outlet, and at least one interior reservoir defined by an interior wall and disposed in a fluid path between the inlet and the outlet, the cell culture vessel being configured allow fluid to flow in from the inlet, through the interior reservoir in a bulk flow direction, and out via the outlet; and a cell culture matrix disposed in the reservoir, the cell culture matrix comprising a plurality of substrates configured for adhering cells thereto, the plurality of substrates being stacked in a stacking direction parallel to the bulk flow direction, wherein the cell culture matrix comprises a matrix width extending across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted, and wherein the plurality of substrates is under compression with a compression force in a compression direction that is substantially parallel to the stacking direction and substantially perpendicular to the reservoir width.

2. The bioreactor system of claim 1, wherein the compression force is at least 1 lb / in2, at least 1.5 lb / in2, greater than or equal to 1 lb / in2and less than or equal to 3 lb / in2, less than or equal to 2 lb / in2, less than or equal to 3 lb / in2, greater than or equal to 1.5 lb / in2and less than or equal to 3 lb / in2, greater than or equal to 1 lb / in2and less than or equal to 2 lb / in2, greater than or equal to 1.5 lb / in2and less than or equal to 2 lb / in2, greater than 0 lb / in2and less than or equal to 3 lb / in2, or greater than 0 lb / in2and less than or equal to 2 lb / in2.

3. The bioreactor system of claim 1 or claim 2, wherein the cell culture matrix comprises a height measured in the stacking direction, and the height is compressed by at least 10% compared to the plurality of substrates when not subject to the compression force.

4. The bioreactor system of claim 3, wherein the height is compressed by at least 20% compared to the plurality of substrates when not subject to the compression force.

5. The bioreactor system of any of the preceding claims, wherein the compression force produces a radial force exerted by the plurality of substrates on the interior wall, the radial force being orthogonal to the compression force.

6. The bioreactor system of any of the preceding claims, further comprising an O-ring disposed at a top or a bottom of the cell culture matrix in the stacking direction, the O-ring being disposed along a circumference of the interior wall.

7. The bioreactor system of claim 6, wherein the O-ring comprises an elastomeric material.

8. The bioreactor system of claim 6 or claim 7, wherein the O-ring is disposed between the cell culture matrix and a sealing surface, the O-ring being compressed between the cell culture matrix and the sealing surface.

9. The bioreactor system of claim 8, wherein the sealing surface is a fluid flow distribution plate, a cell culture matrix support member, a top wall of the interior reservoir, or a bottom wall of the interior reservoir.

10. The bioreactor system of any of the preceding claims, wherein at least one substrate of the plurality of substates comprises a peripheral edge surrounding an interior of the at least one substrate, the peripheral edge comprising an edge thickness that is greater than a thickness of the interior of the at least one substrate, and wherein the thickness of the interior and the edge thickness are measured in a direction parallel to the stacking direction.

11. The bioreactor system of claim 10, wherein the peripheral edge is compressible in the edge thickness direction.

12. The bioreactor system of any one of claim 1-11, wherein two or more consecutive layers of the plurality of substrates are joined at peripheral edges of the two or more consecutive layers forming a joined layer.

13. The bioreactor system of claim 12, wherein the plurality of substrates comprises a plurality of joined layers.

14. The bioreactor system of claim 12 or claim 13, wherein the peripheral edges are joined by at least one of an adhesive material, a molded material, and a melted edge of one or more of the peripheral edges.

15. The bioreactor system of any of claims 12-14, wherein the joined layer comprises layers of differing properties.

16. The bioreactor system of claim 15, wherein the differing properties comprises at least one of a physical dimension, a material composition, a stiffness, and a porosity of the two or more consecutive layers forming the joined layer.

17. The bioreactor system of claim 16, wherein the plurality of substates comprises fibers and pores separated by the fibers, and the physical dimension comprises at least one of a fiber diameter and a pore diameter.

18. The bioreactor system of any of the preceding claims, wherein the plurality of substrates comprises a substrate layer comprising: a first major surface; a second major surface separated from the first major surface by a thickness; a plurality of fibers comprising a fiber diameter; anda plurality of pores comprising a pore diameter, each separated from others of the plurality of pores by the plurality of fibers and passing through the substrate layer from the first major surface to the second major surface.

19. The bioreactor system of claim 18, wherein the fiber diameter is greater than 0 pm and is less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, or less than or equal to 30 pm.

20. The bioreactor system of claim 18 or claim 19, wherein the opening diameter is greater than 0 pm and is less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, less than or equal to 30 pm, or less than or equal to 10 pm.

21. The bioreactor system of any of claims 1-20, wherein the cell culture vessel is configured to flow fluid into the interior reservoir via the inlet, through the cell culture matrix, and out of the interior reservoir via the outlet in the bulk flow direction such that the fluid flows substantially in a linear path between the inlet and the outlet.

22. The bioreactor system of any of the preceding claims, wherein the cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix, such that the uniform flow satisfies the following expressions: f^ E(0)d0 = P,A0 = 02 - 0i, andA0 <N, wherein P is greater than or equal to at least one of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 0.95, and wherein N is less than or equal to at least one of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0.

23. The bioreactor system of claim 22, wherein the uniform flow is maintained through a bed height of the fixed bed, the bed height being greater than or equal to 10 cm; 20 cm; 30 cm; 40 cm; 50 cm; 60 cm; 70 cm; 80 cm; 90 cm; or 100 cm.

24. The bioreactor system of any of the preceding claims, wherein the cell culture matrix is configured to maintain a uniform flow of liquid media through the cell culture matrix, such that E(0) is less than or equal to 0.2 or less than or equal to 0.1, when 0 is less than or equal to 0.8, 0.75, 0.7, 0.6, or 0.5.

25. A bioreactor system for culturing cells, the system comprising: a cell culture vessel comprising an inlet, an outlet, and at least one interior reservoir defined by an interior wall and disposed in a fluid path between the inlet and the outlet, the cell culture vessel being configured allow fluid to flow in from the inlet, through the interior reservoir in a bulk flow direction, and out via the outlet; and a cell culture matrix disposed in the reservoir, the cell culture matrix comprising a plurality of substrates configured for adhering cells thereto, the plurality of substrates comprising a substrate layer comprising: a first major surface; a second major surface separated from the first major surface by a thickness of the substrate layer; and a peripheral edge at the boundary for the first major surface and the second major surface, wherein the plurality of substrates are stacked in a stacking direction parallel to the bulk flow direction, and wherein the plurality of substrates comprises two or more of the substrate layers that are attached to each other at the peripheral edges of the two or more substrate layers forming a joined layer.

26. The bioreactor system of claim 25, wherein the two or more substrate layers of the joined layer are not attached to each other at the first major surface and / or the second major surface within the peripheral edges.

27. The bioreactor system of claim 25 or claim 26, wherein the cell culture matrix comprises a matrix width extending across a reservoir width of the interior reservoir such that fluid flow between the cell culture matrix and the interior wall is restricted.

28. The bioreactor system of any of claims 25-27, wherein the plurality of substrates comprises a plurality of joined layers.

29. The bioreactor system of any of claim 25-28, wherein the peripheral edges are joined by at least one of an adhesive material, a molded material, and a melted edge of one or more of the peripheral edges.

30. The bioreactor system of any of claims 25-29, wherein the joined layer comprises two or more substrate layers of differing properties.

31. The bioreactor system of claim 30, wherein the differing properties comprises at least one of a physical dimension, a material composition, a stiffness, and a porosity of the two or more consecutive layers forming the jointed layer.

32. The bioreactor system of claim 30 or claim 31, wherein the plurality of substates comprises fibers and pores separated by the fibers, and the physical dimension comprises at least one of a fiber diameter and a pore diameter.

33. The bioreactor system of any of claims 25-32, wherein a thickness of the peripheral edge of the substrate layer is greater than a thickness of an interior of the substrate layer located radially within the peripheral edge.

34. The bioreactor system of any of claims 25-33, wherein the cell culture matrix is under compression with a compression force in a compression direction that is substantially parallel to the stacking direction and substantially perpendicular to the reservoir width.

35. The bioreactor system of any of claims 25-34, wherein the joined layer has a rigidity that is greater than a rigidity of a same number of substrate layers that are not joined, the rigidity being measured with respect to a force applied in a direction of perpendicular to the first major surface.

36. The bioreactor system of any of claims 25-35, wherein individual substrate layers of the joined layer are oriented with respect to each other to achieve maximum packing density in the joined layers.

37. The bioreactor system of claim 36, wherein the individual substrate layers of the joined layer are oriented with respect to each other by rotating one or more of the substrate layers about a rotational axis that is perpendicular to the first major surface.

38. The bioreactor system of claim 37, wherein the degree of rotation of the one or more substrate layers achieves an interlocking of fibers of the one or more substrate layers.

39. The bioreactor system of any of claims 25-38, wherein the joined layer comprises two or more different types of substrate layers arranged in an alternating order in the stack of the joined layer.

40. The bioreactor system of claim 39, wherein the two or more different types of substrate layers comprise substrates having different fiber diameters, different pore diameters, or different weave patterns.

41. The bioreactor system of any of claims 25-40, wherein the joined layer comprises an even number of substrate layers.

42. The bioreactor system of any of claims 25-41, wherein the plurality of substrates comprise one or more substrate layers comprising a fiber diameter that is greater than 0 pm and is less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, or less than or equal to 30 pm.

43. The bioreactor system of any of claims 25-42, wherein the plurality of substrates comprise one or more substrate layers comprising an opening diameter that is greater than 0 pm and is less than or equal to 250 pm, less than or equal to 200 pm, less than or equal to 150 pm, less than or equal to 100 pm, less than or equal to 75 pm, less than or equal to 55 pm, less than or equal to 40 pm, less than or equal to 30 pm, or less than or equal to 10 pm.

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