Uniform flow roll-like adherent cell culture substrate for use in fixed bed reactors

Abstract

A cell culture substrate for culturing cells in a fixed bed reactor is provided. The cell culture substrate includes a first substrate material having an ordered and regular array of openings through a layer, the openings being separated by the substrate material, the substrate material having a substantially regular and homogeneous physical structure and the physical structure being configured to grow cells thereon. The physical structure and array of openings are configured such that when the first substrate material is rolled into a roll-like substrate matrix and the roll-like substrate matrix comprises a roll-like layer of the first substrate material, the roll-like layer has a change in packing density of less than about 20 x, less than about 10 x, less than about 5 x, or less than about 2 x throughout the roll-like substrate matrix.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119 to U.S. Provisional Application Serial No. 63 / 395,409 filed on August 5, 2022 and U.S. Provisional Application Serial No. 63 / 394,848 filed on August 3, 2022. This application is based on the contents of both and the entire text of each is incorporated herein by reference. Technical Field

[0002] The present disclosure generally relates to a substrate for culturing cells, and a fixed bed bioreactor comprising the substrate. In particular, the present disclosure relates to a fixed bed having a rolled substrate exhibiting uniform fluid flow characteristics, and a bioreactor having the fixed bed. Background Art

[0003] In the bioprocessing industry, cells are cultured on a large scale for the production of hormones, enzymes, antibodies, vaccines, and cell therapies. The cell and gene therapy market is growing rapidly, with promising treatments entering clinical trials and moving quickly toward commercialization. However, a cell therapy dose may require billions of cells or trillions of viruses. Therefore, being able to provide large quantities of cell or virus products in a short period of time is critical to clinical success.

[0004] Most cells used in bioprocessing are anchorage-dependent, meaning that the cells need a surface to attach to in order to grow and function. Traditionally, the culture of adherent cells is performed on two-dimensional (2D) surfaces to which the cells adhere, which are contained in one of a variety of container formats such as T-flasks, petri dishes, cell factories, cell stack containers, roller bottles, and These approaches can have significant disadvantages, including difficulty in achieving high enough cell densities to make them suitable for large-scale production of cells or therapeutics.

[0005] Alternative methods have been proposed to increase the volume density of cultivated cells. These include microcarrier cultures carried out in stirred tanks. In this way, cells attached to the microcarrier surface are subjected to constant shear stress, resulting in a significant impact on proliferation and culture performance. Another example of a high-density cell culture system is a hollow fiber bioreactor, in which cells can form large three-dimensional aggregates when they proliferate in the interstitial space of the fibers. However, cell growth and performance are greatly inhibited because of a lack of nutrients. In order to alleviate this problem, these bioreactors are made smaller 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 type of bioreactor, a cell substrate is randomly packed into the bioreactor container and used to provide a surface for adherent cells to attach. The culture medium is perfused along the surface or through the packed bed to provide nutrients and oxygen required for cell growth. For example, a packed bed bioreactor system comprising a packed bed for capturing cells has been previously disclosed in U.S. Pat. Nos. 4,833,083; 5,501,971 and 5,510,262. The packed bed matrix is ​​often made of non-woven microfibers of porous particles or polymers used as a substrate. This bioreactor acts as a recirculating flow bioreactor. One of the major problems of this bioreactor is the uneven distribution of cells in the packed bed. For example, the packed bed is used as a deep filter, and cells are mainly captured at the inlet area, resulting in a cell distribution gradient during the inoculation step. In addition, due to the random packing of the fibers, the flow resistance and cell capture efficiency of the packed bed cross section are uneven. For example, the culture medium flows quickly through areas with low cell packing density, while flowing slowly through areas with higher resistance due to a higher number of trapped cells. This creates a channeling effect, where nutrients and oxygen are more efficiently delivered to areas with lower cell volume density, while areas with higher cell density remain under suboptimal culture conditions.

[0007] Another significant disadvantage of the packed bed systems disclosed in the prior art is the inability to efficiently harvest intact live cells at the end of the culture process. Cell harvesting is important if the final product is cells, or if the bioreactor is used as part of a "seed training" where a cell population is grown in one container and then transferred to another container for further cell population growth. U.S. Patent No. 9,273,278 discloses a bioreactor design for increasing the efficiency of recovering cells from a packed bed during a cell harvesting step. It is based on loosening the packed bed matrix and agitating or stirring the packed bed particles so that the porous matrix collides and thereby debonds the cells. However, this method is laborious and can cause significant cell damage, thereby reducing overall cell viability.

[0008] For example, some packed-bed bioreactors use small strips of cell substrate material that consists of randomly oriented fibers arranged in a nonwoven arrangement. These strips are packed into a container to create a packed bed. However, uneven packing of the substrate strips can create visible channels within the packed bed, resulting in preferential and uneven distribution of medium flow and nutrients through the packed bed. Studies of such systems have noted “systematic uneven distribution of cells, with cell numbers increasing from the top to the bottom of the fixed bed,” as well as “nutrient gradients…resulting in limited cell growth and proliferation,” all of which lead to “uneven cell distribution, which can 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 can improve dispersion, but can also have other disadvantages (i.e., “agitation necessary for better dispersion during inoculation and transfection can lead to increased shear stress, which in turn can result in reduced cell viability.” Ibid.). Another study noted that uneven distribution of cells makes it difficult to monitor cell populations using biomass sensors (“…if cells are unevenly distributed, the biomass signal from cells on the top carriers may not be indicative of the bioreactor population.” Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale. Human Gene Therapy, Vol. 26, No. 8, 2015).

[0009] Furthermore, due to the random arrangement of fibers in the substrate strips and the variability of strip packing between one packed bed and another given packed bed reactor system, it can be difficult for the user to predict cell culture performance because the substrate is not only non-uniform within a given bioreactor vessel, but also varies between cell culture runs with different reactors or different packed beds. Additionally, the packing substrates of many existing bioreactors make it very difficult or impossible to efficiently harvest cells because it is believed that the cells are trapped by the packed bed.

[0010] A platform capable of producing a greater number of high-quality cell and virus products is needed to reach a later stage commercial production scale. In particular, a cell culture substrate and / or matrix, bioreactor, system and method capable of culturing cells in a high density format, with uniform cell distribution, improved and uniform fluid flow characteristics, and easy access and increased harvest yield is needed. Summary of the invention

[0001] According to an embodiment of the present disclosure, a cell culture matrix for culturing cells in a fixed bed bioreactor is provided. A cell culture matrix for culturing cells in a fixed bed reactor is provided. The cell culture matrix comprises a first substrate material having an ordered and regular array of openings passing through a layer, the openings being separated by a substrate material, the substrate material having a substantially regular and uniform physical structure and the physical structure being configured to allow cells to grow thereon. The physical structure and the array of openings are configured such that when the first substrate material is rolled into a roll-like substrate matrix and the roll-like substrate matrix comprises a roll-like layer of the first substrate material, in the entire roll-like substrate matrix, the packing density variation of the roll-like layer is less than about 20x, less than about 10x, less than about 5x or less than about 2x.

[0002] The following is a description of various aspects of various embodiments 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 embodiments are intended to illustrate several aspects of the disclosed subject matter and should not be considered as a comprehensive or exhaustive description of all possible embodiments.

[0003] Aspect 1 relates to a cell culture substrate for culturing cells in a fixed bed reactor, the cell culture substrate comprising a first substrate material constituting a first layer, the first layer having opposing faces separated by layer thickness, an ordered and regular first array of openings passing through the first layer, and a first physical structure that is substantially regular and uniform and configured to allow cells to grow thereon. The first physical structure separates the openings of the first array of openings from one another. The cell culture substrate also comprises a second substrate material constituting a second layer, the second layer having opposing faces separated by layer thickness, an ordered and regular second array of openings passing through the second layer, and a second physical structure that is substantially regular and uniform and configured to allow cells to grow thereon, wherein the second physical structure separates the openings of the second array of openings from one another. The first substrate material and the second substrate material are rolled together to form a rolled cell culture bed. The first physical structure is different from the second physical structure, whereby the packing density of the first substrate material and the second substrate material is substantially uniform throughout the rolled cell culture bed.

[0004] Aspect 2 relates to the cell culture substrate of aspect 1, wherein the packing density of the first substrate material and the second substrate material varies by less than about 20x, less than about 10x, less than about 5x, or less than about 2x throughout the rolled cell culture bed.

[0005] Aspect 3 relates to the cell culture substrate of aspect 1 or aspect 2, wherein the first substrate material includes a first plurality of fibers defining a first array of openings, and the second substrate material includes a second plurality of fibers defining a second array of openings, wherein the first physical structure includes a first spacing of the first substrate material, the first spacing being the sum of the fiber diameters of the first plurality of fibers and the opening diameters of the first array of openings, and wherein the second physical structure includes a second spacing of the second substrate material, the second spacing being the sum of the fiber diameters of the second plurality of fibers and the opening diameters of the second array of openings.

[0006] Aspect 4 relates to the cell culture substrate of aspect 3, wherein the second spacing is different from the first spacing.

[0007] Aspect 5 relates to the cell culture substrate of Aspect 4, wherein the second spacing is within about 100% of the first spacing, the second spacing is within about 90% of the first spacing, the second spacing is within about 75% of the first spacing, the second spacing is within about 50% of the first spacing, the second spacing is within about 25% of the first spacing, the second spacing is within about 15% of the first spacing, the second spacing is within about 10% of the first spacing, or the second spacing is within about 50% of the first spacing.

[0008] Aspect 6 relates to the cell culture substrate of any one of aspects 1-5, wherein at least one of the first substrate material and the second substrate material comprises at least one of a molded polymer lattice, a 3D printed lattice, and a woven mesh.

[0009] Aspect 7 relates to the cell culture substrate of any of the preceding aspects, wherein at least one of the first substrate material and the second substrate material is a woven substrate.

[0010] Aspect 8 relates to the cell culture substrate of any of the preceding aspects, wherein at least one of the first substrate material and the second substrate material comprises at least one of the following: simple weave, twill weave, Dutch weave, satin weave, regular matte weave, irregular matte rib weave, irregular weft rib weave, regular weft rib weave, regular warp rib weave, irregular warp rib weave, satin weave, 2 / 2 twill weave, 3 / 3 twill weave, basket weave, mountain twill weave, royal oxford weave, houndstooth weave, herringbone weave, or five-needle weave.

[0011] Aspect 9 relates to the cell culture substrate of any of the preceding aspects, wherein the first substrate material and the second substrate material are arranged as alternating layers of a rolled cell culture bed.

[0012] Aspect 10 relates to the cell culture substrate of any of the preceding aspects, wherein the first substrate material and the second substrate material are in direct physical contact with each other.

[0013] Aspect 11 relates to the cell culture substrate of any of the preceding aspects, wherein the first substrate material and the second substrate material are not separated from each other by a spacer material or barrier.

[0014] Aspect 12 relates to the cell culture substrate of any of the preceding aspects, further comprising a third substrate material constituting a third layer, the third layer having opposing faces separated by the thickness of the third layer, an ordered and regular third array of openings passing through the third layer, and a third physical structure that is substantially regular and uniform and configured to allow cells to grow thereon, the third physical structure separating the openings of the third array of openings from each other, wherein the first substrate material, the second substrate material, and the third substrate material are rolled together to form a rolled cell culture bed.

[0015] Aspect 13 relates to the cell culture substrate of any of the preceding aspects, wherein the rolled cell culture bed has no spacing material or barrier between subsequent substrate layers of the rolled cell culture bed.

[0016] Aspect 14 relates to the cell culture substrate of any of the preceding aspects, wherein the first substrate material and the second substrate material differ from each other in at least one physical dimension.

[0017] Aspect 15 relates to the cell culture substrate of Aspect 1, wherein the at least one physical dimension is at least one of a diameter of the opening, a thickness of the physical structure, a pattern of the physical structure, a weave type, and a spacing of the physical structures on either side of the opening.

[0018] Aspect 16 relates to the cell culture substrate of any of the preceding aspects, wherein the variation in flow rate of the fluid flowing through the cell culture substrate is uniform across the width of the cell culture substrate, the width being a direction perpendicular to the direction of fluid flow.

[0019] Aspect 17 relates to the cell culture substrate of any of the preceding aspects, wherein the plurality of substrate layers comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0020] Aspect 18 relates to the cell culture substrate of any of the preceding aspects, wherein the plurality of fibers comprises first fibers having a first fiber diameter, the first fiber diameter being about 30 μm to about 1000 μm, about 30 μm to about 600 μm, about 30 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0021] Aspect 19 relates to the cell culture substrate of any of the preceding claims, wherein the openings comprise a diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

[0022] Aspect 20 relates to a bioreactor system for culturing cells, the system comprising a cell culture vessel comprising at least one internal reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir; and a cell culture matrix according to any one of aspects 1-19, the cell culture matrix being disposed in the reservoir.

[0023] Aspect 21 relates to the bioreactor system of aspect 20, wherein the bioreactor system is configured to pass a perfusion flow through the cell culture matrix during cell culture.

[0024] Aspect 22 relates to the bioreactor system of aspect 20 or aspect 21, wherein the bioreactor system is configured to harvest living cells from the cell culture matrix in the reservoir.

[0025] Aspect 23 relates to a cell culture substrate for culturing cells in a fixed bed reactor, the cell culture substrate comprising a first substrate material constituting a first layer; and a second substrate material constituting a second layer, the first layer having opposing faces separated by a layer thickness, a first array of openings passing through the first layer, and a first physical structure comprising a first fiber array, the first physical structure being configured to allow cells to grow thereon and to separate the openings of the first array of openings from one another; the second substrate material having opposing faces separated by a layer thickness, a second array of openings passing through the second layer, and a second physical structure comprising a second fiber array, the second physical structure being configured to allow cells to grow thereon and to separate the openings of the second array of openings from one another, wherein the first substrate material and the second substrate material are rolled together to form a rolled cell culture bed, wherein the first physical structure is different from the second physical structure.

[0026] Aspect 24 relates to the cell culture substrate of aspect 23, wherein the packing density of the first substrate material and the second substrate material is substantially uniform throughout the rolled cell culture bed due to the first physical structure being different from the second physical structure.

[0027] Aspect 25 relates to the cell culture substrate of aspect 23 or aspect 24, wherein the first array of openings is in an ordered and regular first array.

[0028] Aspect 26 relates to the cell culture substrate of any one of aspects 23-25, wherein the second array of openings is in an ordered and regular second array.

[0029] Aspect 27 relates to the cell culture substrate of any one of aspects 23-26, wherein the first physical structure and the second physical structure are substantially regular and uniform.

[0030] Aspect 28 relates to the cell culture substrate of any of aspects 23-27, wherein the first physical structure and the second physical structure comprise fibers in a non-random arrangement.

[0031] Aspect 29 relates to a cell culture substrate for culturing cells in a fixed bed reactor, the cell culture substrate comprising a first substrate material constituting a first layer, the first layer having opposing faces separated by the thickness of the first layer, an ordered and regular array of openings through the first layer, and a physical structure that is substantially regular and uniform and configured to allow cells to grow thereon, the physical structure separating the openings from each other, wherein the first substrate material has a three-dimensional surface defined by the array of openings and the physical structure, and wherein the three-dimensional surface is configured to prevent the first layer of substrate material from nesting with a second layer of substrate material, the second layer being adjacent to the first layer and in direct physical contact with the first layer.

[0032] Aspect 30 relates to the cell culture substrate of aspect 29, wherein the first substrate material is a single piece constituting the first layer and the second layer.

[0033] Aspect 31 relates to the cell culture substrate of aspect 30, wherein the single sheet is rolled up to form a roll-shaped cell culture bed, wherein the rolling up forms a plurality of radially arranged roll layers in the roll-shaped cell culture bed, the plurality of roll layers comprising a first layer and a second layer.

[0034] Aspect 32 relates to the cell culture substrate of any of aspects 29-31, wherein the first substrate material is a woven substrate comprising a plurality of fibers woven together in a predetermined pattern.

[0035] Aspect 33 relates to the cell culture substrate of aspect 32, wherein the woven substrate comprises a twill weave, a Dutch weave, a satin weave, a regular matte weave, an irregular matte rib weave, an irregular weft rib weave, a regular weft rib weave, a regular warp rib weave, an irregular warp rib weave, a satin weave, a 2 / 2 twill weave, a 3 / 3 twill weave, a basket weave, a mountain twill weave, a royal oxford weave, a houndstooth weave, a herringbone weave, or a five-needle weave.

[0036] Aspect 34 relates to the cell culture substrate of any of Aspects 29-33, further comprising a second substrate material having an ordered and regular array of openings through the layer, the openings being separated by the substrate material, the substrate material having a substantially regular and uniform physical structure and the physical structure being configured to allow cells to grow thereon, wherein the second substrate material constitutes a second layer, and wherein the first substrate material and the second substrate material have the same physical structure and the same array of openings.

[0037] Aspect 35 relates to the cell culture substrate of aspect 34, wherein the first substrate layer and the second substrate layer are stacked in an alternating arrangement.

[0038] Aspect 36 relates to the cell culture substrate of aspect 34, wherein the first substrate material and the second substrate material are rolled up to form a rolled cell culture bed, wherein the rolling up forms a plurality of radially arranged roll layers in the rolled cell culture bed, the plurality of roll layers including a first layer and a second layer.

[0039] Aspect 37 relates to the cell culture substrate of any of Aspects 29-33, further comprising a second substrate material having an ordered and regular array of openings through the layer, the openings being separated by the substrate material, the substrate material having a substantially regular and uniform physical structure and the physical structure being configured to allow cells to grow thereon, wherein the second substrate material constitutes a second layer, and wherein the first substrate material and the second substrate material differ from each other in at least one physical dimension, the first substrate material and the second substrate material being arranged as alternating layers of the cell culture substrate.

[0040] Aspect 38 relates to the cell culture substrate of any of Aspects 34-37, wherein the second substrate material comprises a twill weave, a Dutch weave, a satin weave, a regular matte weave, an irregular matte rib weave, an irregular weft rib weave, a regular weft rib weave, a regular warp rib weave, an irregular warp rib weave, a satin weave, a 2 / 2 twill weave, a 3 / 3 twill weave, a basket weave, a mountain twill weave, a royal oxford weave, a houndstooth weave, a herringbone weave, a five-needle weave, a micromesh, or a mesh net.

[0041] Aspect 39 relates to the cell culture substrate of any of Aspects 37-38, wherein the at least one physical dimension is at least one of a diameter of the opening, a thickness of the physical structure, a pattern of the physical structure, and a spacing of the physical structures on either side of the opening.

[0042] Aspect 40 relates to the cell culture substrate of any of aspects 34-39, wherein the first substrate material and the second substrate material comprise at least one of a molded polymer lattice, a 3D printed lattice, and a woven mesh.

[0043] Aspect 41 relates to the cell culture substrate of any of aspects 29-40, wherein the degree of relative rotation between the first substrate material and the second substrate material is random.

[0044] Aspect 42 relates to the cell culture substrate of any of aspects 29-41, wherein the variation in flow rate of the fluid flowing through the cell culture substrate is uniform across the width of the cell culture substrate, the width being a direction perpendicular to the direction of fluid flow.

[0045] Aspect 43 relates to the cell culture substrate of any one of aspects 31-42, wherein the rolled layers are not separated by spacers or barriers, or they are in physical contact with each other.

[0046] Aspect 44 relates to the cell culture substrate of any of aspects 29-43, wherein the first substrate material comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0047] Aspect 45 relates to the cell culture substrate of any of Aspects 32-44, wherein the plurality of fibers comprises first fibers having a first fiber diameter of about 30 μm to about 1000 μm, about 30 μm to about 600 μm, about 30 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0048] Aspect 46 relates to the cell culture substrate of any of aspects 29-45, wherein the openings comprise a diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

[0049] Aspect 47 relates to the cell culture substrate of any of Aspects 29-46, wherein the physical structure and the array of openings are configured such that when the first substrate material is rolled into a roll-like substrate matrix and the roll-like substrate matrix comprises a roll-like layer of the first substrate material, the packing density of the roll-like layer varies by less than about 20x, less than about 10x, less than about 5x or less than about 2x throughout the roll-like substrate matrix.

[0050] Aspect 48 relates to a bioreactor system for culturing cells, the system comprising a cell culture vessel comprising at least one internal reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir; and a cell culture matrix according to any one of Aspects 29-47, the cell culture matrix being disposed in the reservoir.

[0051] Aspect 49 relates to the bioreactor system of aspect 48, wherein the bioreactor system is configured to pass a perfusion flow through the cell culture matrix during cell culture.

[0052] Aspect 50 relates to the bioreactor system of aspect 48 or aspect 49, wherein the bioreactor system is configured to harvest living cells from the cell culture matrix in the reservoir. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1A According to one or more embodiments of the present disclosure, a perspective view of a three-dimensional model of a cell culture substrate is shown.

[0012] Figure 1B yes Figure 1A Two-dimensional plan view of the substrate.

[0013] Figure 1C yes Figure 1B A cross section of the substrate along line AA.

[0014] Figure 2 A schematic diagram of a cell culture system having a fixed bed cell culture matrix is ​​shown according to one or more embodiments.

[0015] Figure 3A According to one or more embodiments of the present disclosure, a plan view of a molded multilayer woven mesh cell culture substrate in a staggered or close packed arrangement is shown.

[0016] Figure 3B According to one or more embodiments of the present disclosure, it is shown Figure 3A Side cross-sectional view of a multilayer woven mesh cell culture substrate.

[0017] Figure 4A According to one or more embodiments of the present disclosure, a plan view of a molded multilayer woven mesh cell culture substrate in an aligned or loose fill arrangement is shown.

[0018] Figure 4B According to one or more embodiments of the present disclosure, it is shown Figure 4A Side cross-sectional view of a multilayer woven mesh cell culture substrate.

[0019] Figure 5A Shows Figure 3A and 3BThe molded empty space in the dotted volume shown in .

[0020] Figure 5B Shows Figure 4A and 4B The molded empty space in the dotted volume shown in .

[0021] Fig. 6A According to an embodiment of the present disclosure, a schematic plan view of two layers of substrates stacked or superimposed in an aligned arrangement with a relative rotation of 0° between the layers is shown.

[0022] Figure 6B According to an embodiment of the present disclosure, a schematic plan view of two substrate layers stacked or superimposed in a staggered arrangement with the layers relatively rotated 0° with respect to each other is shown.

[0023] Figure 6C According to an embodiment of the present disclosure, a schematic plan view of two superimposed or stacked substrates with the layers relatively rotated 5° is shown.

[0024] Fig.6D According to an embodiment of the present disclosure, a schematic plan view of two superimposed or stacked substrates with the layers relatively rotated by 10° is shown.

[0025] Fig. 6E According to an embodiment of the present disclosure, a schematic plan view of two layers of substrates stacked or superimposed with the layers relatively rotated by 15° is shown.

[0026] Fig. 6F According to an embodiment of the present disclosure, a schematic plan view of two superimposed or stacked substrates with the layers relatively rotated by 20° is shown.

[0027] Figure 6G According to an embodiment of the present disclosure, a schematic plan view of two superimposed or stacked substrates with the layers relatively rotated by 25° is shown.

[0028] Figure 6H According to an embodiment of the present disclosure, a schematic plan view of two superimposed or stacked substrates with the layers relatively rotated by 30° is shown.

[0029] Fig.6I According to an embodiment of the present disclosure, a schematic plan view of two superimposed or stacked substrates with the layers relatively rotated by 35° is shown.

[0030] Figure 6J According to an embodiment of the present disclosure, a schematic plan view of two superimposed or stacked substrates with the layers relatively rotated 40° is shown.

[0031] Figure 6KAccording to an embodiment of the present disclosure, a schematic plan view of two superimposed or stacked substrates with the layers relatively rotated by 45° is shown.

[0032] Fig. 7A is a schematic diagram of fluid flow rates before and after passing through a substrate layer according to an embodiment of the present disclosure.

[0033] Figure 7B According to an embodiment of the present disclosure, Fig. 7A A graph of the flow rate of a fluid flowing through a substrate layer.

[0034] Figure 8 According to an embodiment of the present disclosure, Fig. 7A and 7B Graph of the resistance of a medium porous substrate to fluid flow.

[0035] Fig. 9 is a three-dimensional illustration of a simplified model of a substrate material for modeling flow through a two-layer substrate, according to an embodiment.

[0036] Fig.10 is a graphical representation of simulated flow velocity variation along a woven web surface using a sinusoidal model, according to an embodiment of the present disclosure.

[0037] Fig.11A is a graphical representation of simulated flow rate variation along the surface of a two-layer woven mesh stack using a sinusoidal model for the substrate layer and having a staggered arrangement and a relative rotation of 0° between the layers according to an embodiment of the present disclosure.

[0038] Fig. 11B is a graphical representation of simulated flow rate variation along the surface of a two-layer woven mesh stack using a sinusoidal model for the substrate layer and having an aligned arrangement and a relative rotation of 0° between the layers in accordance with an embodiment of the present disclosure.

[0039] Fig. 12A According to the embodiments of the present disclosure, it is shown that Fig. 6A and 6B Two graphical representations of simulated flow rate variations along the surface of a stack of two identical woven web substrate layers for the boundary conditions of perfect alignment and staggered alignment shown, with a relative rotation of 0° between the layers.

[0040] Fig. 12B A graphical representation of simulated flow rate variation along the surface of a stack of two identical woven mesh substrate layers is shown, in accordance with an embodiment of the present disclosure, wherein the layers are rotated 5° relative to each other.

[0041] Fig. 12CAccording to an embodiment of the present disclosure, a graphical representation of simulated flow rate variation along the surface of a stack of two identical woven mesh substrate layers is shown, wherein the layers are rotated 10° relative to each other.

[0042] Fig.12D According to an embodiment of the present disclosure, a graphical representation of simulated flow rate variation along the surface of a stack of two identical woven mesh substrate layers is shown, wherein the layers are rotated 20° relative to each other.

[0043] Fig.12E According to an embodiment of the present disclosure, a graphical representation of simulated flow rate variation along the surface of a stack of two identical woven mesh substrate layers is shown, wherein the layers are rotated 30° relative to each other.

[0044] Fig.12F According to an embodiment of the present disclosure, a graphical representation of simulated flow rate variation along the surface of a stack of two identical woven mesh substrate layers is shown, wherein the layers are rotated 45° relative to each other.

[0045] Fig.13A A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.2 and a relative rotation of 0° between the layers is shown, according to an embodiment of the present disclosure.

[0046] Fig. 13B A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.2 and a relative rotation of 5° between the layers is shown, according to an embodiment of the present disclosure.

[0047] Fig. 13C A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.2 and a relative rotation of 10° between the layers is shown, according to an embodiment of the present disclosure.

[0048] Fig.13D A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.2 and a relative rotation of 20° between the layers is shown, according to an embodiment of the present disclosure.

[0049] Fig.13E A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.2 and a relative rotation of 30° between the layers is shown, according to an embodiment of the present disclosure.

[0050] Fig.13FA graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.2 and with a relative rotation of 45° between the layers is shown, according to an embodiment of the present disclosure.

[0051] Fig.14A A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.4 and a relative rotation of 0° between the layers is shown, according to an embodiment of the present disclosure.

[0052] Fig. 14B A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.4 and a relative rotation of 5° between the layers is shown, according to an embodiment of the present disclosure.

[0053] Fig. 14C A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.4 and a relative rotation of 10° between the layers is shown, according to an embodiment of the present disclosure.

[0054] Fig.14D A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.4 and a relative rotation of 20° between the layers is shown, according to an embodiment of the present disclosure.

[0055] Fig.14E A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.4 and a relative rotation of 30° between the layers is shown, according to an embodiment of the present disclosure.

[0056] Fig.14F A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.4 and a relative rotation of 45° between the layers is shown, according to an embodiment of the present disclosure.

[0057] Fig.15A A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.6 and a relative rotation of 0° between the layers is shown, according to an embodiment of the present disclosure.

[0058] Fig. 15BA graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.6 and a relative rotation of 5° between the layers is shown, according to an embodiment of the present disclosure.

[0059] Fig. 15C A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.6 and a relative rotation of 10° between the layers is shown, according to an embodiment of the present disclosure.

[0060] Fig.15D A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.6 and a relative rotation of 20° between the layers is shown, according to an embodiment of the present disclosure.

[0061] Fig.15E A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.6 and a relative rotation of 30° between the layers is shown, according to an embodiment of the present disclosure.

[0062] Fig.15F A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.6 and with a relative rotation of 45° between the layers is shown, according to an embodiment of the present disclosure.

[0063] Fig.16A A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.8 and a relative rotation of 0° between the layers is shown, according to an embodiment of the present disclosure.

[0064] Fig. 16B A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.8 and a relative rotation of 5° between the layers is shown, according to an embodiment of the present disclosure.

[0065] Fig. 16C A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.8 and a relative rotation of 10° between the layers is shown, according to an embodiment of the present disclosure.

[0066] Fig.16DA graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.8 and a relative rotation of 20° between the layers is shown, according to an embodiment of the present disclosure.

[0067] Fig.16E A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.8 and a relative rotation of 30° between the layers is shown, according to an embodiment of the present disclosure.

[0068] Fig.16F A graphical representation of simulated flow rate variation along the surface of a stack of two woven mesh substrate layers having different inter-filament spacings in a ratio of 1:1.8 and with a relative rotation of 45° between the layers is shown, according to an embodiment of the present disclosure.

[0069] Fig.17 is a graphical representation showing RTD measurements of four different fixed bed bioreactors according to an embodiment of the present disclosure.

[0070] Fig.18A is an illustration of a cross-sectional view of a web substrate and a corresponding sinusoidal diagram of the substrate according to an embodiment of the present disclosure.

[0071] Fig.18B is an illustration of cross-sectional views of mesh substrates and their corresponding sinusoidal graphs showing different packing densities according to embodiments of the present disclosure.

[0072] Fig.19 is an illustration of a cross-sectional view of a mesh substrate in a sinusoidal diagram in a coiled packed bed arrangement according to an embodiment of the present disclosure.

[0073] Fig. 20 is an illustration of a cross-sectional view of a mesh substrate in a sinusoidal diagram in a coiled packed bed arrangement according to an embodiment of the present disclosure.

[0074] Fig.21 is a graphical representation showing RTD measurements of three different fixed bed bioreactors according to an embodiment of the present disclosure.

[0075] Fig. 22 is a graphical representation showing RTD measurements of three different fixed bed bioreactors employing a hybrid mesh substrate bed, according to an embodiment of the present disclosure. DETAILED DESCRIPTION

[0076] Various embodiments of the present disclosure are described in detail below with reference to the accompanying drawings (if any). Reference to various embodiments does not limit the scope of the present invention, which is limited only by the scope of the appended claims. In addition, any examples listed in this specification are not limiting and only list some of the many possible embodiments of the claimed invention.

[0077] Embodiments of the present disclosure include fixed bed cell culture substrates and cell culture or bioreactor systems comprising the substrates. Substrates and bioreactor systems comprising the substrates exhibit improved flow characteristics through substrates. For example, a more uniform flow through substrates is achieved, and the non-uniform flow caused by channel effects or turbulence is reduced or eliminated. Compared to alternative solutions, the flow "dead zone" in the substrate or fixed bed of a bioreactor is significantly reduced or eliminated. The result is that the substrate or fixed bed obtained makes uniform perfusion through the entire substrate or fixed bed, which promotes cell health during cell culture, and not only to the cultivation of cells, but also to the harvesting of cell inoculation and cells or cell byproducts, facilitates an effective cell culture process.

[0078] Embodiments of the present disclosure also include fixed bed substrates and bioreactors that enable simplified and more efficient manufacturing and assembly. For example, embodiments include fixed bed cell culture substrates that need to be assembled from one or more substrates, and bioreactors in which the fixed bed cell culture substrates are placed. Aspects of embodiments of the present disclosure allow the assembly of the fixed bed and / or the placement of the fixed bed in the reactor to be simplified by reducing the degree to which each cell culture substrate needs to be aligned with each other, which reduces the need for complex procedures for handling and assembling the fixed bed, or the need for complex mechanisms for maintaining a specific orientation or alignment of the fixed bed in the bioreactor. This reduction in complexity can be converted into faster and cheaper manufacturing, transportation and assembly, and more reliable bioreactors.

[0079] Aspects of the embodiments also include fixed bed substrate matrices and bioreactors that provide a more uniform fluid flow through a fixed bed of cell culture substrate.

[0080] In conventional large-scale cell culture bioreactors, different types of fixed bed or packed bed bioreactors have been used. Typically, these packed beds contain porous matrices to retain adherent or suspended cells, as well as to support growth and proliferation. The packed bed matrix provides a high surface area / volume ratio, so the cell density can be higher than in other systems. However, the packed bed often acts as a deep filter, in which cells are physically trapped in the tangled fibers of the matrix. Therefore, due to the linear flow of the cell inoculum through the packed bed, the cells are subjected to uneven distribution in the packed bed, causing the cell density to change in the depth or width of the packed bed. For example, the cell density may be higher at the inlet area of ​​the bioreactor and significantly lower at the outlet closer to the bioreactor. This uneven distribution of cells in the packed bed greatly hinders the scalability and predictability of such bioreactors in bioprocess manufacturing, and may even lead to a reduction in the cell growth or viral vector production efficiency of the packed bed per unit surface area or volume.

[0081] Another problem encountered in the packed bed bioreactor disclosed in the prior art is the channel effect. Due to the random nature of the nonwoven fibers filled, the local fiber density at any given cross section of the packed bed is uneven. The liquid culture medium flows rapidly in the area of ​​low fiber density (high bed permeability), and flows significantly slower in the area of ​​high fiber density (lower bed permeability). The uneven culture medium perfusion obtained on the entire packed bed produces a channel effect, which is manifested as a significant nutrient and metabolite gradient, which adversely affects the overall cell culture and bioreactor performance. Cells located in the low culture medium perfusion area will starve and often die due to metabolite poisoning or lack of nutrients. The flow may be very uneven, so that there is an effective flow "dead zone" in the packed bed, in which no perfusion occurs, and the delivery of any nutrients to the cells in these areas is limited to the diffusion mechanism in the culture medium.

[0082] Cell harvesting is another problem encountered when using bioreactors filled with nonwoven fiber skeletons. Since the packed bed acts as a depth filter, the cells released at the end of the cell culture process are retained in the packed bed and the cell recovery rate is very low. This greatly limits the use of such bioreactors in bioprocesses where living cells are the product or where it is necessary to harvest living cells for further processing to capture cell byproducts. Therefore, the non-uniformity causes various areas to be exposed to flow and shear to varying degrees, effectively reducing the available cell culture area, causing non-uniform culture, and interfering with transfection efficiency and cell release.

[0083] To address these and other problems with existing cell culture solutions, embodiments of the present disclosure provide cell growth substrates, fixed bed assemblies of these substrates, and / or bioreactor systems using these substrates, which enable efficient and high-yield cell culture of anchorage-dependent cells, as well as production of cell products (e.g., proteins, antibodies, viral particles) while simplifying the manufacture and assembly of the fixed bed. Embodiments include a porous cell culture substrate made of an ordered and regular array of porous substrate materials, the porous cell culture substrate enabling uniform cell inoculation and culture medium / nutrient perfusion, as well as efficient cell harvesting. Embodiments can also enable scalable cell culture solutions, wherein the substrate and bioreactor can inoculate and grow cells and / or harvest cell products from process development scale to full production size scale without sacrificing the uniform performance of the embodiments. For example, in some embodiments, the bioreactor can be easily scaled up from process development scale to production scale, and throughout the production scale, the substrate has a comparable viral genome (VG / cm 2 ). The harvestability and scalability of the embodiments herein enable efficient seed training to grow cell populations at multiple scales on the same cell substrate. In addition, the embodiments herein provide a cell culture substrate having a high surface area, which combined with the other features described herein enables a high yield cell culture solution. In some embodiments, for example, the cell culture substrate and / or bioreactor described herein can produce 10 16 Up to 10 18 A viral genome (VG).

[0084] In one embodiment, the matrix has a structurally defined surface area for adherent cells to adhere and proliferate, and when assembled in a fixed bed or other bioreactor, the matrix has good mechanical strength and forms a highly uniform multiple interconnected fluid network. For example, in an embodiment, the structurally defined substrate has sufficient mechanical strength so that it does not deform at the perfusion flow rate used in the bioreactor system. That is, the substrate can be completely non-deformed, or deformed in a significant manner that will affect the flow of the fluid through the fixed bed. In a specific embodiment, a mechanically stable non-degradable woven mesh can be used as a substrate to support adherent cell production. The cell culture matrix disclosed herein supports the attachment and proliferation of anchorage-dependent cells in the form of high volume density. This matrix can achieve uniform cell inoculation, as well as efficient harvesting of cells or other products of the bioreactor. In addition, embodiments of the present disclosure support this cell culture to provide uniform cell distribution during the inoculation step and achieve fused monolayer or multilayer adherent cells on the disclosed matrix, and can avoid the formation of large and / or uncontrollable 3D cell aggregates with limited nutrient perfusion and increased metabolite concentrations. Therefore, the matrix eliminates diffusion limitations during bioreactor operation. Furthermore, the matrix enables easy and efficient harvesting of cells from a bioreactor.The structurally defined matrix of one or more embodiments enables complete recovery of cells from a packed bed of a bioreactor and consistent cell harvesting.

[0085] Different from the cell culture substrate (that is, the nonwoven substrate of randomly arranged fibers) currently used for cell culture bioreactors, the embodiments of the present disclosure include cell culture substrates with limited and ordered structures. This limited and ordered structure allows for consistent and predictable cell culture results. In addition, the substrate has an open porous structure, which prevents cell retention and can achieve uniform flow through the packed bed. This structure can improve cell inoculation, nutrient delivery, cell growth and cell harvesting. According to one or more specific embodiments, the fixed bed matrix is ​​formed by at least one substrate material, and the substrate material has a thin sheet structure, with a first side and a second side, and the first side and the second side are separated by a relatively small thickness, so that the thickness of the sheet is relatively small relative to the width and / or length of the first side and the second side of the substrate. In addition, a plurality of holes or openings through the thickness of the substrate are formed. The size and geometry of the substrate material between the openings allow cells to adhere to the surface of the substrate material, as if it is approximately two-dimensional (2D) surface, while also allowing enough fluid to flow around the substrate material and flow through the openings. In some embodiments, the substrate is a polymer-based material, and it can be formed as a molded polymer sheet; a polymer sheet with openings through the thickness; a plurality of filaments fused into a mesh layer; a 3D printed substrate; or a plurality of filaments woven into a mesh layer. The physical structure of the fixed bed matrix has a high surface / volume ratio for culturing anchorage-dependent cells. According to various embodiments, the fixed bed matrix can be arranged or filled in a bioreactor in certain ways described herein for uniform cell seeding and growth, uniform culture medium perfusion, efficient cell harvesting, and simplified manufacturing and packaging.

[0086] Embodiments of the present disclosure enable a practical-sized viral vector platform that can produce viral genomes at a scale of greater than about 10 14 Viral genomes, each batch is greater than about 10 15 Viral genomes, each batch is greater than about 10 16 Viral genomes, each batch is greater than about 10 17 viral genomes, or up to or greater than about g10 per batch 16 In some embodiments, the production rate is about 10 per batch. 15 to about 10 18 For example, in some embodiments, the viral genome yield can be about 10 per batch. 15 to about 10 16 viral genomes, or about 10 per batch 16 to about 10 19 viral genomes, or about 10 per batch 16 -10 18 viral genomes, or about 10 per batch 17to about 10 19 viral genes, or about 10 per batch 18 to about 10 19 viral genomes, or about 10 per batch 18 or more viral genomes.

[0087] In addition, the embodiments disclosed herein can not only realize that cells are attached to cell culture substrates and grow, but also can harvest cultured cells alive. It is a major disadvantage of existing platforms that living cells cannot be harvested, and it causes difficulty in constructing and maintaining a sufficient number of cells for production capacity. According to one aspect of the embodiments disclosed herein, living cells can be harvested from cell culture substrates, including 80% to 100% living cells, or about 85% to about 99% living cells, or about 90% to about 99% living cells. For example, at least 80% of the harvested cells are alive, at least 85% are alive, at least 90% are alive, at least 91% are alive, at least 92% are alive, at least 93% are alive, at least 94% are alive, at least 95% are alive, at least 96% are alive, at least 97% are alive, at least 98% are alive, or at least 99% are alive. Cells can be released from cell culture substrates using, for example, trypsin, TrypLE or Accutase enzymes.

[0088] Figure 1A and 1B According to an example of one or more embodiments of the present disclosure, a three-dimensional (3D) perspective view and a two-dimensional (2D) plan view of a cell culture substrate 100 are shown, respectively. 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 first direction and the second direction have a defined relationship to each other. For example, in Figure 1A and 1B , the first direction and the second direction are perpendicular to each other. However, embodiments include other defined non-random relative directions such that the first direction and the second direction can be separated by an angle less than 90°, including, for example, 30°, 45°, 60°, or 75°, or any other defined angle. The first plurality of fibers 102 are spaced apart from each other by a first fiber spacing S1, and the second plurality of fibers 104 are spaced apart from each other by a second fiber spacing S2. The first fiber spacing 102 and the second fiber spacing 104 are defined by the vertical distance from the centerline of one of the first plurality of fibers 102 and the second plurality of fibers 104 to the centerline of an adjacent or nearest fiber, respectively. According to various embodiments, the first fiber spacing S1 and the second fiber spacing S2 can be equal or unequal, as discussed below.

[0089] The woven fibers of the substrate 100 form a plurality of openings 106, which may be defined by one or more widths or diameters (e.g., D1, D2). The size and shape of the openings may vary depending on the type of weave (e.g., the number, shape, and size of the filaments; the angle between intersecting filaments, etc.). A woven web may be characterized as a two-dimensional sheet or layer on a macroscopic scale. However, a closer look at the woven web reveals that it has a three-dimensional structure due to the rise and fall of the intersecting fibers of the web. Thus, as Figure 1C As shown, the thickness T of the woven mesh 100 can be thicker than the thickness of a single fiber (e.g., t1). As used herein, the thickness T is the maximum thickness between the first side 108 and the second side 110 of the woven mesh. Without being bound by theory, it is believed that the three-dimensional structure of the structure 100 is advantageous because 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 can achieve uniform fluid flow.

[0090] exist Figure 1B In the embodiment of the present invention, the opening 106 has a diameter D1 and a diameter D2, wherein the diameter D1 is defined as the distance between the relative fibers of the first plurality of fibers 102, and the diameter D2 is defined as the distance between the relative fibers of the second plurality of fibers 104. Depending on the braiding geometry, D1 and D2 may be equal or unequal. In the case where D1 and D2 are unequal, the larger one may be referred to as the major diameter, and the smaller one may be referred to as the minor diameter. In some embodiments, the diameter of the opening may refer to the widest part of the opening. Unless otherwise specified, the opening diameter used herein shall refer to the distance between the parallel fibers on the opposite sides of the opening.

[0091] A given fiber in the first plurality of fibers 102 has a thickness t1, and a given fiber in the second plurality of fibers 104 has a thickness t2. In the case of a fiber of circular cross-section, such as Figure 1A or other three-dimensional cross-sectional views, thickness t1 and t2 are the maximum diameters or thicknesses of the fiber cross-sections. According to some embodiments, the first plurality of fibers 102 all have the same thickness t1, and the second plurality of fibers 104 all have the same thickness t2. In addition, t1 and t2 may be equal. However, in one or more embodiments, t1 and t2 are not equal, for example, when the first plurality of fibers 102 and the second plurality of fibers 104 are different. In addition, each of the first plurality of fibers 102 and the second plurality of fibers 104 may include two or more fibers of different thicknesses (e.g., t 1a ,t 1b etc. and t 2a ,t 2bAccording to an embodiment, the thicknesses t1 and t2 are large relative to the size of the cells cultured thereon, so that from the perspective of the cells, the fibers provide a generally flat surface, which enables better cell attachment and growth compared to some other solutions where the fiber size is small (e.g., the scale of the cell diameter). Due to the three-dimensional nature of the woven mesh, such as Figures 1A-1C As shown, the 2D surface area of ​​the fibers available for cell attachment and proliferation exceeds the surface area for attachment on a comparable planar 2D surface.

[0092] In one or more embodiments, the diameter of the fiber can be in the following range: about 10 μm to about 1000 μm; about 30 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μm to about 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400 μm; about 200 μm to about 300 μm; or about 150 μm to about 300 μm. At the microscopic scale level, since the fiber is compared to the scale of the cell (e.g., the fiber diameter is larger than the cell), the surface of the monofilament fiber exists as a roughly 2D surface for adherent cells to attach and proliferate. The fiber can be woven into a mesh, and the opening is in the range of about 100 μm x 100 μm to about 1000 μm x 1000 μm. In some embodiments, the diameter of the openings can be from about 30 μm to about 1000 μm; from about 100 μm to about 750 μm; from about 125 μm to about 600 μm; from about 150 μm to about 500 μm; from about 200 μm to about 400 μm; or from about 200 μm to about 300 μm. These ranges of filament diameters and opening diameters are examples of some embodiments, but are not intended to limit the possible characteristic dimensions of the mesh of all embodiments. The combination of fiber diameter and opening diameter is selected to provide efficient and uniform fluid flow through the substrate, for example, when the fixed bed cell culture matrix includes multiple adjacent mesh layers (e.g., a stack of individual layers or a rolled mesh layer).

[0093] Factors such as fiber diameter, opening diameter and / or fiber spacing and weaving type / pattern will determine the surface area that can be used for cell attachment and growth. In addition, when the cell culture matrix includes a stack, roll or other arrangement of overlapping substrates, the packing density of the cell culture matrix will affect the surface area of ​​the fixed bed matrix, and the fluid flow characteristics by the fixed bed. Packing density can change with the packing thickness (for example, the space required for the layer of substrate) of the substrate material. For example, if the stack of the cell culture matrix has a certain height, each layer of the stack can be said to have a packing thickness, which is determined by dividing the total height of the stack by the number of layers in the stack. The packing thickness will change based on fiber diameter and weaving conditions, but it can also change based on the alignment of the adjacent layers in the stack. Due to the three-dimensional properties of the substrate (for example, woven layer or three-dimensional porous sheet) in the disclosed embodiments, a certain amount of interlocking or overlapping can occur between them in adjacent layers based on their alignment to each other. In the first alignment, adjacent layers may be nested closely together, but in the second alignment, adjacent layers may have zero overlap, for example, when the lowest point of the upper layer is in direct contact with the highest point of the lower layer. In practice, the amount of nesting can be affected by both the translational and rotational alignment of the fiber patterns in adjacent layers. For certain applications, it may be desirable to provide a cell culture matrix with a lower density of layer filling (for example, when higher permeability is a priority), or it may be desirable to provide a cell culture matrix with a higher density of filling (for example, when maximizing the surface area of ​​the substrate is a priority). According to one or more aspects of the embodiments of the present invention, the filling thickness can be about 30 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μm to about 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400 μm; about 200 μm to about 300 μm. These ranges are provided by way of example only, and embodiments may include packing thicknesses outside of these ranges based on the substrate selected, the fixed bed packing, the bioreactor design, and the application.

[0094] The above structural factors can determine the available surface area of ​​the cell culture substrate, whether it is a cell culture substrate with a single layer of cell culture substrate or a cell culture substrate with multiple substrate layers. As used herein, "effective surface area" is the total surface area of ​​fibers in a portion of the substrate material that can be used for cell attachment and growth. Unless otherwise specified, "surface area" refers to this effective surface area. According to an embodiment, for example, a single woven mesh substrate layer with a diameter of 6 cm can have a surface area of ​​about 50 cm. 2 To about 90cm 2 ; About 53cm 2 To about 81cm 2 ; About 68cm 2 ; About 75cm 2 ; or about 81cm2 The effective surface area of ​​the substrate can be determined by the size of the fibers, the arrangement of the fibers / openings in the substrate, and of course the size of the substrate layer itself. These ranges of effective surface areas are provided for illustration only, and some embodiments may have different effective surface areas. The cell culture substrate can also be characterized in terms of porosity, as discussed in the Examples herein.

[0095] The substrate mesh can be made of monofilament or multifilament fibers of a polymer material compatible in cell culture applications, such as polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole and polypropylene oxide. The mesh substrate can have different patterns or weaves, such as knitting, warp knitting or weaving (e.g., plain weave, twill weave, Dutch weave, satin weave, regular matte weave, irregular matte rib weave, irregular weft rib weave, regular weft rib weave, regular warp rib weave, irregular warp rib weave, satin weave, 2 / 2 twill weave, 3 / 3 twill weave, basket weave, mountain twill weave, royal oxford weave, houndstooth weave, herringbone weave, five-needle weave, micro-mesh or mesh netting). Aspects of the embodiments also include filaments made from other suitable materials that are suitable for forming into a porous structure and subsequently coated with a material that is compatible with cell culture applications.

[0096] The surface chemistry of the mesh filaments may need to be modified to provide the desired cell adhesion properties. These modifications may be achieved by chemical treatment of the polymer material of the mesh, or by grafting cell adhesion molecules onto the surface of the filaments. Alternatively, a biocompatible hydrogel (e.g., comprising collagen or ) thin layer coating the mesh. Alternatively, the filament fiber surface of the mesh can be provided with cell adhesion properties by treatment processes using various types of plasmas, process gases and / or chemicals known in the industry. However, in one or more embodiments, the mesh can provide an effective cell growth surface without the need for surface treatment.

[0097] Compared to a planar 2D surface of comparable size, the three-dimensional quality of the multiple substrates described in accordance with embodiments of the present disclosure provides increased surface area for cell attachment and proliferation. This increased surface area contributes to the scalable performance achieved by embodiments of the present disclosure. For process development and process validation studies, small-scale bioreactors are often required in order to save reagent costs and increase experimental throughput. Embodiments of the present disclosure can be applied to such small-scale studies, but can also be scaled up to industrial or production scales. Due to the plugged perfusion flow in the packed bed, the same flow rate can be used in smaller and larger scale bioreactor formats, which is measured in ml / min / cm2 (cm 2 The larger surface area allows for higher seeding density and higher cell growth density. According to one or more embodiments, the cell culture substrate described herein exhibits a cell seeding density as high as 22,000 cells / cm 2 or greater. For reference, Corning's Approximately 20,000 cells / cm on a 2D surface 2 As used herein, "plugged perfusion flow" or "plugged flow" refers to laminar flow through a bioreactor having a fixed bed of embodiments herein, wherein flow through any cross section of the fixed bed perpendicular to the direction of flow travels at the same rate across the cross section.

[0098] Another advantage of higher surface area and high cell seeding or growth density is that the cost of the embodiments disclosed herein can be the same or less than competitive solutions. Specifically, the cost per cell product (e.g., per cell or per viral genome) can be equal to or less than the cell product cost of other packed bed bioreactors.

[0099] Therefore, multiple factors of the fixed bed substrate can affect the cell culture process, including packing density, surface chemistry and effective surface area, and the characteristics of fluid flow through and within the packed bed. By using a culture substrate with a sufficiently rigid structure, high uniformity of flow resistance is achieved on the substrate or packed bed. According to various embodiments, the substrate can be deployed with a flexible and scalable multi-layer substrate arrangement. This flexibility eliminates diffusion restrictions and provides uniform nutrient and oxygen delivery for cells attached to the substrate. In addition, the open substrate does not have any cell retention area in the packed bed configuration, allowing cell harvesting to be completed with high vitality at the end of the culture. The substrate also provides packing uniformity for the packed bed and realizes direct scalability from process development units to large-scale industrial bioprocessing units. The ability to harvest cells directly from the packed bed eliminates the need to resuspend the substrate in a stirred or mechanically shaken container, which increases complexity and may cause harmful shear stress to the cells. Further, the high packing density of the cell culture substrate produces high bioprocess productivity in a manageable volume on an industrial scale.

[0100] The geometry of the mesh substrate layer is designed to allow efficient and uniform flow through one or more substrate layers. In addition, the structure of the substrate can allow fluid to flow through the substrate in multiple orientations. For example, the overall fluid flow direction can be perpendicular to the main side surfaces of the first and second substrate layers. However, the substrate can also be oriented relative to the flow so that the relative side or face of the substrate layer is parallel to the overall flow direction. In addition to the fluid flow perpendicular or parallel to the first and second sides of the mesh layer, the substrate can be arranged so that multiple substrate sheets are at intermediate angles, or even randomly arranged relative to the fluid flow. The flexibility of this orientation is achieved by the basic isotropic flow behavior of uniform, structurally defined substrates. In contrast, the substrates of adherent cells in existing bioreactors do not show this behavior, on the contrary, their packed beds often produce preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the substrate disclosed herein allows it to be used in various applications and bioreactor or container designs, while achieving better and more uniform permeability throughout the bioreactor container.

[0101] According to one or more embodiments, the cell culture substrate can be used in a bioreactor container. For example, the substrate can be used in a packed bed bioreactor configuration, or other configurations in a three-dimensional culture chamber. However, embodiments are not limited to three-dimensional culture spaces, and it is expected that the substrate can be used in a configuration that can be considered as a two-dimensional culture surface configuration, wherein one or more layers of the substrate are laid flat, for example, in a flat-bottomed culture dish, to provide a culture substrate for cells. For concerns about contamination, the container can be a disposable container and can be discarded after use.

[0102] According to various embodiments, a cell culture system is provided in which a cell culture matrix is ​​used within a culture chamber of a bioreactor vessel. Figure 2An example of a cell culture system 200 is shown, which includes a bioreactor container 202 having a cell culture chamber 204 in the interior of the bioreactor container 202. Within the cell culture chamber 204 is a cell culture substrate 206, which is made of a stack of substrate layers 208. The substrate layers 208 are stacked, and the first side or second side of the substrate layer faces the first side or second side of the adjacent substrate layer. The bioreactor container 200 has an inlet 210 at one end, which is used to input culture medium, cells and / or nutrients into the culture chamber 204, and an outlet 212 at the opposite end, which is used to remove culture medium, cells or cell products from the culture chamber 204. By stacking the substrate layers in this manner, the system can be easily scaled up, and due to the defined structure and the efficient flow of fluid through the stacked substrates, the system has no adverse effects on cell attachment and proliferation. Although the container 200 may be generally described as having an inlet 210 and an outlet 212, some embodiments may utilize one or both of the inlet 210 and the outlet 212 to allow medium, cells, or other contents to flow into and out of the cell culture chamber 204. For example, during the cell inoculation, perfusion, and / or culture phases, the inlet 210 may be used to allow medium or cells to flow into the culture chamber 204, but it may also be used during the harvest phase to remove one or more of the medium, cells, or cell products through the inlet 112. Thus, the terms "inlet" and "outlet" are not intended to limit the functionality of these openings.

[0103] exist Figure 2 In the embodiment, the overall flow direction is in the direction from the inlet 210 to the outlet 212, and in this example, the first major side and the second major side of the substrate layer 208 are perpendicular to the overall flow direction. According to some aspects of the embodiments, the substrate may be different from Figure 2 Other construction settings shown, Figure 2 Shown only as an example. In the bioreactor system 200, the size and shape of the substrate 208 is adjusted to fill the interior space defined by the culture chamber 204, so that the culture space in the container is filled for cell growth surface, thereby maximizing efficiency in terms of cells / unit volume. Although Figure 3 shows a single inlet 210 and a single outlet 212, it is contemplated that the system 200 can be fed by multiple inlets and have multiple outlets. According to embodiments of the present invention, a distribution plate can be used to help distribute culture medium, cells or nutrients across a cross-section of the packed bed and thereby improve the uniformity of fluid flow through the packed bed.

[0104] Depending on the desired system, the cell culture substrate can be arranged in the culture chamber with a variety of configurations. For example, in one or more embodiments, the system includes one or more substrate layers, and its width extends over the entire width of the cell culture space of the limitation in the culture chamber. Multiple substrate layers can be stacked to a predetermined height in this way. As described above, the substrate layer can be arranged so that the first side and the second side of one or more layers are perpendicular to the overall flow direction of the culture medium through the limited culture space in the culture chamber, or the first and second sides of one or more layers can be parallel to the overall flow direction. In one or more embodiments, the cell culture substrate includes one or more substrate layers that are in a first orientation relative to the overall flow, and one or more other layers that are in a second orientation different from the first orientation. For example, each layer can have a first side and a second side, which are parallel or perpendicular to the overall flow direction, or are in a certain angle between parallel and vertical.

[0105] In one or more embodiments, the cell culture system includes a plurality of discrete cell culture substrate sheets in a packed bed configuration, wherein the length and / or width of the substrate sheet is relatively small relative to the culture chamber. As used herein, when the length and / or width of the substrate sheet is equal to or less than about 50% of the length and / or width of the culture space, the substrate sheet is considered to have a length and / or width relatively small relative to the culture chamber. Therefore, the cell culture system may include a plurality of substrate sheets filled into the culture space in a desired arrangement. The arrangement of the substrate sheets may be random or semi-random, or may have a predetermined order or alignment form, for example, the substrate sheets are oriented with substantially similar orientations (e.g., horizontally, vertically, or at an angle between 0 ° and 90 ° relative to the overall flow direction).

[0106] As used herein, "defined culture space" refers to the space in a culture chamber occupied by a cell culture matrix and in which cell inoculation and / or culture will occur. The defined culture space may fill approximately the entire culture chamber, or may occupy a portion of the space within the culture chamber. As used herein, "overall flow direction" is defined as the direction of the overall mass flow of fluid or culture medium through or on a cell culture matrix during cell culture, and / or during flow of culture medium into or out of the culture chamber.

[0107] In one or more embodiments, the cell culture matrix is ​​stabilized in the culture chamber by a fixing mechanism. The fixing mechanism can stabilize a portion of the cell culture matrix to the culture chamber wall surrounding the matrix, or stabilize to the chamber wall at one end of the culture chamber. In some embodiments, the fixing mechanism adheres a portion of the cell culture matrix to a member that travels through the culture chamber, for example, parallel to the member that travels along the longitudinal axis of the culture chamber, or adheres to the member that travels perpendicularly to the longitudinal axis. However, in one or more other embodiments, the cell culture matrix can be contained in the culture chamber and is not fixedly attached to the wall of the chamber or bioreactor container. For example, the matrix can be contained in the room by the boundary of the culture chamber or other structural members, so that the matrix is ​​maintained in the predetermined area of ​​the bioreactor container and the matrix is ​​not fixedly stabilized to these boundaries or structural members.

[0108] When multiple substrate layers such as Figure 2 When stacked face-to-face as shown, the packing density of the layers can vary based on the alignment and degree of nesting or overlap between the fibers of adjacent layers. For example, assuming no relative rotation between adjacent layers, in which case the first plurality of fibers of one layer are parallel to the first plurality of fibers of another layer, then the packing density boundary condition is defined by the amount of nesting or overlap between adjacent layers. According to one or more embodiments, if the fibers of adjacent layers are not perfectly aligned, then due to the three-dimensional nature of the substrate, there is some degree of nesting between the fibers of one layer and the fibers of the adjacent layer. Figure 3A and 3B The plan view and cross-sectional view of this situation are shown respectively. Specifically, the fibers of the first layer running in the first direction are displaced midway between the fibers of the adjacent second layer running in the first direction, so that the fibers of the first layer are located in the openings of the second layer (see Figure 3A ). This represents the highest degree of overlap that can occur between adjacent layers, and therefore the highest degree of compaction (without some additional external force pressing the layers together). At the other extreme of the range, Figure 4A and 4B A similar view of a stack of substrate layers is shown, where the fibers of one substrate layer running in a first direction are all perfectly aligned with the fibers of the other substrate layers running in the first direction. This represents the lowest degree of overlap (i.e., zero overlap), and therefore also represents the lowest degree of compaction, although the layers are still in contact with each other.

[0109] for Figures 3A-4B For each modeling configuration in FIG. 1 , sample cells 300, 400 were defined that enclosed the same volume of mesh material to analyze the porosity in the volume of each sample cell 300, 400. The modeled volume of open space within each cell is shown in FIG. Figure 5A (for tightly packed stacks) and Figure 5B(For loosely packed stacks). Porosity, in terms of the percentage of open space, is about 40.8% for loosely packed cells and 61.4% for tightly packed cells. Figures 3A-4B The modeled stacks in represent the most densely and loosely packed configurations for a given mesh material, so the porosities of 40.8% and 61.4% are the upper and lower limits of the porosity for this particular mesh material. Depending on the alignment and the actual packing density when using this mesh material, the porosity can fall between these limits. However, embodiments of the present disclosure are not limited to this porosity range, as variations in mesh size and placement of the substrate within the cell culture container can result in different porosity ranges.

[0110] In addition to the modeled porosity range, a real packed bed with a PET woven mesh substrate was measured. The measurements were performed using 100 discs stacked in random alignment, each with a diameter of 22.4 mm. The total weight of the stack of 100 discs was 5.65 ± 0.2 g. The PET density was assumed to be 1.38 g / cm 3 , use the following formula to calculate the volume of the PET material in the stack: V PET =(total weight of the stack) / (density of PET) Equation 1 Therefore, the PET volume V of 5.65 g PET (100 disks with a diameter of 22.4 mm) is calculated PET The total volume V of the stack is then calculated using the following formula 总 ——Including PET volume V PET And the volume of the open space within the stack: V 总 =π×(0.5×dish diameter)×(height of stacked bed) Equation 2 The stack height of a stack of 100 disks is 25 ± 1 mm. Therefore, for a disk diameter of 22.4 mm, V is found to be 总 is 9.85 ml. Therefore, the porosity of the stacked bed can be calculated using the following formula: Porosity = (V 总 –V PET ) / V 总 Equation 3

[0111] Using Equation 3 and the above values, the porosity was calculated to be 58.4%, which is within the range predicted by the model.

[0112] As described above, in a loose-pack configuration, the fibers in the layers of the fixed bed are aligned. It follows that if the substrate material in the layers is the same, the openings in the layers will also be aligned. Therefore, if a stack of substrates in this loose-pack configuration is viewed in plan view, it will be possible to see through the aligned openings in the stack. Fig. 6A Such a stack of loosely stacked substrate layers 600 is shown, having at least two layers 602, 604. On the other hand, in a tightly stacked configuration, it is not possible to see through the stack when viewed from above, because the fibers of one layer overlap with the openings of an adjacent layer. Figure 6B 600′ has at least two layers 602′, 604′ that are perfectly nested. For fixed beds made of woven mesh, it has been shown that the alignment of the fibers between adjacent layers has a significant effect on the uniformity of flow through the fixed bed. For layers with a rotational alignment of 0° offset, such as Figure 4A The difference in permeability of the fully interlocked stacked substrate layers shown can be as follows: Figure 3A Ten times (10x) larger than when fully overlapped is shown.

[0113] However, in addition to translational displacement between layers in a fixed bed, rotational misalignment may also occur. It should be noted that both translational and rotational misalignment can be a manufacturing defect of the substrate material itself (e.g., when the individual layers are cut from a larger sheet) or displacement between the layers in the fixed bed (e.g., displacement that may occur during the handling or assembly of the bioreactor, or displacement that may occur after the fixed bed bioreactor is fully assembled). Although tight manufacturing and assembly tolerances can accommodate and reduce such displacement, such precision increases the complexity and cost of the process.

[0114] To understand how the rotational alignment of the substrate layer can affect fixed bed properties, Figure 6C-6K Shown are substrate stacks with various degrees of rotation between the two layers and the possible effect of such rotation on fluid flow properties due to the degree to which the stack has uniform openings available for fluid flow. Figure 6C In the stack 610, layer 612 is rotated 5° relative to layer 614; Fig.6D In the stack 620, layer 622 is rotated 10° relative to layer 624; Fig. 6E In the stack 630, layer 634 is rotated 15° relative to layer 632; Fig. 6F In the stack 640, layer 642 is rotated 20° relative to layer 644; Figure 6G In the stack 650, layer 652 is rotated 25° relative to layer 654; Figure 6H In the stack 660, layer 662 is rotated 30° relative to layer 664; Fig.6I In the stack 670, layer 672 is rotated 35° relative to layer 674; Figure 6J In, layer 682 in stack 680 is rotated 40° relative to layer 684; and Figure 6K , layer 692 in stack 690 is rotated 45° relative to layer 694.

[0115] If there is a rotation angle between the two layers, especially for Figure 6C and 6D At lower angles such as 5° to 10° in the stack, some clear patterns can be observed in the openings of the stack. There are clear dark and light areas, which represent significant differences in permeability between these areas. For example, the brighter areas are due to the overlap of continuous openings with openings in adjacent layers. Therefore, it is expected that these areas will have less flow resistance. In other areas where the continuous openings are blocked by fibers in adjacent layers, the stack looks dark, and people would expect these dark areas to have greater resistance to flow. This suggests that these different areas will have different flow rates. Different flow rates can potentially cause different cell inoculations and nutrient supplies, and therefore cause uneven cell growth. It may also affect the acquisition of transfection reagents during transfection and shear forces during harvesting. It is challenging to directly measure or quantify this uneven flow distribution. However, in these cases, after staining the mesh after cell culture, uneven cell distribution was observed. The staining images show similar patterns, suggesting that uneven flow does exist due to different alignment of the layers and can adversely affect cell culture uniformity. To reduce this non-uniformity, it is preferred to have a relatively uniform alignment of dark-light areas. Figures 6A-6K , a rotation angle of about 30° to about 45° is preferred (see Figures 6H-6K ), and 45°( Figure 6K ) is a particularly preferred embodiment.

[0116] However, the above-mentioned precise degree of rotational alignment can be difficult. It requires some steps to be taken during reactor assembly to align the mesh and some mechanical features to maintain the alignment during handling, transportation and application. These additional steps and mechanical features will add additional costs and risks. Therefore, embodiments of the present disclosure include fixed beds for cell culture that achieve uniform flow without the need for these costly measures to ensure precise rotational alignment.

[0117] According to an embodiment of the present disclosure, a fixed bed is provided which has at least two different types of substrate materials stacked together in the same cell culture bed. In an embodiment, the at least two different substrates are alternately stacked (e.g., substrate A, substrate B, substrate A, substrate B, etc.). The at least two different types of substrate materials may be different in one or more physical dimensions. For example, fiber diameter, opening diameter, fiber spacing or fiber direction may be different. Embodiments using different types of substrate materials provide several advantages, including: no specific alignment is required during reactor assembly; no mechanical features are required in the container to hold the net in place; minimized packed bed porosity or density changes; and improved flow uniformity in a packed bed reactor. The terms "mixed substrate", "mixed net" or "mixed fixed bed" are sometimes used herein to refer to a substrate matrix or fixed bed comprising at least two different types of substrate materials as described above.

[0118] In one or more preferred embodiments, the two different types of substrates have different fiber spacings. The relationship between the fiber spacings of two different types of substrates can be expressed as the ratio of the fiber spacing of the first web to the fiber spacing of the second web. Embodiments include at least two types of web substrates, and have a fiber spacing ratio of at least about 1.1 and at most about 2.0, 2.5, 3.5, 4.0, 4.5 and 5.0; about 1.2 to about 4.0; or about 1.2 to about 2.0. The fiber spacing ratio. According to the embodiment with the fiber spacing ratio, it is not necessary to control or keep the layer to maintain specific alignment. This can be demonstrated by modeling the flow pattern through the substrate layer stack with different fiber spacing ratios, for example, as discussed below.

[0119] Example

[0120] To demonstrate the effect of web alignment on flow uniformity and the benefit of using a hybrid web, a simplified sinusoidal model can simulate the periodic variation of flow produced through a web layer. The web is a weave pattern of two sets of parallel fibers (a first set of fibers running parallel in a first direction and a second set of fibers running parallel in a second direction). As a basic model, a series of parallel cylinders are used to simulate the first set of fibers running in a first direction. Fig. 7A A cross-sectional view of a series of such fibers 700 is shown. In this example, the diameter of the cylinder is 160 μm (the fiber diameter) and the diameter of the opening between each cylinder is 250 μm. Water 702 flows to the cylinder at an average rate of 30 ml / min, which is within the typical range commonly used in cell culture. The water passes through the series of cylinders 700 and will have a different velocity distribution 704 on the other side of the cylinder due to the obstruction and resistance of the cylinder. Using a computational fluid dynamics (CFD) model, the water velocity follows a sine function at 160 μm on the other side of the array, as shown in Figure 7B shown.

[0121] The resistance of a porous material (e.g., a series of cylinders 700) to fluid flow can be calculated from the flow rate using Equation 4, where R is the flow resistance, P is the pressure, and U is the flow rate. The inverse of this sine equation is used to represent the relative change in flow resistance at different locations along the cylinder array. Figure 8 A graphical representation of the resistance obtained by applying Equation 4 to a sinusoidal flow rate of FIG. 7 is shown.

[0122] To construct the model, a woven web 900 is modeled with two cylinder arrays 902, 904 that are stacked on top of each other and run orthogonally to each other, such as Fig. 9 As shown. It is assumed that the resistance at each position is a linear combination of the resistances from the two arrays at the same position. Therefore, the change in flow velocity over the simulated mesh layer can be calculated as the inverse function of the final resistance represented at each position, as Fig.10 Using this approach, the effect of web alignment on flow rate distribution can be simulated. For example, when two such webs are stacked and perfectly rotationally aligned (0° rotation relative to each other), there are two boundary conditions: (i) the fibers from the two webs are completely interlocked ( Figure 3A and 3B and (ii) the fibers from the two webs are perfectly aligned ( Figure 4A and 4B loose filling structure in the Fig.11A and 11B The modeled flow rate distributions for these close-packed configurations and loose-packed configurations are shown separately. Based on the CFD model results, the close-packed configuration shows the highest resistance to flow and the lowest flow rate. In the sinusoidal model simulation, the flow rate is also lower. Locally, there are small flow velocity oscillations, such as Fig.11A As shown, this corresponds to the pattern of the mesh. Over a longer range, the flow rate distribution is roughly uniform. According to the CFD model, the flow resistance is lowest and the flow rate is highest in the aligned (loose packing) configuration. In the sinusoidal model simulation, the flow rate is also higher. In fact, the permeability in this aligned configuration is about 10 times that in the dense packing configuration. Similarly, there are only local oscillations of the flow rate corresponding to the mesh pattern, and over a larger range, the flow rate is consistent. This means that in both cases, different areas of the mesh can obtain similar flows.

[0123] The same approach can be applied to simulate the flow rate distribution when the two webs have different degrees of rotation relative to each other. Figures 12A-12F Shown are simulated flow rate profiles through a stack of two web layers having identical physical web geometry (ie, fiber spacing, etc.). Fig. 12A The two boundary conditions mentioned above are shown with a relative rotation of 0°. Fig. 12B and 12C Relative rotations of 5° and 10° are shown, respectively, and reveal that some regions have significantly higher flow rates than other regions, which is consistent with Figure 6C and 6D The pattern of the opening area in the figure matches. Fig.12D , 12E 12F and 12F show relative rotations of 20°, 30°, and 45°, respectively. For alignments of about 20° or greater, the non-uniform flow pattern begins to disappear, with about 30° to about 45° approaching a certain level of uniformity. That is, the amount or pattern of flow variation becomes more similar in all areas of the web stack, although significant non-uniformity still exists.

[0124] To address this non-uniformity issue, embodiments of the present disclosure include combining at least two different types of mesh in a stack. For example, as described herein, the at least two mesh materials may be stacked in an alternating manner. To demonstrate the flow uniformity improvement of such embodiments, the same simulation as described above may be employed. For example, Figures 13A-16F Shown is the flow pattern when two different types of mesh layers are stacked together. Figures 13A-13F The cases of two web layers having a fiber pitch ratio of 1.2 with rotational orientations of 0°, 5°, 10°, 20°, 30° and 45° are shown. Figures 14A-14F The cases of two web layers having a fiber pitch ratio of 1.4 with rotational orientations of 0°, 5°, 10°, 20°, 30° and 45° are shown. Figures 15A-15F The rotational orientations of the two web layers with a fiber spacing ratio of 1.6 are shown to be 0°, 5°, 10°, 20°, 30°, and 45°. Figures 16A-16F The cases of two web layers having a fiber pitch ratio of 1.8 with rotational orientations of 0°, 5°, 10°, 20°, 30° and 45° are shown.

[0125] When the fiber spacing ratio between the two web layers is 1.2, Figures 13A-13F As shown, when the web is aligned at a lower angle (e.g. 5° or 10°), inhomogeneities are still observed, but at greater degrees of rotation, the inhomogeneities are again slightly reduced. Figures 14A-14F ) or greater, especially 1.6( Figures 15A-15F ) or larger (e.g. Figures 16A-16FWhen the ratio of the mesh material to the fixed bed is about 1.8, there are few discernible areas that show significantly higher flow rates than other areas. This means that the use of a heterogeneous fixed bed and a mesh material with a fiber spacing ratio of greater than or equal to about 1.4 should effectively eliminate the effect of alignment on flow uniformity. This will make it easier to control the fixed bed filling process of a fixed bed reactor during manufacturing, transportation and end use.

[0126] As discussed herein, embodiments of the present disclosure include fixed bed substrates having a plurality of cell culture substrates arranged in a stacked manner, or one or more roll-like substrate layers. Embodiments of the present disclosure provide solutions for improving the flow uniformity in a bioreactor (and therefore improving cell inoculation, cultivation and harvesting performance). Aspects of embodiments include combining multiple substrates in a single fixed bed, wherein the multiple substrates include substrates with different geometries, including the braiding geometry of a weaving or knitting substrate. The multiple substrates, for example, may include at least two substrates with different weaving patterns.

[0127] In embodiments where one or more rolls of substrate are used to form a fixed bed, the use of substrates having one geometry type (e.g., two or more woven substrates having the same weave type) can result in inherent variations in packing density, which adversely affects flow uniformity and, therefore, results in suboptimal cell seeding, oxygen supply, culture health, and harvest. However, the use of a fixed bed made from a mixed substrate roll (i.e., two or more different substrates, such as two substrates of different weaves) can eliminate flow non-uniformity and, therefore, improve cell seeding, growth, and harvest.

[0128] Embodiments of the present disclosure are also advantageous for manufacturing and assembling the fixed bed and bioreactor system disclosed herein. A rolled fixed bed with a single type of substrate (e.g., a single woven net) is susceptible to changes in the rolling process, such as rolling tension and rolling alignment. On the other hand, in the roll of a hybrid net, once the layers are in close contact with each other, the effects from the rolling tension will be minimized. This will make the rolling and assembly process more robust. In addition to these advantages, the rolled fixed bed design can be assembled using existing industrial equipment, and does not require individual cutting or stacking of layers as in the stacked substrate embodiment, thus greatly reducing manufacturing costs.

[0129] According to embodiments using a fixed bed made from a roll-like substrate, aspects of the embodiments also help minimize fluid (or cell, medium, etc.) bypass and reduce the need for high precision in vessel design and manufacturing in terms of tolerances between the vessel and the substrate. The roll-like substrate design may also have better performance in terms of the effect of bubbles formed in the bioreactor system on flow uniformity.

[0130] Example: Packing density and RTD measurement of a roll substrate

[0131] Fig.17 Shown are the flow uniformity of RTD measurements in four fixed bed bioreactors, two with stacked mesh fixed beds and two with rolled mesh fixed beds, all with a single type of substrate. Fig.17 The total surface area used is shown to be 2.5m 2 and 5m 2 RTD measurements of multiple stacked woven web substrate layers with a total surface area of ​​2.5 m 2 and 5m 2 RTD measurements of two bioreactors with two rolls of substrate. Fig.17 Compared to the above, embodiments of the present disclosure can improve flow uniformity and the resulting RTD measurements. Fig.17 To account for the relative inhomogeneity in the web, a sinusoidal curve can be used to simulate the maximum and minimum widths along a cross section of a woven web, where the sinusoidal peaks represent the thickest parts of the web and the valleys represent the openings between the fibers, as Fig.18A That is, the web spacing (the distance between two parallel fibers) is represented by the period, and the web thickness is represented by the amplitude. When two or more layers of substrate are stacked, the fiber alignment and packing changes can be visualized by overlapping sinusoidal curves, such as Fig.18B The loosely and densely packed regions are also associated with different patterns of sinusoids between adjacent layers, as shown in Fig.18B For example, Fig.18B The top example in shows perfect alignment of the two layers, resulting in a loose fill. Fig.18B The bottom example also shows loose filling when adjacent layers are offset by one cycle of the sine curve. Fig.18B The middle example in shows the tight packing resulting from an offset of 0.5 cycles between layers.

[0132] When the substrate is rolled up, the packing density of the roll can be visualized by observing the sinusoidal curve of the roll, e.g. Fig.19 shown. Fig.19 A cross section of a rolled web (using 250 / 37 web) is shown as a sinusoidal curve in a polar coordinate system. Variations in the pattern can be seen in the large regions around the cross section. These patterns are associated with three loosely packed regions and two tightly packed regions in the web substrate assembly, as shown in FIG. Fig.19 The loosely packed areas have higher permeability and lower flow resistance, thus resulting in fluid bypass. On the other hand, the densely packed areas have low permeability and high flow resistance, thus resulting in dead zones. Based on CFD simulations, the permeability difference between the loosely packed areas and the densely packed areas can be as high as 10 times. This can explain the difference in permeability when using a single type of substrate web. Fig.17 The RTD curve in has a wider distribution, indicating a relatively non-uniform flow.

[0133] In contrast, if two different types of webs are rolled together in a fixed bed, different results are achieved, e.g. Fig. 20 shown. Fig. 20 Overlapping sinusoids with two different mesh types (250 / 37 and 300 / 35 mesh) and alternating layers with different spacings (as shown in Table 1 below) are shown. Fig. 20 As shown, there are no large regions with relatively loose packing or relatively dense packing patterns. This means that the permeability is likely to be consistent throughout the fixed bed. Net type 250 / 37 300 / 35 thickness 0.300mm 0.405mm spacing 0.410mm 0.505mm Table 1: Mesh types and geometries in the mixed mesh volume.

[0134] To test the above concept, the two types of nets shown in Table 1 were stacked together and placed at 5 m 2 Bioreactor vessel (using FBR). Fig.21 The RTD measurements are shown. The RTD peak for the hybrid roll web is narrower than that for the single web roll and is closer to the RTD peak for the stacked disk. This demonstrates the uniform flow predicted above. The bypass is much smaller compared to the stacked substrate design. This hybrid roll design also demonstrates an additional advantage in how to handle bubbles. After introducing a large amount of air into the bed, no significant effect of bubbles was observed, as shown in Figure 2. Fig. 22 As shown. It can be expected that air bubbles can be pushed out of the bed more easily between the mesh layers in the roll design than in the mesh disk in the stacked disk design. The exemplary mesh used here only had about a 10% difference between the two types of mesh, but was still effective in preventing large areas of different packing density. This shows that even if the difference between the substrates is small (e.g., 10%), it can be beneficial. As used herein, the percentage difference between the mesh substrate layers refers to the difference in the spacing of the meshes, where the spacing is defined as the fiber diameter plus the opening diameter. Therefore, a 10% difference is equivalent to the sum of the fiber diameter and the opening diameter of one mesh being 10% larger or smaller than the sum of the fiber diameter and the opening diameter of the other mesh.

[0135] According to an embodiment, the cell culture substrate comprises a first substrate material and a second substrate material, wherein the first substrate material has a first plurality of fibers defining a first array of openings, and the second substrate material has a second plurality of fibers defining a second array of openings. The first physical structure comprises a first spacing of the first substrate material, the first spacing being the sum of the fiber diameters in the first plurality of fibers and the opening diameters of the first array of openings, and the second physical structure comprises a second spacing of the second substrate material, the second spacing being the sum of the fiber diameters in the second plurality of fibers and the opening diameters of the second array of openings. As one aspect of the embodiment, the second spacing is not the same as the first spacing. For example, in an embodiment, the second spacing is within about 100% of the first spacing, within about 90% of the first spacing, within about 75% of the first spacing, within about 50% of the first spacing, within about 25% of the first spacing, within about 15% of the first spacing, within about 10% of the first spacing, or within about 50% of the first spacing.

[0136] In addition, according to embodiments of the present disclosure, interlocking of layers can be prevented based on the structure of the structurally defined substrate. For example, certain weave types can prevent interlocking of adjacent mesh layers in a stacked mesh or rolled mesh design, or a single mesh layer in a rolled design. It is expected that plain weaves can more easily produce interlocking and create local low permeability zones, while some other weave patterns can help minimize interlocking and thus provide a more uniform fixed bed structure when used as stacked layers or rolled together for a fixed bed reactor. Twill weave is one of the examples that can help with application, but embodiments are not limited thereto and can include other weave types, including those mentioned in the present disclosure.

[0137] In some embodiments, interlocking can be prevented by wrapping the film with the web. The film will trigger on all the high points and will eliminate interlocking. Although the film will have a smaller surface area, it can also be very thin (possibly as low as 10um) and cheap, and still provide some surface area. In some embodiments, a post-heat treatment can be used, whereby the web is calendered to deform the high points, making them less likely to lock and more likely to wrap like a film. Moreover, the web can be hot-calendered onto a roller to form a monolith. Alternatively, a molten fiber web can be used.

[0138] The bioreactor container optionally includes one or more outlets, which can be attached to an import and / or an outlet device. Through the one or more outlets, liquid, culture medium or cells can be supplied to the chamber, or liquid, culture medium or cells can be removed from the chamber. A single port in the container can act as both an import and an outlet, or multiple ports can be provided for special import and export.

[0139] The fixed bed cell culture substrate of the disclosed embodiment can be composed of a cell culture woven mesh substrate, and any other form of cell culture substrate is not provided therein or any other form of cell culture substrate interlaced with the cell culture substrate. That is, the cell culture woven mesh substrate of the disclosed embodiment is an effective cell culture substrate, and does not require the irregular, non-woven substrate type used in existing solutions, or does not require the use of any separate or different structures or spacers between layers (e.g., those for establishing fluid flow channels between layers of the substrate). This enables a cell culture system with a simplified design and construction, while providing other advantages described herein about flow uniformity, harvestability, etc. to the high-density cell culture substrate.

[0140] As described herein, cell culture substrates and bioreactor systems provide multiple advantages. For example, embodiments of the present disclosure can support the production of any of many viral vectors (e.g., AAV (all serotypes) and lentiviruses), and can be applied to in vivo and in vitro gene therapy applications. Uniform cell inoculation and distribution maximize the viral vector yield per container, and the design enables the harvesting of live cells, which can be useful for seed domestication consisting of multiple amplification stages using the same platform. In addition, the embodiments herein can be expanded 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 cell culture processes to maximize vector yield and improve reproducibility. Finally, the production-level scale (e.g., 10 per batch) of viral vectors can be greatly reduced compared to other cell culture solutions. 16 Up to 10 18 The number of containers required for each AAV VG).

[0141] Embodiments are not limited to container rotation around a central longitudinal axis. For example, the container can rotate around an axis that is not located at the center relative to the container. In addition, the rotation axis can be a horizontal or vertical axis. definition

[0142] "Fully synthetic" or "all synthetic" means that the cell culture article (such as the surface of a microcarrier or culture vessel) is composed entirely of synthetically derived materials and does not have any animal-derived or animal-derived materials. The disclosed fully synthetic cell culture articles eliminate the risk of xenogeneic contamination.

[0143] The terms "include", "comprising" or similar terms mean including but not limited to, ie, inclusive rather than exclusive.

[0144] According to the embodiments of the present invention, "user" refers to a person who uses the systems, methods, products or kits disclosed herein, including a person who cultures cells to harvest cells or cell products, or a person who uses the cultured and / or harvested cells or cell products.

[0145] "About" used to describe modifications of embodiments of the present disclosure, such as the amount of an ingredient in a composition, concentration, volume, process temperature, process time, yield, flow rate, pressure, viscosity, and other numerical values ​​and their ranges, or component size, and other numerical values ​​and their ranges, refers to variations in quantity that may occur, for example, in typical assays and processing steps used to prepare materials, compositions, compounds, concentrates, component parts, manufactured articles, or applied formulations; inadvertent errors in these procedures; in differences in the manufacture, source, or purity of the starting materials or ingredients used to perform the method; and similar considerations. The term "about" also includes amounts that differ due to aging of a composition or formulation having a specific initial concentration or mixture, as well as amounts that differ due to mixing or processing a composition or formulation having a specific initial concentration or mixture.

[0146] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0147] As used herein, the indefinite article "a" or "an" and the corresponding definite article "the" mean at least one, or one or more, unless stated otherwise.

[0148] Abbreviations familiar to those of ordinary skill in the art may be used (e.g., "h" or "hrs" for hours; "g" or "gm" for grams; "mL" for milliliters; "rt" for room temperature; "nm" for nanometers, and the like).

[0149] The specific and preferred numerical values ​​and ranges disclosed in components, ingredients, additives, scales, conditions and the like are only for illustration, and they do not exclude other limited numerical values ​​or other numerical values ​​within the limited range. The systems, kits and methods disclosed herein may include any numerical value or any combination of each numerical value, specific numerical value, more specific numerical value and preferred numerical value described herein, including explicit or implicit intermediate numerical values ​​and intermediate ranges.

[0150] Unless otherwise stated, it is not intended that any method described herein be construed as requiring that its steps be performed in a specific order. Therefore, when a method claim does not actually state that its steps follow a certain order or it does not specifically indicate in any other way in the claims or description that the steps are limited to a specific order, it is not intended to imply any particular order.

[0151] It will be apparent to those skilled in the art that various modifications and variations may be made without departing from the spirit or scope of the disclosed embodiments. Because those skilled in the art may make various improvements, combinations, sub-combinations and variations to the disclosed embodiments in combination with the spirit and substance of the embodiments, the embodiments of the present disclosure shall be deemed to include all of the contents within the scope of the attached claims and their equivalents.

Claims

1. A cell culture matrix for culturing cells in a fixed bed reactor, the cell culture matrix comprising: a first substrate material constituting a first layer having opposing faces separated by a layer thickness, an ordered and regular first array of openings through the first layer, and a first physical structure that is substantially regular and uniform and configured to allow cells to grow thereon, the first physical structure separating the openings of the first array of openings from one another; and a second substrate material constituting a second layer having opposing faces separated by a layer thickness, an ordered and regular second array of openings through the second layer, and a second physical structure that is substantially regular and uniform and configured to allow cells to grow thereon, the second physical structure separating the openings of the second array of openings from one another, wherein the first substrate material and the second substrate material are rolled together to form a rolled cell culture bed, and The first physical structure is different from the second physical structure, and thus the filling density of the first substrate material and the second substrate material is substantially uniform on the entire rolled cell culture bed.

2. The cell culture matrix according to claim 1, wherein The packing density of the first substrate material and the second substrate material varies by less than about 20x, less than about 10x, less than about 5x, or less than about 2x throughout the rolled cell culture bed.

3. The cell culture matrix according to claim 1 or claim 2, wherein: the first substrate material comprises a first plurality of fibers defining a first array of openings, and the second substrate material comprises a second plurality of fibers defining a second array of openings, wherein the first physical structure comprises a first pitch of the first substrate material, the first pitch being the sum of a fiber diameter of the first plurality of fibers and an opening diameter of the first array of openings, and The second physical structure includes a second spacing of the second substrate material, and the second spacing is the sum of the fiber diameter of the second plurality of fibers and the opening diameter of the second opening array.

4. The cell culture matrix according to claim 3, wherein The second pitch is different from the first pitch.

5. The cell culture matrix according to claim 4, wherein The second spacing is within about 100% of the first spacing, the second spacing is within about 90% of the first spacing, the second spacing is within about 75% of the first spacing, the second spacing is within about 50% of the first spacing, the second spacing is within about 25% of the first spacing, the second spacing is within about 15% of the first spacing, the second spacing is within about 10% of the first spacing, or the second spacing is within about 50% of the first spacing.

6. The cell culture matrix according to any one of claims 1 to 5, wherein At least one of the first substrate material and the second substrate material comprises at least one of a molded polymer lattice, a 3D printed lattice, and a woven mesh.

7. A cell culture matrix according to any one of the preceding claims, wherein At least one of the first substrate material and the second substrate material is a woven substrate.

8. A cell culture matrix according to any one of the preceding claims, wherein At least one of the first substrate material and the second substrate material includes at least one of the following: simple weave, twill weave, Dutch weave, satin weave, regular matte weave, irregular matte rib weave, irregular weft rib weave, regular weft rib weave, regular warp rib weave, irregular warp rib weave, weft satin weave, 2 / 2 twill weave, 3 / 3 twill weave, square plain weave, mountain twill weave, royal oxford weave, houndstooth weave, herringbone weave, or five-needle weave.

9. A cell culture matrix according to any one of the preceding claims, wherein The first substrate material and the second substrate material are arranged as alternating layers of the rolled cell culture bed.

10. A cell culture matrix according to any one of the preceding claims, wherein The first substrate material and the second substrate material are in direct physical contact with each other.

11. A cell culture matrix according to any one of the preceding claims, wherein The first substrate material and the second substrate material are not separated from each other by a spacing material or barrier.

12. The cell culture substrate of any of the preceding claims, further comprising a third substrate material constituting a third layer having opposing faces separated by the thickness of the third layer, an ordered and regular third array of openings through the third layer, and a substantially regular and uniform third physical structure configured to allow cells to grow thereon, the third physical structure separating the openings of the third array of openings from one another, in, The first substrate material, the second substrate material and the third substrate material are rolled together to form a rolled cell culture bed.

13. A cell culture matrix as claimed in any one of the preceding claims, wherein The rolled cell culture bed has no spacer material or barrier between subsequent substrate layers of the rolled cell culture bed.

14. A cell culture matrix as claimed in any one of the preceding claims, in, The first substrate material and the second substrate material differ from one another in at least one physical dimension.

15. The cell culture matrix according to claim 1, wherein The at least one physical dimension is at least one of a diameter of the opening, a thickness of the physical structure, a pattern of the physical structure, a weave type, and a spacing of the physical structure on either side of the opening.

16. A cell culture matrix as claimed in any one of the preceding claims, wherein The variation in flow rate of a fluid flowing through the cell culture matrix is ​​uniform across the width of the cell culture matrix, which is a direction perpendicular to the direction of fluid flow.

17. A cell culture matrix as claimed in any one of the preceding claims, wherein The plurality of substrate layers include at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

18. A cell culture matrix according to any one of the preceding claims, wherein The plurality of fibers include first fibers having a first fiber diameter of about 30 μm to about 1000 μm, about 30 μm to about 600 μm, about 30 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

19. A cell culture matrix according to any one of the preceding claims, wherein The opening comprises a diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

20. A bioreactor system for culturing cells, the system comprising: a cell culture container comprising at least one internal reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir; and The cell culture matrix according to any one of claims 1 to 19, wherein the cell culture matrix is ​​disposed in a reservoir.

21. The bioreactor system of claim 20, wherein: The bioreactor system is configured to pass a perfusion flow through a cell culture matrix during cell culture.

22. The bioreactor system of claim 20 or 21, wherein: The bioreactor system is configured to harvest living cells from a cell culture matrix in a reservoir.

23. A cell culture matrix for culturing cells in a fixed bed reactor, the cell culture matrix comprising: a first substrate material comprising a first layer having opposing faces separated by a layer thickness, a first array of openings through the first layer, and a first physical structure comprising a first array of fibers, the first physical structure being configured to grow cells thereon and to separate the openings of the first array of openings from one another; and a second substrate material constituting a second layer, the second substrate material having opposing faces separated by the thickness of the layer, a second array of openings through the second layer, and a second physical structure comprising a second array of fibers, the second physical structure being configured to allow cells to grow thereon and to separate the openings of the second array of openings from one another, wherein the first substrate material and the second substrate material are rolled together to form a rolled cell culture bed, and The first physical structure is different from the second physical structure.

24. The cell culture matrix according to claim 23, wherein By virtue of the first physical structure being different from the second physical structure, the packing density of the first substrate material and the second substrate material is substantially uniform throughout the rolled cell culture bed.

25. The cell culture matrix according to claim 23 or claim 24, wherein The first array of openings is in an ordered and regular first array.

26. The cell culture matrix according to any one of claims 23 to 25, wherein The second array of openings is in an ordered and regular second array.

27. The cell culture matrix according to any one of claims 23 to 26, wherein The first physical structure and the second physical structure are substantially regular and uniform.

28. The cell culture matrix according to any one of claims 23 to 27, wherein The first physical structure and the second physical structure include fibers in a non-random arrangement.

29. A cell culture matrix for culturing cells in a fixed bed reactor, the cell culture matrix comprising: a first substrate material constituting a first layer having opposing faces separated by the thickness of the first layer, an ordered and regular array of openings through the first layer, and a substantially regular and uniform physical structure configured to allow cells to grow thereon, the physical structure separating the openings from one another, wherein the first substrate material has a three-dimensional surface defined by an array of openings and a physical structure, and The three-dimensional surface is configured to prevent a first layer of substrate material from nesting with a second layer of substrate material, the second layer being adjacent to and in direct physical contact with the first layer.

30. The cell culture matrix of claim 29, wherein The first substrate material is a single piece that constitutes the first layer and the second layer.

31. The cell culture matrix of claim 30, wherein: The single sheet is rolled up to form a roll-shaped cell culture bed, wherein the rolling up forms a plurality of radially arranged roll layers in the roll-shaped cell culture bed, the plurality of roll layers comprising a first layer and a second layer.

32. The cell culture matrix according to any one of claims 29 to 31, wherein The first substrate material is a woven substrate comprising a plurality of fibers woven together in a predetermined pattern.

33. The cell culture matrix of claim 32, wherein The woven substrate includes twill weave, Dutch weave, satin weave, regular matte weave, irregular matte rib weave, irregular weft rib weave, regular weft rib weave, regular warp rib weave, irregular warp rib weave, weft satin weave, 2 / 2 twill weave, 3 / 3 twill weave, square plain weave, mountain twill weave, royal oxford weave, houndstooth weave, herringbone weave, or five-needle weave.

34. The cell culture substrate of any one of claims 29-33, further comprising a second substrate material having an ordered and regular array of openings through the layer, the openings being separated by the substrate material, the substrate material having a substantially regular and uniform physical structure and the physical structure being configured to allow cells to grow thereon, in, The second substrate material constitutes the second layer, and The first substrate material and the second substrate material have the same physical structure and the same opening array.

35. The cell culture matrix of claim 34, wherein The first substrate layers and the second substrate layers are stacked in an alternating arrangement.

36. The cell culture matrix of claim 34, wherein The first substrate material and the second substrate material are rolled up to form a rolled cell culture bed, wherein the rolling up forms a plurality of radially arranged roll layers in the rolled cell culture bed, the plurality of roll layers including a first layer and a second layer.

37. The cell culture substrate of any one of claims 29-33, further comprising a second substrate material having an ordered and regular array of openings through the layer, the openings being separated by the substrate material, the substrate material having a substantially regular and uniform physical structure and the physical structure being configured to allow cells to grow thereon, in, The second substrate material constitutes the second layer, and Wherein the first substrate material and the second substrate material differ from each other in at least one physical dimension, and the first substrate material and the second substrate material are arranged as alternating layers of the cell culture matrix.

38. The cell culture matrix according to any one of claims 34 to 37, wherein The second substrate material includes twill weave, Dutch weave, satin weave, regular matte weave, irregular matte rib weave, irregular weft rib weave, regular weft rib weave, regular warp rib weave, irregular warp rib weave, weft satin weave, 2 / 2 twill weave, 3 / 3 twill weave, square plain weave, mountain twill weave, royal oxford weave, houndstooth weave, herringbone weave, five-needle weave, micro mesh or mesh netting.

39. The cell culture matrix of claim 37 or claim 38, wherein The at least one physical dimension is at least one of a diameter of the opening, a thickness of the physical structure, a pattern of the physical structure, and a spacing of the physical structures on either side of the opening.

40. The cell culture matrix according to any one of claims 34 to 39, wherein The first substrate material and the second substrate material comprise at least one of a molded polymer lattice, a 3D printed lattice, and a woven mesh.

41. The cell culture matrix according to any one of claims 29 to 40, wherein The degree of relative rotation between the first substrate material and the second substrate material is random.

42. The cell culture matrix according to any one of claims 29 to 41, wherein The variation in flow rate of a fluid flowing through the cell culture matrix is ​​uniform across the width of the cell culture matrix, which is a direction perpendicular to the direction of fluid flow.

43. The cell culture matrix according to any one of claims 31 to 42, wherein The roll-like layers are not separated by spacers or barriers, or they are in physical contact with each other.

44. The cell culture matrix according to any one of claims 29 to 43, wherein The first substrate material includes at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

45. The cell culture matrix of any one of claims 32-44, wherein The plurality of fibers include first fibers having a first fiber diameter of about 30 μm to about 1000 μm, about 30 μm to about 600 μm, about 30 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

46. ​​The cell culture matrix of any one of claims 29-45, wherein The opening comprises a diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

47. The cell culture matrix of any one of claims 29-46, wherein The physical structure and the array of openings are configured such that when the first substrate material is rolled into a roll-like substrate matrix and the roll-like substrate matrix contains a roll-like layer of the first substrate material, the packing density of the roll-like layer varies by less than about 20x, less than about 10x, less than about 5x or less than about 2x throughout the roll-like substrate matrix.

48. A bioreactor system for culturing cells, the system comprising: a cell culture container comprising at least one internal reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir; and The cell culture matrix of any one of claims 29-47, wherein the cell culture matrix is ​​disposed in a reservoir.

49. The bioreactor system of claim 48, wherein: The bioreactor system is configured to pass a perfusion flow through a cell culture matrix during cell culture.

50. A bioreactor system as claimed in claim 48 or claim 49, wherein: The bioreactor system is configured to harvest living cells from a cell culture matrix in a reservoir.

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