Roll-shaped adherent cell culture substrate for uniform flow in a fixed bed reactor

Abstract

A cell culture matrix for culturing cells in a fixed bed reactor is provided. The cell culture matrix includes a first substrate material having a regular and orderly array of openings passing through the layer, the openings being separated by a substrate material having a physical structure that is substantially regular and uniform and configured such that cells grow thereon. The physical structure and array of the openings are configured such that when the first substrate material is rolled into a roll-shaped substrate matrix including a roll-shaped layer of the first substrate material, the variation in the packing density of the roll-shaped layer is less than about 20 times, less than about 10 times, less than about 5 times, or less than about 2 times throughout the roll-shaped 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 Patent Application No. 63 / 395,409, filed on August 5, 2022, and U.S. Provisional Patent Application No. 63 / 394,848, filed on August 3, 2022, the contents of which are relied upon and incorporated herein by reference in their entirety.

Technical Field

[0002] The present disclosure generally relates to substrates for culturing cells and fixed - bed bioreactors incorporating such substrates. In particular, the present disclosure relates to fixed beds having roll - shaped substrates exhibiting uniform fluid flow characteristics and bioreactors equipped with such fixed beds.

Background Art

[0003] In the bioprocess industry, large - scale culturing of cells is carried out for the production of hormones, enzymes, antibodies, vaccines, and cell therapies. The markets for cell therapy and gene therapy are growing rapidly, and promising therapies are moving into clinical trials and rapidly advancing towards commercialization. However, a single administration of cell therapy may require billions of cells or trillions of viruses. Therefore, for clinical success, it is important to be able to provide large quantities of cell products or virus products in a short period of time.

[0004] A significant portion of the cells used in bioprocesses are substrate - dependent, meaning that cells require a surface to which they can adhere in order to grow and function. Conventionally, the culture of adherent cells is carried out on two - dimensional (2D) cell - adhesion surfaces incorporated into one of a number of container formats, such as T - flasks, Petri dishes, cell factories, cell stack containers, roller bottles, and HYPER - STACK (registered trademark) containers. These methods may have significant drawbacks, including difficulty in achieving cell densities high enough to enable large - scale production of therapeutic agents or cells.

[0005] Alternative methods for increasing the volumetric density of cultured cells have been proposed. These include microcarrier culture carried out in stirred tanks. In this technique, cells adhered to the surface of the microcarriers are exposed to a certain shear stress, which as a result has a significant impact on growth and culture performance. Another example of a high-density cell culture system is the hollow fiber bioreactor, where cells can form large three-dimensional aggregates when growing in the inter-fiber space. However, when nutrients are insufficient, cell growth and performance are significantly inhibited. To mitigate this problem, these bioreactors are made small and are not suitable for large-scale manufacturing.

[0006] Another example of a high-density culture system for anchorage-dependent cells is the packed bed bioreactor system. In this type of bioreactor, a cell substrate is randomly packed into the bioreactor vessel and used to provide a surface for the attachment of adherent cells. The medium is perfused along the surface or through the packed bed to supply the nutrients and oxygen necessary for cell growth. For example, packed bed bioreactor systems including a packed bed for capturing cells are disclosed in Patent Documents 1 to 3. The packed bed matrix is usually made of porous particles or non-woven microfibers of a polymer as a substrate. Such a bioreactor functions as a recirculation flow-through bioreactor. One of the major problems with such bioreactors is the non-uniform cell distribution within the packed bed. For example, the packed bed functions as a depth filter, and cells are mainly captured in the inlet region, resulting in a gradient of cell distribution during the inoculation step. In addition, due to the random packing of the fibers, the flow resistance and cell capture efficiency across the cross-section of the packed bed are not uniform. For example, the medium flows fast in regions with a low cell packing density and slowly in regions with a high resistance where a large number of cells are captured. This results in a channeling effect where nutrients and oxygen are more efficiently supplied to regions with a low volumetric cell density and maintained under sub-optimal culture conditions in regions with a high cell density.

[0007] Another significant drawback of the packed bed systems disclosed in the prior art is the inability to efficiently recover intact live cells at the end of the culture process. Cell recovery is important when the final product is cells, or when the bioreactor is used as part of a "seed train" that grows a cell population in one vessel and then transfers the cell population to another vessel for further growth. Patent Document 4 discloses the design of a bioreactor for improving cell recovery efficiency from a packed bed during the cell harvesting step. This is based on loosening the packed bed matrix, agitating the packed bed particles, and causing the porous matrix to collide, thereby separating the cells. However, this method is laborious and can cause significant cell damage, resulting in a decrease in overall cell viability.

[0008] For example, some packed-bed bioreactors use small strips of a cell substrate material consisting of randomly oriented fibers in a non-woven configuration. These strips are packed into a vessel to create a packed bed. However, due to the non-uniform packing of the substrate strips, visible channels are created within the packed bed, resulting in a preferential and non-uniform flow of medium and distribution of nutrients throughout the packed bed. Studies of such systems have pointed out "a systematic non-uniform distribution of cells that increases in number from the top to the bottom of the fixed bed", as well as "nutrient gradients that lead to limitations in cell growth and production", all of which lead to "a non-equal distribution of cells that can impair transfection efficiency." (Rational plasmid design and bioprocess optimization for enhancing the productivity of recombinant adeno-associated virus (AAV) in mammalian cells (Non-Patent Document 1). The study pointed out that agitation of the packed bed would improve dispersion but would have other drawbacks (i.e., "necessary agitation for better dispersion during inoculation and transfection would induce increased shear stress, in turn leading to reduced cell viability." (Non-Patent Document 1). Another study pointed out that it is difficult to monitor cell populations using a biomass sensor because the cells are non-uniformly distributed ("... if the cells are unevenly distributed, the biomass signal from the cells on the top carriers may not show the general view of the entire bioreactor." Non-Patent Document 2).

[0009] In addition, due to the random arrangement of the fibers within the substrate strip and the variation in the packing of the strips between one packed bed and another in a given packed bed reactor system, the substrate is not only non-uniform within a given bioreactor vessel but also varies between different reactors or different packed beds during the implementation of cell culture, making it difficult for the user to predict cell culture performance. Furthermore, since the packed substrates of many existing bioreactors are thought to trap cells within the packed bed, efficient cell recovery is made very difficult or impossible.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0012] To reach late-stage commercial production scale, a platform is needed that can produce higher quantities of high-quality cell and viral products. In particular, there is a need for cell culture substrates and / or matrices, bioreactors, systems, and methods that enable cell culture in a high-density format with uniform cell distribution, improved uniform fluid flow characteristics, and easily achievable and increased harvests.

Means for Solving the Problems

[0013] According to an embodiment of the present disclosure, a cell culture matrix for culturing cells in a fixed bed reactor is provided. The cell culture matrix includes a first substrate material having an ordered regular array of openings passing through a layer, the openings being substantially regular and uniform and separated by a substrate material having a physical structure configured for cells to grow thereon. The physical structure and array of the openings are configured such that when the first substrate material is rolled into a roll-shaped substrate matrix including a roll-shaped layer of the first substrate material, the variation in the packing density of the roll-shaped layer is less than about 20-fold, less than about 10-fold, less than about 5-fold, or less than about 2-fold throughout the roll-shaped substrate matrix.

[0014] The following is an explanation of various aspects of the implementation forms 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 implementation forms are intended to explain some aspects of the disclosed subject matter and should not be regarded as an inclusive or exhaustive description of all possible implementation forms.

[0015] Aspect 1 relates to a cell culture matrix for culturing cells in a fixed bed reactor, comprising a first substrate material including a first layer having opposing surfaces separated by a layer thickness, a first ordered regular array of openings passing through the first layer, and a first physical structure that is substantially regular and uniform and configured such that cells grow thereon, the first physical structure separating the openings of the first opening array. The cell culture matrix also includes a second substrate material including a second layer having opposing surfaces separated by a layer thickness, a second ordered regular array of openings passing through the second layer, and a second physical structure that is substantially regular and uniform and configured such that cells grow thereon, the second physical structure separating the openings of the second opening array. The first substrate material and the second substrate material are rolled together to form a roll-shaped cell culture bed. The first physical structure is different from the second physical structure, whereby the packing densities of the first substrate material and the second substrate material are substantially uniform throughout the roll-shaped cell culture bed.

[0016] Aspect 2 relates to the cell culture matrix according to Aspect 1, wherein the variation in the packing density of the first substrate material and the second substrate material is less than about 20 times, less than about 10 times, less than about 5 times, or less than about 2 times throughout the roll-shaped cell culture bed.

[0017] Aspect 3 relates to the cell culture matrix according to Aspect 1 or Aspect 2, wherein the first substrate material includes a first plurality of fibers defining the first opening array, the second substrate material includes a second plurality of fibers defining the second opening array, wherein the first physical structure includes a first spacing of the first substrate material, the first spacing being the sum of the fiber diameter of the first plurality of fibers and the opening diameter of the first opening array, and the second physical structure includes a second spacing of the second substrate material, the second spacing being the sum of the fiber diameter of the second plurality of fibers and the opening diameter of the second opening array.

[0018] Aspect 4 relates to the cell culture matrix according to Aspect 3, wherein the second interval is different from the first interval.

[0019] Aspect 5 relates to the cell culture matrix according to Aspect 4, wherein the second interval is within about 100% of the first interval, within about 90% of the first interval, within about 75% of the first interval, within about 50% of the first interval, within about 25% of the first interval, within about 15% of the first interval, within about 10% of the first interval, or within about 5% of the first interval.

[0020] Aspect 6 relates to the cell culture matrix according to any one of Aspects 1 to 5, wherein at least one of the first base material and the second base material includes at least one of a molded polymer lattice, a 3D printed lattice, and a woven mesh.

[0021] Aspect 7 relates to the cell culture matrix according to any one of Aspects 1 to 6, wherein at least one of the first base material and the second base material is a textile substrate.

[0022] Aspect 8 relates to the cell culture matrix according to any one of Aspects 1 to 7, wherein at least one of the first base material and the second base material includes at least one of plain weave, twill weave, Dutch weave, crepe weave, regular mat weave, irregular mat 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, point twill weave, royal oxford weave, houndstooth check weave, herringbone weave, or five needle weave.

[0023] Aspect 9 relates to the cell culture matrix according to any one of Aspects 1 to 8, wherein the first base material and the second base material are arranged as alternating layers of a roll-shaped cell culture bed.

[0024] Aspect 10 relates to the cell culture matrix according to any one of Aspects 1 to 9, wherein the first base material and the second base material are in direct physical contact with each other.

[0025] Aspect 11 relates to the cell culture matrix according to any one of Aspects 1 to 10, in which the first base material and the second base material are not separated from each other by a spacer material or a barrier.

[0026] Aspect 12 further includes a third base material including a third layer having opposing surfaces separated by the thickness of the third layer, a regular and orderly third array of openings passing through the third layer, and a third physical structure that is substantially regular and uniform and on which cells are configured to grow, the third physical structure separating the openings of the third opening array from each other, and the first base material, the second base material, and the third base material are rolled together to form a roll-shaped cell culture bed, and relates to the cell culture matrix according to any one of Aspects 1 to 11.

[0027] Aspect 13 relates to the cell culture matrix according to any one of Aspects 1 to 12, in which there is no spacer material or barrier between subsequent base material layers of the roll-shaped cell culture bed.

[0028] Aspect 14 relates to the cell culture matrix according to any one of Aspects 1 to 13, in which the first and second base materials differ from each other in at least one physical dimension.

[0029] Aspect 15 relates to the cell culture matrix according to Aspect 1, in which at least one physical dimension is at least one of the diameter of the opening, the thickness of the physical structure, the pattern of the physical structure, the weaving method, and the interval of the physical structures on both sides of the opening.

[0030] Aspect 16 relates to the cell culture matrix according to any one of Aspects 1 to 15, in which the change in the flow rate of the fluid flowing through the cell culture matrix is uniform across the width of the cell culture matrix, and the width is in a direction perpendicular to the direction of the fluid flow.

[0031] Aspect 17 relates to the cell culture matrix according to any one of Aspects 1 to 16, wherein the plurality of base material layers includes at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrroles, and polypropylene oxide.

[0032] Aspect 18 relates to the cell culture matrix according to any one of Aspects 1 to 17, wherein the plurality of fibers includes 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.

[0033] Aspect 19 relates to the cell culture matrix according to any one of Aspects 1 to 18, wherein the opening has 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.

[0034] Aspect 20 relates to a bioreactor system for culturing cells, comprising a cell culture vessel including 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 to 19, disposed within the reservoir.

[0035] Aspect 21 relates to the cell culture matrix according to Aspect 20, wherein the bioreactor system is configured for perfusion flow through the cell culture matrix during cell culture.

[0036] Aspect 22 relates to the cell culture matrix according to Aspect 20 or Aspect 21, wherein the bioreactor system is configured to recover live cells from the cell culture matrix within the reservoir.

[0037] Aspect 23 relates to a cell culture matrix for culturing cells in a fixed bed reactor, the cell culture matrix comprising a first substrate material including a first layer having opposing surfaces separated by a layer thickness, a first array of openings passing through the first layer, and a first physical structure including a first array of fibers configured to grow cells thereon and separate the openings of the first array of openings from each other; and a second substrate material including a second layer having opposing surfaces separated by a layer thickness, a second array of openings passing through the second layer, and a second physical structure including a second array of fibers configured to grow cells thereon and separate the openings of the second array of openings from each other, wherein the first substrate material and the second substrate material are rolled together to form a roll-shaped cell culture bed, and the first physical structure is different from the second physical structure.

[0038] Aspect 24 relates to the cell culture matrix according to Aspect 23, wherein the packing density of the first substrate material and the second substrate material is substantially uniform throughout the roll-shaped cell culture bed due to a first physical structure that is different from the second physical structure.

[0039] Aspect 25 relates to the cell culture matrix according to Aspect 23 or Aspect 24, wherein the first array of openings forms an ordered regular first array.

[0040] Aspect 26 relates to the cell culture matrix according to any one of Aspects 23 to 25, wherein the second array of openings forms an ordered regular second array.

[0041] Aspect 27 relates to the cell culture matrix according to any one of Aspects 23 to 26, wherein the first physical structure and the second physical structure are substantially regular and uniform.

[0042] Aspect 28 relates to the cell culture matrix according to any one of Aspects 23 to 27, wherein the first physical structure and the second physical structure include fibers in a non-random arrangement.

[0043] Embodiment 29 relates to a cell culture matrix for culturing cells in a fixed-bed reactor, the cell culture matrix comprising a first substrate material including: a first layer with opposing surfaces separated by a thickness of the first layer; an ordered and regular array of openings through the first layer; and a substantially regular and uniform physical structure upon which cells are configured to grow, the physical structure separating the openings from one another, wherein the first substrate material has a three-dimensional surface defined by the array of openings and the physical structure, the three-dimensional surface configured to prevent nesting of the first layer with a second layer of substrate material adjacent to and in direct physical contact with the first layer.

[0044] Example 30 relates to the cell culture matrix of example 29, wherein the first substrate material is a single sheet comprising a first layer and a second layer.

[0045] Example 31 relates to the cell culture matrix of Example 30, wherein a single sheet is rolled to form a rolled cell culture bed, and the rolling forms a plurality of radially arranged roll layers within the rolled cell culture bed, the plurality of roll layers comprising a first layer and a second layer.

[0046] Example 32 relates to the cell culture matrix of any of Examples 29 to 31, wherein the first substrate material is a woven substrate, the woven substrate comprising a plurality of fibers interwoven in a predetermined manner.

[0047] Example 33 relates to the cell culture matrix of example 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 point twill weave, a royal oxford weave, a houndstooth weave, a herringbone weave, or a five needle weave.

[0048] Aspect 34 further includes a second substrate material having an orderly and regular array of openings passing through the layer, the openings being separated by a substrate material having a physical structure that is substantially regular and uniform and configured such that cells grow thereon, the second substrate material including a second layer, and the first and second substrate materials having the same physical structure and the same opening array, and relates to the cell culture matrix according to any one of Aspects 29 to 33.

[0049] Aspect 35 relates to the cell culture matrix according to Aspect 34, in which the first and second substrate layers are laminated in an alternating arrangement.

[0050] Aspect 36 relates to the cell culture matrix according to Aspect 34, in which the first substrate material and the second substrate material are rolled to form a roll-shaped cell culture bed, and by being rolled, a plurality of roll layers arranged radially in the roll-shaped cell culture bed are formed, and the plurality of roll layers include a first layer and a second layer.

[0051] Aspect 37 further includes a second substrate material having an orderly and regular array of openings passing through the layer, the openings being separated by a substrate material having a physical structure that is substantially regular and uniform and configured such that cells grow thereon, the second substrate material including a second layer, the first and second substrate materials being different from each other in at least one physical dimension, and the first substrate material and the second substrate material being arranged as alternating layers of the cell culture matrix, and relates to the cell culture matrix according to any one of Aspects 29 to 33.

[0052] Aspect 38 relates to the cell culture matrix according to any one of Aspects 34 to 37, wherein the second base material includes twill weave, Dutch weave, crepe weave, regular mat weave, irregular mat rib weave, irregular weft rib weave, regular weft rib weave, regular warp rib weave, irregular warp rib weave, damask weave, 2 / 2 twill weave, 3 / 3 twill weave, basket weave, point twill weave, royal oxford weave, houndstooth check weave, herringbone weave, five - needle knitting, micro - mesh, or mesh netting.

[0053] Aspect 39 relates to the cell culture matrix according to Aspect 37 or 38, wherein at least one physical dimension is at least one of the diameter of the opening, the thickness of the physical structure, the pattern of the physical structure, and the distance between the physical structures on both sides of the opening.

[0054] Aspect 40 relates to the cell culture matrix according to any one of Aspects 34 to 39, wherein the first base material and the second base material include at least one of a molded polymer lattice, a 3D - printed lattice, and a woven mesh.

[0055] Aspect 41 relates to the cell culture matrix according to any one of Aspects 29 to 40, wherein the degree of relative rotation between the first base material and the second base material is random.

[0056] Aspect 42 relates to the cell culture matrix according to any one of Aspects 29 to 41, wherein the change in the flow rate of the fluid flowing through the cell culture matrix is uniform across the width of the cell culture matrix, and the width is in a direction perpendicular to the direction of the fluid flow.

[0057] Aspect 43 relates to the cell culture matrix according to any one of Aspects 31 to 42, wherein the roll - shaped layers are not separated by a spacer material or a barrier, or are in physical contact with each other.

[0058] Aspect 44 relates to the cell culture matrix according to any of Aspects 29 to 43, wherein the first base material includes at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrroles, and polypropylene oxide.

[0059] Aspect 45 relates to the cell culture matrix according to any of Aspects 32 to 44, wherein the plurality of fibers includes first fibers having a first fiber diameter of from about 30 μm to about 1000 μm, from about 30 μm to about 600 μm, from about 30 μm to about 400 μm, from about 100 μm to about 325 μm, or from about 150 μm to about 275 μm.

[0060] Aspect 46 relates to the cell culture matrix according to any of Aspects 29 to 45, wherein the opening includes a diameter of from about 100 μm to about 1000 μm, from about 200 μm to about 900 μm, or from about 225 μm to about 800 μm.

[0061] Aspect 47 relates to the cell culture matrix according to any of Aspects 29 to 46, wherein when the first base material is rolled into a roll-shaped base matrix including a roll-shaped layer of the first base material, the physical structure and array of the openings are configured such that the variation in the packing density of the roll-shaped layer is less than about 20 times, less than about 10 times, less than about 5 times, or less than about 2 times throughout the roll-shaped base matrix.

[0062] Aspect 48 relates to a bioreactor system for culturing cells, the system including at least one internal reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir; and a cell culture container including a cell culture matrix according to any of Embodiments 29 to 47, the cell culture matrix being disposed within the reservoir.

[0063] Aspect 49 relates to the cell culture matrix according to Aspect 48, wherein the bioreactor system is configured for perfusion flow through the cell culture matrix during cell culture.

[0064] Aspect 50 relates to the cell culture matrix according to aspect 48 or aspect 49, wherein the bioreactor system is configured to recover live cells from the cell culture matrix in the reservoir. BRIEF DESCRIPTION OF THE DRAWINGS

[0065]

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DETAILED DESCRIPTION OF THE INVENTION

[0066] Various embodiments of the present disclosure will be described in detail with reference to the drawings, if any. References to various embodiments are not intended to limit the scope of the invention, which is limited only by the appended claims. Additionally, the examples described herein are not limiting and merely describe some of the many possible embodiments of the claimed invention.

[0067] Embodiments of the present disclosure include fixed bed cell culture substrates, as well as cell culture systems or bioreactor systems incorporating such substrates. The substrate, and the bioreactor system incorporating it, exhibit improved flow characteristics through the substrate. For example, a more uniform flow is achieved through the substrate, and non-uniform flow due to channeling or turbulence is reduced or eliminated. The "dead zones" of the flow within the substrate or fixed bed of the bioreactor are significantly reduced or eliminated as compared to alternative solutions. As a result, a substrate or fixed bed is obtained that enables uniform perfusion throughout the substrate or fixed bed, which promotes the health of the cells during cell culture and also promotes an efficient cell culture process with respect to not only cell culture but also seeding of cells and recovery of cells or cell by-products.

[0068] 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 substrate pieces, and bioreactors in which such fixed bed cell culture substrates are disposed. Aspects of embodiments of the present disclosure enable simplifying such assembly of the fixed bed and / or placement of the fixed bed into the reactor by reducing the degree to which fragments of the cell culture substrate need to be aligned with each other, thereby reducing the need for complex procedures for handling and assembling the fixed bed or the need for complex mechanisms within the bioreactor to maintain a particular orientation or alignment of the fixed bed. This reduction in complexity can lead to faster and less expensive manufacturing, shipping, and assembly, as well as a more reliable bioreactor.

[0069] Aspects of embodiments also include fixed bed substrates and bioreactors that provide a more uniform fluid flow through the fixed bed of the cell culture substrate.

[0070] In conventional large-scale cell culture bioreactors, various types of fixed-bed or packed-bed bioreactors have been used. Typically, these packed beds contain a porous matrix to retain adherent or suspended cells and support their growth and proliferation. The packed-bed matrix has a high surface area-to-volume ratio and can achieve a higher cell density than other systems. However, packed beds often function as depth filters where cells are physically trapped in the intertwined fibers of the matrix. Therefore, due to the linear flow of the cell inoculum material through the packed bed, cells are non-uniformly distributed within the packed bed, leading to variations in cell density across the depth or width of the packed bed. For example, the cell density is higher in the inlet region of the bioreactor and significantly lower closer to the outlet of the bioreactor. This non-uniform distribution of cells within the packed bed significantly hinders the scalability and predictability of such bioreactors in bioprocess manufacturing and can even lead to a decrease in the efficiency of cell growth or virus vector production per unit surface area or volume of the packed bed.

[0071] Another problem encountered in packed-bed bioreactors disclosed in the prior art is the channeling effect. Due to the random nature of the packed non-woven fibers, the local fiber density in any cross-section of the packed bed is not uniform. The liquid medium flows rapidly in regions with low fiber density (high bed permeability) but very slowly in regions with high fiber density (low bed permeability). The resulting non-uniform medium perfusion across the packed bed creates the channeling effect, which manifests as significant nutrient and metabolite gradients that negatively impact overall cell culture and bioreactor performance. Cells located in regions with low medium perfusion become starved and often die due to nutrient deprivation or metabolite toxicity. Because the flow can be highly non-uniform, there are effectively "dead zones" of flow within the packed bed where perfusion does not occur, and nutrient delivery to the cells in that region is limited to diffusion kinetics within the medium.

[0072] When using a bioreactor filled with a non-woven fiber scaffold, cell recovery encounters yet another problem. Since the packed bed functions as a depth filter, the cells released at the end of the cell culture process are trapped within the packed bed, resulting in a very low cell recovery rate. This significantly limits the use of such bioreactors in bioprocesses where live cells are the product or where live cells need to be harvested for further processing to capture cell by-products. Thus, due to the non-uniformity, the regions exposed to flow and shear are different, effectively reducing the available cell culture area, making the culture non-uniform, and hindering transfection efficiency and cell release.

[0073] To address these and other problems of existing cell culture solutions, embodiments of the present disclosure provide a cell growth substrate, a fixed bed assembly of such a substrate, and / or a bioreactor system using such a substrate that simplifies the manufacture and assembly of fixed beds while enabling efficient and high-yield cell culture and production of cell products (e.g., proteins, antibodies, virus particles) of scaffold-dependent cells. Embodiments include a porous cell culture matrix made from a regularly ordered configuration of a porous substrate material that enables uniform cell seeding and perfusion of media / nutrients, as well as efficient cell recovery. Embodiments also enable an expandable cell culture solution with a substrate and bioreactor that can seed and grow cells and / or recover cell products from the process development scale to the 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 the process development scale to the product scale, and the virus genome per unit surface area of the substrate (VG / cm 2) is equivalent. The recoverability and scalability of the embodiments herein enable the use in an efficient seed train for growing cell populations at multiple scales on the same cell substrate. Additionally, the embodiments herein provide a cell culture matrix with a high surface area that, in combination with the other features described, enables a high-yield cell culture solution. In some embodiments, for example, the cell culture substrates and / or bioreactors discussed herein can generate from 10 16 to 10 18 viral genomes (VG) per batch.

[0074] In one embodiment, the matrix is provided with a structurally defined surface area for adherent cells to adhere and grow thereon, which has good mechanical strength and forms a very uniform number of interconnected fluid networks when assembled in a packed bed or other bioreactor. For example, in an embodiment, the structurally defined substrate has sufficient mechanical strength such that it does not deform at the perfusion flow rates used in bioreactor systems. That is, the substrate will either not deform at all or will not deform significantly enough to affect the flow of fluid through the fixed bed. In certain embodiments, a mechanically stable and non-degradable woven mesh can be used as the substrate to assist in the production of adherent cells. The cell culture matrix disclosed herein supports the adhesion and growth of anchorage-dependent cells in a high volume density format. Uniform cell seeding onto such a matrix can be achieved, and efficient recovery of cells or other products of the bioreactor is also possible. Additionally, embodiments of the present disclosure provide a uniform cell distribution during the seeding step, support cell culture to achieve a confluent monolayer or multilayer of adherent cells on the disclosed matrix, and avoid the formation of large and / or uncontrollable 3D cell aggregates where nutrient diffusion is limited and metabolite concentrations increase. Thus, the matrix eliminates diffusion limitations during bioreactor operation. Additionally, the matrix enables easy and efficient cell recovery from the bioreactor. The structurally defined matrix of one or more embodiments enables complete cell recovery and consistent cell recovery from the packed bed of the bioreactor.

[0075] In contrast to existing cell culture substrates used in cell culture bioreactors (e.g., randomly configured fibrous nonwoven substrates), embodiments of the present disclosure include cell culture substrates having a defined regular structure. The defined regular structure provides consistent and predictable cell culture results. Additionally, the substrate has an open porous structure that prevents cell entrapment and allows for a uniform flow through the packed bed. This structure enables improved cell seeding, nutrient delivery, cell growth, and cell recovery. According to one or more particular embodiments, the fixed bed matrix is formed of at least one substrate material having a thin sheet-like structure with a first surface and a second surface separated by a relatively thin thickness such that the thickness of the sheet is small compared to the width and / or length of the first and second surfaces of the substrate. Additionally, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is sized and shaped such that cells can adhere to the surface of the substrate material as if it were a substantially two-dimensional (2D) surface while simultaneously allowing for proper fluid flow around and through the substrate material and the openings. In some embodiments, the substrate is a polymer-based material and can be formed as a molded polymer sheet; a polymer sheet with openings perforated throughout the thickness; a plurality of filaments fused to a mesh-like 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 area to volume ratio for culturing anchorage-dependent cells. According to various embodiments, the fixed bed matrix can be arranged or packed within a bioreactor in certain ways discussed herein for uniform cell seeding and growth, uniform media perfusion, efficient cell recovery, and simplified manufacturing and packing.

[0076] Embodiments of the present disclosure provide greater than about 10 14 supervirus genomes per batch, greater than about 10 15 supervirus genomes per batch, greater than about 10 16 supervirus genomes per batch, greater than about 10 17 supervirus genomes per batch, or up to about 10 16or a viral vector platform of a practical size capable of producing viral genomes on a scale of the viral genome or more. In some embodiments, the production is about 10 per batch 15 to about 10 18 or more viral genomes. For example, in some embodiments, the viral genome yield is about 10 per batch 15 to about 10 16 of viral genomes, or about 10 per batch 16 to about 10 19 of viral genomes, or about 10 per batch 16 ~10 18 of viral genomes, or about 10 per batch 17 to about 10 19 of viral genomes, or about 10 per batch 18 to about 10 19 of viral genomes, or about 10 per batch 18 or more viral genomes.

[0077] In addition, the embodiments disclosed herein enable not only the adhesion and growth of cells to a cell culture substrate but also the viable recovery of cultured cells. The inability to recover live cells is a significant drawback of current platforms and makes it difficult to build and maintain a sufficient number of cells for production capacity. According to one aspect of the embodiments of the present disclosure, live cells can be recovered from a cell culture substrate, including 80% to 100% survival, or about 85% to about 99% survival, or about 90% to about 99% survival. For example, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the recovered cells survive. The cells can be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0078] Figures 1A and 1B respectively show a three-dimensional (3D) perspective view and a two-dimensional (2D) plan view of a cell culture substrate 100 according to an example of one or more embodiments of the present disclosure. The cell culture substrate 100 is a woven mesh layer composed of a first plurality of fibers 102 extending in a first direction and a second plurality of fibers 104 extending in a second direction. The first direction and the second direction have a defined relationship with each other. For example, in Figures 1A and 1B, the first direction and the second direction are perpendicular to each other. However, embodiments include other defined non-random relative directions, and the first direction and the second direction can be separated at 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 at a first fiber interval S1, and the second plurality of fibers 104 are spaced apart from each other at a second fiber interval S2. The first and second fiber intervals 102 and 104 are each defined by the perpendicular distance from the center line of one fiber within the first and second pluralities of fibers 102 and 104 to the center line of the adjacent or closest fiber. According to various embodiments, the first and second fiber intervals S1 and S2 may or may not be equal, as discussed below.

[0079] The woven fibers of the substrate 100 form a plurality of apertures 106 that can be defined by one or more widths or diameters (e.g., D1, D2). The size and shape of the apertures can vary based on the type of weave (e.g., the number, shape, and size of the filaments; the angle between intersecting filaments, etc.). The woven mesh can be characterized as a two-dimensional sheet or layer at the macroscale. However, upon closer examination of the woven mesh, a three-dimensional structure becomes apparent due to the up-and-down movement of the intersecting mesh fibers. Thus, as shown in FIG. 1C, the thickness T of the woven mesh 100 can be greater than the thickness of a single fiber (e.g., t1). As used herein, the thickness T is the maximum thickness between the first surface 108 and the second surface 110 of the woven mesh. Without being bound by theory, the three-dimensional structure of the substrate 100 is advantageous because it provides a large surface area for culturing adherent cells, and it is believed that the structural rigidity of the mesh can provide a consistent and predictable cell culture matrix structure that allows for uniform fluid flow.

[0080] In FIG. 1B, the aperture 106 has a diameter D1 defined as the distance between opposing fibers of the first plurality of fibers 102 and a diameter D2 defined as the distance between opposing fibers of the second plurality of fibers 104. D1 and D2 may or may not be equal, depending on the shape of the weave. When D1 and D2 are not equal, the larger one is referred to as the major axis and the smaller one as the minor axis. In some embodiments, the aperture diameter can refer to the widest part of the aperture. Unless otherwise specified, the aperture diameter as used herein refers to the distance between parallel fibers on opposing surfaces of the aperture.

[0081] A given fiber of the first plurality of fibers 102 has a thickness t1, and a given fiber of the second plurality of fibers 104 has a thickness t2. In the case of fibers with a circular cross-section as shown in FIG. 1A, or fibers with other three-dimensional cross-sections, the thicknesses t1 and t2 are the maximum diameter or thickness of the fiber cross-section. According to some embodiments, all of the first plurality of fibers 102 have the same thickness t1, and all of the second plurality of fibers 104 have the same thickness t2. Further, 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 are different from the second plurality of fibers 104. In addition, each of the first plurality of fibers 102 and the second plurality of fibers 104 may include fibers of two or more different thicknesses (e.g., t 1a , t 1b , etc., and t 2a , t 2b , etc.). According to an embodiment, the thicknesses t1 and t2 are large compared to the size of the cells cultured thereon, such that the fibers provide a substantially flat surface from the perspective of the cells, thereby enabling better cell adhesion and proliferation compared to some other solutions where the fiber size is small (e.g., on the scale of the cell diameter). As shown in FIGS. 1A - 1C, due to the three-dimensional nature of the woven mesh, the 2D surface area of the fibers available for cell adhesion and proliferation exceeds the surface area for adhesion in an equivalent planar 2D surface.

[0082] In one or more embodiments, the fibers can have a diameter in the range of from about 10 μm to about 1000 μm; 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; from about 200 μm to about 300 μm; or from about 150 μm to about 300 μm. At the microscale level, due to the scale of the fibers compared to cells (e.g., the fiber diameter is larger than the cell), the surface of the monofilament fiber is presented as an approximation of a 2D surface for adherent cells to adhere and proliferate. The fibers can be woven into a mesh having openings in the range of from about 100 μm × 100 μm to about 1000 μm × 1000 μm. In some embodiments, the openings can have a diameter of 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 diameter and opening diameter are examples of some embodiments, but are not intended to limit the possible feature sizes of the mesh according to all embodiments. The combination of fiber diameter and opening diameter is selected, for example, to provide an efficient and uniform fluid flow through the substrate when the fixed bed cell culture matrix includes a number of adjacent mesh layers (e.g., a stack of individual layers or a roll of mesh layers).

[0083] The surface area available for cell adhesion and growth is determined by factors such as fiber diameter, aperture diameter and / or fiber spacing, and the type / pattern of the weave. In addition, if the cell culture matrix includes a stack, roll, or other configuration of overlapping substrates, the packing density of the cell culture matrix will affect the surface area of the fixed bed matrix and the flow characteristics of the fluid through the fixed bed. The packing density can vary depending on the packing thickness of the substrate material (e.g., the space required for a layer of the substrate). For example, if a stack of cell culture matrices has a particular height, it can be said that each layer of the stack has a packing thickness determined by dividing the total height of the stack by the number of layers in the stack. The packing thickness varies based on fiber diameter and weaving method, but can also vary depending on the arrangement of adjacent layers within the stack. Due to the three-dimensional nature of the substrate (e.g., a fabric layer or a three-dimensional porous sheet) in embodiments of the present disclosure, based on the alignment between adjacent layers, there may be a certain amount of connection or overlap between adjacent layers. In a first alignment, adjacent layers can be placed firmly, but in a second alignment, the overlap between adjacent layers can be zero, such as when the lowermost point of the upper layer is in direct contact with the uppermost point of the lower layer. In practice, the amount of nesting can be affected by both translational alignment and rotational alignment of the fiber patterns of adjacent layers. For certain applications, it may be desirable to provide a cell culture matrix where the packing density of the layer is low (e.g., when higher permeability is prioritized) or the packing density is high (e.g., when maximizing the substrate surface area is prioritized). According to one or more aspects of the embodiments described herein, the packing thickness 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; from about 200 μm to about 300 μm. These ranges are provided by way of example only, and embodiments can include packing thicknesses outside these ranges based on the selected substrate, packing of the fixed bed, design of the bioreactor, and application.

[0084] Based on the above structural factors, whether it is a single-layer cell culture substrate or a cell culture matrix having multiple layers of the substrate, the available surface area of the cell culture matrix can be determined. As used herein, "effective surface area" refers to the total surface area of the fibers in a part of the substrate material available for cell adhesion and proliferation. Unless otherwise specified, references to "surface area" refer to this effective surface area. According to an embodiment, for example, a single woven mesh substrate layer having a diameter of 6 cm can have an effective surface area of about 50 cm 2 to about 90 cm 2 ; about 53 cm 2 to about 81 cm 2 ; about 68 cm 2 ; about 75 cm 2 ; or about 81 cm 2 . These ranges of effective surface area are provided for illustrative purposes only, and some embodiments may have different effective surface areas. The cell culture matrix can also be characterized from the perspective of porosity, as discussed in the examples herein.

[0085] The substrate mesh can be made from monofilament or multifilament fibers of a polymer material suitable for cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrroles, and polypropylene oxide. The mesh substrate can have various patterns or weaves, including, for example, knitting, warp knitting, or weaving (e.g., plain weave, twill weave, Dutch weave, crepe weave, regular mat weave, irregular mat 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, point twill weave, royal oxford weave, houndstooth check weave, herringbone weave, 5-harness knitting, micro-mesh, or mesh netting). Aspects of embodiments also include filaments made of any other suitable material to form a porous structure and then coated with a material suitable for cell culture applications.

[0086] The surface chemistry of the mesh filaments may need to be modified to provide the desired cell adhesion properties. Such modification can be done through chemical treatment of the polymer material of the mesh or by grafting cell adhesion molecules onto the filament surface. Alternatively, the mesh can be coated with a thin layer of a biocompatible hydrogel demonstrating cell adhesion properties, including, for example, collagen or Matrigel®. Alternatively, the surface of the filament fibers of the mesh can be imparted with cell adhesion properties through a treatment process using various types of plasmas, treatment gases, and / or chemicals known in the art. However, in one or more embodiments, the mesh can provide an efficient cell growth surface without surface treatment.

[0087] Due to the three-dimensional quality of the substrate according to embodiments of the present disclosure, an increase in surface area for cell adhesion and growth is provided as compared to a planar 2D surface of equivalent size. This increased surface area serves the scalable performance achieved by embodiments of the present disclosure. In process development and process validation studies, small-scale bioreactors are often required to save reagent costs and improve experimental throughput. Embodiments of the present disclosure are applicable to such small-scale studies, but can likewise be scaled up to industrial or production scale. By plug-type perfusion flow in a packed bed, the same flow rate expressed in ml / min / cm of the cross-sectional surface area of the packed bed can be used in both small-scale and large-scale versions of the bioreactor. The larger surface area enables higher seeding density and higher cell growth density. According to one or more embodiments, the cell culture substrates described herein have demonstrated cell seeding densities of up to 22,000 cells / cm 2 or greater. For reference, the seeding density on the two-dimensional surface of a Corning HyperFlask® is on the order of 20,000 cells / cm 2 or so. As used herein, "plug-type perfusion flow" or "plug flow" refers to laminar flow through a bioreactor having a fixed bed according to embodiments of the present specification, and the flow through any cross-section of the fixed bed perpendicular to the flow direction proceeds at the same velocity across the entire cross-section. 2 Another advantage of the higher surface area and high cell seeding or growth density is that the cost of the embodiments disclosed herein can be the same as or lower than competing solutions. Specifically, the cost per cell product (e.g., per cell, or per viral genome) can be made equal to or lower than that of other packed bed bioreactors.

[0088]

[0089] Thus, there are multiple factors in fixed bed substrates that can affect the cell culture process, including packing density, surface chemistry, and effective surface area, as well as the nature of the fluid flow through / within the packed bed. By using a structurally defined culture matrix with sufficient rigidity, a high uniformity of flow resistance across the matrix or packed bed is achieved. According to various embodiments, the matrix can be deployed in a flexible and expandable multi-layer substrate arrangement. This flexibility eliminates diffusion limitations and provides uniform supply of nutrients and oxygen to cells adhered to the matrix. Additionally, the open matrix lacks the cell capture regions of the packed bed configuration, enabling complete cell recovery at high viability at the end of the culture. The matrix also provides uniformity in the packing of the packed bed, allowing for direct scale-up from process development units to large-scale industrial bioprocess units. The ability to directly recover cells from the packed bed eliminates the need to resuspend the matrix in a stirred or mechanically vibrated vessel, which would be increasingly complex and could impose shear stress harmful to the cells. Furthermore, the high packing density of the cell culture matrix results in high bioprocess productivity in industrially manageable amounts.

[0090] The shape of the mesh substrate layer is designed to allow for an efficient and uniform flow through one or more substrate layers. Further, the structure of the matrix can adapt to the flow of fluid through the matrix in multiple orientations. For example, the direction of the bulk fluid flow can be perpendicular to the major surfaces of the first and second substrate layers. However, the matrix can also be oriented with respect to the flow such that the opposing sides or faces of the substrate layer are parallel to the bulk flow direction. In addition to the fluid flow being perpendicular or parallel to the first and second faces of the mesh layer, the matrix can arrange a plurality of substrate pieces at intermediate angles or can be arranged randomly with respect to the fluid flow. This flexibility in orientation is made possible by the essentially isotropic flow behavior of the homogeneous and structurally defined substrate. In contrast, existing substrates for adherent cells in bioreactors do not exhibit this behavior and instead tend to have packed beds that create preferential flow paths and substrate materials with anisotropic permeability. The flexibility of the matrix of the present disclosure allows for better and more uniform permeability throughout the bioreactor vessel and enables use in a variety of applications and bioreactor or vessel designs.

[0091] The cell culture substrate can be used within a bioreactor vessel according to one or more embodiments. For example, the substrate can be used in a packed bed bioreactor configuration or in other configurations within a three-dimensional culture chamber. However, the embodiments are not limited to three-dimensional culture spaces and it is contemplated that the substrate can also be used in what can be considered a two-dimensional culture surface configuration, where one or more layers of the substrate are laid flat, such as within a flat-bottomed culture dish, to provide a cell culture substrate. Due to concerns about contamination, the vessel can be a disposable vessel that can be discarded after use.

[0092] According to an embodiment, there is provided a cell culture system in which a cell culture matrix is used within a culture chamber of a bioreactor vessel. FIG. 2 shows an example of a cell culture system 200 including a bioreactor vessel 202 having a cell culture chamber 204 inside. Inside the cell culture chamber 204, there is a cell culture matrix 206 made from a stack of substrate layers 208. The substrate layers 208 are stacked such that the first or second surface faces the first or second surface of an adjacent substrate layer. The bioreactor vessel 200 has an inlet 210 at one end for introducing a medium, cells, and / or nutrients into the culture chamber 204, and an outlet 212 at the opposite end for removing the medium, cells, or cell products from the culture chamber 204. By enabling the stacking of the substrate layers in this way, due to the defined structure and efficient fluid flow through the stacked substrates, the system can be easily scaled up without adversely affecting cell adhesion and growth. The vessel 200 is generally described as having an inlet 210 and an outlet 212, but in some embodiments, one or both of the inlet 210 and the outlet 212 can be used to flow a medium, cells, or other contents into and out of the culture chamber 204. For example, the inlet 210 can be used to flow a medium or cells into the culture chamber 204 during cell seeding, perfusion, or the culture stage, but can also be used to remove one or more of the medium, cells, or cell products through the inlet 210 during the recovery stage. Thus, the terms “inlet” and “outlet” are not intended to limit the functions of those openings.

[0093] In FIG. 2, the bulk flow direction is from the inlet 210 to the outlet 212, and in this example, the first and second main surfaces of the substrate layer 208 are perpendicular to the bulk flow direction. According to some aspects of the embodiments, the substrate can be arranged in a configuration different from the configuration shown in FIG. 2, which is shown by way of example only. In the bioreactor system 200, the substrate 208 is sized and shaped to fill the internal space defined by the culture chamber 204, such that the culture space within the container is filled with a cell growth surface, maximizing efficiency from the perspective of cells per unit volume. Although FIG. 3 shows a single inlet 210 and a single outlet 212, it is contemplated that the system 200 may be supplied from a plurality of inlets and have a plurality of outlets. According to the embodiments herein, the distribution plate can be used to help distribute the medium, cells, or nutrients across the cross-section of the packed bed, and thus improve the uniformity of the fluid flow through the packed bed.

[0094] The cell culture matrix can be arranged in a plurality of configurations within the culture chamber, depending on the desired system. For example, in one or more embodiments, the system includes one or more layers of a substrate having a width that extends across the width of the defined cell culture space within the culture chamber. Thus, a plurality of layers of the substrate can be stacked to a predetermined height. As discussed above, the layers of the substrate can be arranged such that the first and second surfaces of one or more of the layers are perpendicular to the bulk flow direction of the medium through the defined culture space within the culture chamber, or the first and second surfaces of one or more of the layers may be parallel to the bulk flow direction. In one or more embodiments, the cell culture matrix includes one or more substrate layers in a first orientation with respect to the bulk flow and one or more other layers in a second orientation different from the first orientation. For example, the various layers can have first and second surfaces that are parallel or perpendicular to the bulk flow direction, or at some angle therebetween.

[0095] In one or more embodiments, the cell culture system includes a plurality of individual cell culture substrate pieces in a packed bed configuration, where the length and / or width of the substrate pieces are smaller compared to the culture chamber. As used herein, when the length and / or width of the substrate piece is about 50% or less of the length and / or width of the culture space, the substrate piece is considered to have a length and / or width that is smaller compared to the culture chamber. Thus, the cell culture system can include a plurality of substrate pieces filled in the culture space in a desired arrangement. The arrangement of the substrate pieces may be random or semi-random, or may have a predetermined order or configuration, such as when the substrate pieces are oriented in a substantially similar orientation (e.g., horizontal, vertical, or at an angle between 0° and 90° relative to the bulk flow direction).

[0096] As used herein, the "defined culture space" refers to the space within the culture chamber that is occupied by the cell culture matrix and where cell seeding and / or culturing occurs. The defined culture space can fill substantially the entire culture chamber or may occupy a portion of the space within the culture chamber. As used herein, the "bulk flow direction" is defined as the direction of the bulk mass flow of fluid or medium through or over the cell culture matrix during cell culturing and / or during the inflow or outflow of medium into or out of the culture chamber.

[0097] In one or more embodiments, the cell culture matrix is fixed within the culture chamber by a fixing mechanism. The fixing mechanism can fix a part of the cell culture matrix to the wall of the culture chamber surrounding the matrix, or to the chamber wall at one end of the culture chamber. In some embodiments, the fixing mechanism adheres a part of the cell culture matrix to a member passing through the culture chamber, for example, a member running parallel to the longitudinal axis of the culture chamber, or a member running perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture matrix may be contained within the culture chamber without being fixedly attached to the wall of the chamber or bioreactor vessel. For example, the matrix may be accommodated by the boundary of the culture chamber or other structural members within the chamber such that the matrix is held within a predetermined region of the bioreactor vessel without being firmly fixed to those boundaries or structural members.

[0098] When multiple layers of a substrate are stacked facing each other, the packing density of the layers can vary based on the alignment and nesting or overlapping of the fibers between adjacent layers. For example, assuming no relative rotation between adjacent layers, in that case, the first plurality of fibers of one layer will be parallel to the first plurality of fibers of another layer, and the packing density boundary condition is defined by the amount of nesting or overlap between adjacent layers. When the fibers of adjacent layers are not perfectly aligned, according to one or more embodiments, due to the three-dimensional nature of the substrate, the fibers of one layer will be nested to some extent between the fibers of the adjacent layer. FIGS. 3A and 3B respectively show a plan view and a cross-sectional view of this scenario. Specifically, the fibers of the first layer running in the first direction shift to the middle of the fibers of the adjacent second layer running in the first direction such that the fibers of the first layer fit into the openings of the second layer (see FIG. 3A). This represents the highest degree of overlap that can occur between adjacent layers and thus represents the highest degree of compression (without additional external force to compress the layers together). On the opposite side of the spectrum, FIGS. 4A and 4B show similar views of a stack of substrate layers where all the fibers of a substrate layer running in the first direction are perfectly aligned with the fibers of another substrate layer running in the first direction. This represents the lowest degree of overlap (i.e., zero overlap) and thus the lowest degree of compression with the layers remaining in contact with each other.

[0099] For each of the modeled configurations of FIGS. 3A - 4B, sample cells 300, 400 were defined that enclosed the same volume of mesh material in order to analyze the porosity per unit volume of the sample cells 300, 400. The modeled volume of the open space within each cell is shown in FIGS. 5A (for the case of a densely packed stack) and 5B (for the case of a loosely packed stack). The porosity, expressed as a percentage of the open space, was approximately 40.8% for the loosely packed cell and 61.4% for the densely packed cell. Since the modeled stacks of FIGS. 3A - 4B represent the most densely packed configuration and the most loosely packed configuration of a given mesh material, the porosities of 40.8% and 61.4% are the upper and lower limits of the porosity of this particular mesh material. Depending on the arrangement and the actual packing density when using this mesh material, the porosity can fall between these extreme values. However, embodiments of the present disclosure are not limited to this porosity range, as variations in the mesh dimensions and the arrangement of the substrate within the cell culture vessel can result in different ranges of porosity.

[0100] In addition to the modeled porosity range, the porosity was measured using an actual packed bed of a PET woven mesh substrate. The measurement was performed by stacking 100 disks, each having a diameter of 22.4 mm, in a random arrangement. The total weight of the stack of 100 disks was 5.65 ± 0.2 g. The volume of the PET material in the stack was calculated using the following equation, assuming a PET density of 1.38 g / cm 3 : V PET = (total weight of stack) / (PET density) Equation 1 Thus, the PET volume V PET (for 100 disks with a diameter of 22.4 mm) of 5.65 g of PET was calculated to be 4.1 ml. Next, the total volume V PET of the stack, which includes the PET volume V total and the volume of the open space within the stack, was calculated using the following equation: V total = π × (0.5 × disk diameter) × (height of the stacked bed) Equation 2 A stack of 100 discs had a stack height of 25 ± 1 mm. Thus, when the disc diameter was 22.4 mm, V total was found to be 9.85 ml. Thus, the porosity of the packed bed can be calculated using the following: Porosity = (V total - V PET ) / V total Equation 3 Using Equation 3 and the above values, the porosity was calculated to be 58.4%, which is within the range predicted by the model.

[0101] As discussed above, in a loosely packed configuration, the fibers within the fixed bed layer are aligned. Thus, when the base material within the layer is the same, the openings within the layer will also be aligned. Thus, when viewing a stack of the base material in this loosely packed configuration in a plan view, it can be seen through the aligned openings within the stack. FIG. 6A shows a stack 600 of such a loosely packed base material layer having at least two layers 602, 604. On the other hand, in a tightly packed configuration, since the fibers of one layer overlap the openings of the adjacent layer, it is not possible to see through the stack when viewed from above. FIG. 6B represents the configuration of a stack 600' having at least two layers 602', 604' that are completely nested. In a fixed bed made of a woven mesh, it has been shown that the alignment of the fibers between adjacent layers has a significant impact on the uniformity of the flow through the fixed bed. In a layer with a rotational alignment with a 0° offset, the difference in the transmittance of the base material layers that are completely woven and laminated as shown in FIG. 4A can be 10 times (10×) that when the layers completely overlap as shown in FIG. 3A.

[0102] However, in addition to translational shifts between layers within the fixed bed, rotational misalignments can also occur. Note that both translational alignment and rotational alignment can be due to manufacturing products of the base material itself (e.g., when individual layers are cut from large sheets) or misalignments between layers within the fixed bed (e.g., misalignments that can occur during handling or assembly of the bioreactor, or after the fixed bed bioreactor is fully assembled). With strict manufacturing and assembly tolerances, such misalignments can be accommodated and reduced, but such precision increases the complexity and cost of the process.

[0103] To understand the possible effects of rotational alignment of the substrate layers on the fixed bed characteristics, FIGS. 6C - 6K show a substrate stack with different degrees of rotation between two layers and the impact such rotation can have on fluid flow performance based on the degree to which the stack has a uniform opening available for fluid flow. In FIG. 6C, layer 612 is rotated 5° relative to layer 614 of stack 610; in FIG. 6D, layer 622 is rotated 10° relative to layer 624 of stack 620; in FIG. 6E, layer 632 is rotated 15° relative to layer 634 of stack 630; in FIG. 6F, layer 642 is rotated 20° relative to layer 644 of stack 640; in FIG. 6G, layer 652 is rotated 25° relative to layer 654 of stack 650; in FIG. 6H, layer 662 is rotated 30° relative to layer 664 of stack 660; in FIG. 6I, layer 672 is rotated 35° relative to layer 674 of stack 670; in FIG. 6J, layer 682 is rotated 40° relative to layer 684 of stack 680; and in FIG. 6K, layer 692 is rotated 45° relative to layer 694 of stack 690.

[0104] When there is a rotational angle between two layers, especially at small angles such as 5° to 10° in FIGS. 6C and 6D, several distinct patterns are observed in the openings of the stack. There are distinct dark and bright regions, indicating a large difference in transmittance between those regions. For example, the bright regions are due to continuous openings overlapping with the openings of the adjacent layer. Thus, it would be expected that the resistance to flow is reduced in those regions. In other regions where the continuous openings are blocked by the fibers of the adjacent layer, the stack appears dark, and those dark regions would be expected to have a large resistance to flow. This suggests that these different regions would have different flow rates. Different flow rates could result in different cell seeding and nutrient supply, and thus potentially lead to non-uniform cell growth. It could also affect the access to the transfection reagent during transfection and the shear forces during recovery. It is difficult to directly measure or quantify this non-uniform flow distribution. However, a non-uniform distribution of cells has been observed in such situations after staining the mesh after cell culture. The stained images show a similar pattern, suggesting the presence of non-uniform flow due to different alignments of the layers, which could negatively impact the uniformity of cell culture. To reduce such non-uniformity, an alignment with relatively uniform dark and bright regions is preferred. From FIGS. 6A - 6K, a rotational angle of about 30° to about 45° is preferred (see FIGS. 6H - 6K), and 45° (FIG. 6K) is a particularly preferred embodiment.

[0105] However, it may be difficult to accurately set the angle of the above rotational alignment. Steps taken to align the mesh during the assembly of the reactor, as well as mechanical functions to maintain the alignment during handling, shipping, and application are required. These additional procedures and mechanical functions would result in additional cost and risk. Thus, embodiments of the present disclosure include a fixed bed for cell culture that achieves a uniform flow without requiring these costly measures to ensure an accurate rotational alignment.

[0106] According to embodiments of the present disclosure, a fixed bed is provided having at least two different types of substrate materials stacked together on the same cell culture bed. In an embodiment, the at least two different substrates are stacked alternately (e.g., substrate A, substrate B, substrate A, substrate B, etc.). The at least two different types of substrate materials may differ in one or more physical dimensions. For example, they may differ in fiber diameter, aperture diameter, fiber spacing, or fiber direction. Embodiments using different types of substrate materials offer several advantages, such as no need for specific alignment during reactor assembly, no need for mechanical features in the vessel to hold the mesh in place; minimal variation in the porosity or density of the packed bed; and improved flow uniformity within the packed bed reactor. The terms "hybrid substrate", "hybrid mesh", or "hybrid fixed bed" are often used herein to refer to a substrate matrix or fixed bed that includes at least two different types of substrate materials, as discussed above.

[0107] In one or more preferred embodiments, the two different types of substrates have different fiber spacings. The relationship between the two different fiber spacings can be expressed as the ratio of the fiber spacing of the first mesh to the fiber spacing of the second mesh. Embodiments include at least two types of substrates of meshes having a fiber spacing ratio of at least about 1.1, and up to about 2.0, 2.5, 3.5, 4.0, 4.5, and 5.0; a fiber spacing ratio of from about 1.2 to about 4.0; or a fiber spacing ratio of from about 1.2 to about 2.0. According to embodiments having a fiber spacing ratio, there is no need to control or hold the layers to maintain a specific alignment. This can be demonstrated, for example, by modeling the flow pattern through a stack of substrate layers having different fiber spacing ratios, as discussed below.

Examples

[0108] To demonstrate the effect of mesh alignment on flow uniformity and the advantages of using a hybrid mesh, a simplified sine wave model can simulate the periodic changes in flow that occur as the flow passes through the layers of the mesh. The mesh is a weaving pattern of two parallel fiber groups (a first fiber group running parallel in a first direction and a second fiber group running parallel in a second direction). As a basic model, a series of parallel cylinders are used to simulate the first fiber group running in the first direction. FIG. 7A shows a cross-section of such a series of fibers 700. In this example, the cylinders have a diameter of 160 μm (fiber diameter) and an aperture diameter of 250 μm between each cylinder. Water 702 flowing towards the cylinders at an average velocity of 30 ml / min, which is within the typical range commonly used in cell culture, passes through the series of cylinders 700 and will have a different velocity profile 704 on the other side of the cylinders due to the obstruction and resistance of the cylinders. Using computational fluid dynamics (CFD), the velocity of the water follows a sine function at a position 160 μm away from the other side of the array, as shown in FIG. 7B.

[0109] The resistance of a porous material, such as a series of cylinders 700, to fluid flow can be calculated from the flow rate using Equation 4, which is proportional to the reciprocal of the flow rate, where R is the flow resistance, P is the pressure, and U is the flow rate. The reciprocal of this sine equation is used to represent the relative changes in flow resistance at different positions along the cylinder array.

[0110] [Number]

[0111] FIG. 8 shows a resistance graph by applying Equation 4 to the sine wave flow rate in FIG. 7.

[0112] To build the model, the woven mesh 900 is modeled as two cylinder arrays 902, 904 that are stacked on top of each other and run perpendicular to each other, as shown in FIG. 9. The resistance at each location is assumed to be a linear combination of the resistances from both arrays at the same location. Thus, the change in flow velocity across the simulated mesh layer can be calculated as the inverse function of the final resistance represented at each location, as shown in FIG. 10. Using this approach, the effect of mesh alignment on the flow rate distribution can be simulated. For example, when two such meshes are stacked and perfectly aligned in the rotational direction (0° rotation relative to each other), there are two boundary conditions: (i) the fibers from the two meshes are perfectly woven together (the densely packed configuration of FIGS. 3A and 3B); and, (ii) the fibers from the two meshes are perfectly aligned (the loosely packed configuration of FIGS. 4A and 4B). FIGS. 11A and 11B show the modeled flow rate distributions resulting from these densely packed and loosely packed configurations, respectively. The densely packed configuration shows the highest resistance to flow and the lowest flow rate, based on the results of the CFD model. In the sine wave model simulation, the flow rate is also low. Locally, as shown in FIG. 11A, there are small flow velocity oscillations corresponding to the mesh pattern. Over a longer range, the flow rate distribution is generally uniform. In the aligned (loosely packed) configuration, based on the CFD model, the flow resistance is the lowest and the flow rate is the highest. In the sine wave model simulation, the flow rate is higher. In fact, the transmittance in this aligned configuration is about 10 times that in the densely packed configuration. Similarly, there are only local oscillations in the flow rate corresponding to the mesh pattern and a consistent flow rate over a wider range. This means that in both situations, different regions of the mesh have equal access to the flow.

[0113] The same approach can be applied to simulate the flow rate distribution when two meshes have different rotation angles with respect to each other. For example, FIGS. 12A - 12F show the simulated flow rate distributions through a stack of two mesh layers having the same physical mesh shape (i.e., fiber spacing, etc.). FIG. 12A shows the two boundary conditions discussed above with a relative rotation of 0°. FIGS. 12B and 12C show relative rotations of 5° and 10° respectively, and it can be seen that in some regions there is a significantly higher flow rate than in other regions. This is consistent with the patterns in the opening regions of FIGS. 6C and 6D. FIGS. 12D, 12E, and 12F show relative rotations of 20°, 30°, and 45° respectively. At an alignment of about 20° or more, the non-uniform flow pattern begins to disappear, and at about 30° to about 45°, it approaches a certain degree of uniformity. That is, the amount or pattern of flow changes becomes similar in all regions of the mesh stack, but there is still a large non-uniformity.

[0114] To address this non-uniformity issue, embodiments of the present disclosure include combining at least two different types of meshes within the stack. For example, at least two mesh materials can be laminated alternately as discussed herein. To demonstrate the improvement in flow uniformity with such embodiments, the same simulations as discussed above can be used. For example, FIGS. 13A - 16F show the flow patterns when two different types of mesh layers are laminated. FIGS. 13A - 13F show the rotation directions of 0°, 5°, 10°, 20°, 30°, and 45° for two mesh layers with a fiber spacing ratio of 1.2. FIGS. 14A - 14F show the rotation directions of 0°, 5°, 10°, 20°, 30°, and 45° for two mesh layers with a fiber spacing ratio of 1.4. FIGS. 15A - 15F show the rotation directions of 0°, 5°, 10°, 20°, 30°, and 45° for two mesh layers with a fiber spacing ratio of 1.6. Also, FIGS. 16A - 16F show the rotation directions of 0°, 5°, 10°, 20°, 30°, and 45° for two mesh layers with a fiber spacing ratio of 1.8.

[0115] As shown in FIGS. 13A to 13F, when the fiber interval ratio between two mesh layers is 1.2, non-uniformity is observed even when the meshes are aligned at a low angle such as 5° or 10°, but as the rotation angle increases, the non-uniformity decreases somewhat. When the fiber interval ratio is 1.4 (FIGS. 14A to 14F) or more, particularly 1.6 (FIGS. 15A to 15F) or more (for example, 1.8 in FIGS. 16A to 16F), there are almost no distinguishable regions showing a significantly higher flow rate than other regions. This means that when using a hybrid fixed bed using a mesh material having a fiber interval ratio of about 1.4 or more, the influence of alignment on the flow uniformity should be effectively eliminated. Thereby, the manufacture, shipping, and control of the fixed bed filling process during the final use of the fixed bed reactor will be much easier.

[0116] As discussed herein, embodiments of the present disclosure include a plurality of cell culture substrates in a stacked arrangement, or a fixed bed matrix having one or more roll-shaped substrate layers. Embodiments of the present disclosure provide solutions to improve the flow uniformity (and thus the performance of cell seeding, culturing, and recovery) in a bioreactor. Aspects of the embodiments include combining a plurality of substrates in a single fixed bed, and the plurality of substrates include substrates having different shapes, including the weave pattern of a woven or knitted substrate. The plurality of substrates can include, for example, at least two substrates having different weave patterns.

[0117] In embodiments where a fixed bed is formed using one or more roll-shaped substrates, using a single shape type of substrate (for example, two or more woven substrates of the same weave) can cause variations inherent in the packing density, which can adversely affect the flow uniformity, and as a result, cell seeding, oxygen supply, culture integrity, and recovery may not be optimal. However, using a fixed bed made from a mixed substrate roll (that is, two or more different substrates, for example, two or more different woven substrates) eliminates flow non-uniformity, and as a result, cell seeding, growth, and recovery can be improved.

[0118] Embodiments of the present disclosure are also advantageous for the manufacture and assembly of the fixed beds and bioreactor systems disclosed herein. Rolled fixed beds having a single type of substrate (e.g., a single woven mesh) are susceptible to variations in roll processes such as roll tension and roll alignment. On the other hand, in a mixed mesh roll, the influence of roll tension will be minimized when the layers are in close contact with each other. This makes the roll and assembly processes more robust. Despite these advantages, the rolled fixed bed design can be assembled using existing industrial equipment and does not require the layers to be cut or laminated individually as in the case of embodiments of laminated substrates, and thus the manufacturing costs will be significantly reduced.

[0119] According to embodiments that use a fixed bed made from a rolled substrate, aspects of the embodiments also help to minimize bypass of fluid (or cells, media, etc.) and reduce the need for high precision in the design and manufacture of the container with respect to the tolerance between the container and the substrate. The rolled substrate design may also have better performance in terms of the influence on the uniformity of the flow by the bubbles formed in the bioreactor system.

[0120] Example: RTD measurement values and packing density of a roll-shaped substrate Figure 17 shows the flow uniformity by RTD measurement in four fixed bed bioreactors having two laminated mesh fixed beds and two roll mesh fixed beds, all of which use a single type of substrate. Figure 17 shows, respectively, a plurality of laminated woven mesh substrate layers having a total surface area of 2.5 m 2 and 5 m 2 and, respectively, 2.5 m 2 and 5 m 2Shows the RTD measurement values of two bioreactors using two roll-shaped substrates having the total surface area. Embodiments of the present disclosure can improve the flow uniformity and the resulting RTD measurement values as compared to those shown in FIG. 17. To better understand the cause of the relative non-uniformity in FIG. 17, a sine curve can be used to simulate the maximum and minimum widths along the cross-section of the woven mesh. As shown in FIG. 18A, the peaks of the sine curve represent the thickest parts of the mesh, and the valleys represent the openings between the fibers. That is, the mesh spacing (the distance between two parallel fibers) is represented by the period, and the thickness of the mesh is represented by the amplitude. When two or more substrate layers are stacked, as shown in FIG. 18B, the alignment and filling changes of the fibers can be visualized by the overlapping sine curves. Loosely filled zones and densely filled zones also correlate with different patterns of sine curves between adjacent layers as shown in FIG. 18B. For example, the upper example in FIG. 18B shows two completely aligned layers, resulting in loose filling. The lower example in FIG. 18B also shows loose filling when the adjacent layers are offset by one period of the sine curve. The central example in FIG. 18B shows dense filling resulting from an offset of 0.5 periods between the layers.

[0121] When the substrate is rolled, as shown in FIG. 19, the filling density of the roll can be visualized by displaying the rolled sine curve. FIG. 19 shows a cross-section of a roll mesh (using 250 / 37 mesh) represented by a sine curve in a polar coordinate system. Changes in the pattern can be seen in a wide zone around the cross-section. These patterns correlate with three loosely filled zones and two densely filled zones within the mesh substrate assembly, which are circled in FIG. 19. The loosely filled zones have high permeability and low flow resistance, and thus cause fluid bypass. On the other hand, the densely filled zones have low permeability and high flow resistance, and thus cause dead zones. Based on CFD simulations, the difference in permeability between the loosely filled zones and the densely filled zones can be up to 10 times. This can explain the broadening of the RTD profile distribution in FIG. 17 and suggests that a relatively non-uniform flow occurs when using a single type of substrate mesh.

[0122] In contrast, when two different types of meshes are rolled together in a fixed bed, different results are obtained, as shown in FIG. 20. FIG. 20 shows overlapping sine curves from alternating layers of two different mesh types (250 / 37 mesh and 300 / 35 mesh) with different intervals, as shown in Table 1 below. As shown in FIG. 20, there are no large zones with either a relatively loosely filled pattern or a relatively densely filled pattern. This means that the permeability is likely to be consistent throughout the fixed bed.

[0123]

Table 1

[0124] To test the above concept, the two types of meshes shown in Table 1 were laminated to 5m 2Wrapped around the bioreactor vessel (using Ascent® FBR from Corning, Inc.). The RTD measurement results are shown in Figure 21. The RTD peak of the hybrid roll mesh was narrower than that of the single mesh roll and closer to the RTD peak of the stacked disks. This demonstrated the uniform flow predicted above. The bypass was much smaller than that of the stacked substrate design. This hybrid roll design also showed additional advantages in the way air bubbles were handled. As shown in Figure 22, after a large amount of air was introduced to the floor, no significant effect of air bubbles was seen. In the roll design, it is considered easier to push air bubbles out from the floor between the mesh layers of the roll design than to push air bubbles out across the entire mesh disk of the stacked disk design. The example mesh used in this specification has a difference of only about 10% between the two types of meshes, but it was still effective in preventing large regions with different packing densities. This suggests that even when the difference between substrates is small (e.g., 10%), it can be beneficial. As used in this specification, the percentage difference between the mesh substrate layers refers to the difference in the mesh spacing, which is defined as the sum of the fiber diameter and the opening diameter. Thus, a 10% difference corresponds 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.

[0125] According to an embodiment, the cell culture matrix includes a first base material and a second base material. The first base material has a first plurality of fibers defining a first array of openings, and the second base material has a second plurality of fibers defining a second array of openings. The first physical structure includes a first spacing of the first base material, and the first spacing is the sum of the fiber diameter of the first plurality of fibers and the opening diameter of the first array of openings. The second physical structure includes a second spacing of the second base 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 array of openings. As one aspect of the embodiment, the second spacing is different from 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.

[0126] In addition, according to embodiments of the present disclosure, based on the structure of the structurally defined substrate, the interweaving of layers can be prevented. For example, a particular weaving pattern can prevent the interweaving of adjacent mesh layers in a laminated mesh or roll mesh design, or prevent a single mesh layer in a roll design. Plain weave is thought to more easily generate interweaving and may generate local low-permeability regions, but other weaving patterns can help minimize interweaving, so when laminated for a fixed bed reactor or rolled together for installation, a more uniform fixed bed structure can be provided. Twill weave is one example that can be useful for applications, but the embodiments are not limited thereto and may include other weaving patterns including those mentioned in the present disclosure.

[0127] In some embodiments, by winding the film together with the mesh, entanglement can be prevented. The film will touch all the high points and will eliminate entanglement. The surface area of the film will be reduced, but it is very thin (possibly up to 10 μm), inexpensive, and yet a certain amount of surface area will be obtained. In some embodiments, post-heat treatment can be used to calender the mesh, deform the high points, make it less prone to entanglement, and easier to wind like a film. It is also possible to thermally calender the mesh into a roll to produce a monolith. Alternatively, a fused fiber mesh can also be used.

[0128] The bioreactor vessel optionally includes one or more outlets that can be attached to the inlet and / or outlet means. Through the one or more outlets, liquid, medium, or cells can be supplied to or removed from the chamber. A single port within the vessel may function as both an inlet and an outlet, or multiple ports may be provided as dedicated inlets and outlets.

[0129] The fixed bed cell culture matrix of embodiments of the present disclosure can consist of a (one or more) woven cell culture mesh substrate without any other form of cell culture substrate disposed within or dispersed in the cell culture matrix. That is, the woven cell culture mesh substrate of embodiments of the present disclosure does not require the type of irregular non-woven substrate used in existing solutions, or a separate different structure or spacer used between layers (e.g., those used to create fluid flow paths between layers of the substrate, etc.), and is an effective cell culture substrate. This enables a cell culture system with a simplified design and structure while providing a high-density cell culture substrate with other advantages discussed herein related to flow uniformity, recoverability, etc.

[0130] As discussed herein, cell culture substrates and bioreactor systems have provided numerous advantages. For example, embodiments of the present disclosure can assist in the production of any of a number of viral vectors such as AAV (all serotypes) and lentivirus, and can be applied to in vivo and ex vivo gene therapy applications. With uniform cell seeding and distribution, the viral vector yield per container is maximized, and the design enables the recovery of viable cells, which is useful for seed trains consisting of multiple growth periods using the same platform. Additionally, embodiments herein are scalable from process development scale to production scale, ultimately saving development time and cost. The methods and systems disclosed herein also enable the automation and control of the cell culture process, maximizing vector yield and improving reproducibility. Finally, the number of containers required to reach viral vector production levels on the order of (e.g., 10 16 to 10 18 AAV VG per batch) can be significantly reduced compared to other cell culture solutions.

[0131] Embodiments are not limited to rotation of the container about a central longitudinal axis. For example, the container may rotate about an axis that is not centered with respect to the container. Additionally, the axis of rotation may be a horizontal axis or a vertical axis.

[0132] Definitions "Fully synthetic" or "completely synthetic" refers to a cell culture article, such as a microcarrier or the surface of a culture container, that is composed entirely of synthetic raw materials and lacks materials of animal origin or animal derivation. The fully synthetic cell culture articles of the present disclosure eliminate the risk of xenogeneic contamination.

[0133] "Include", or similar terms, means inclusive but not exclusive, i.e., comprehensive and not excluding other elements.

[0134] "User" refers to people who use the systems, methods, articles, or kits disclosed herein, including people who culture cells to recover cells or cell products, or people who use cells or cell products cultured and / or recovered according to the embodiments herein.

[0135] When describing embodiments of the present disclosure, values such as, for example, the amounts, concentrations, volumes, process temperatures, process times, yields, flow rates, pressures, viscosities, etc. of components in a composition, and ranges thereof, or dimensions of components and similar values and ranges thereof, and "about" which changes them, refer to, for example, through general measurement and handling procedures used in the preparation of materials, compositions, composites, concentrates, components, manufactured articles, or use formulations; through inadvertent errors in these procedures; through differences in the manufacture, source, or purity of starting materials or raw materials used to carry out the method; and through similar considerations. The term "about" also encompasses amounts that vary due to degradation of a composition or formulation with a particular initial concentration or mixture, and amounts that vary due to mixing or processing of a composition or formulation with a particular initial concentration or mixture.

[0136] "Optional" or "optionally" means that the event or situation described thereafter may or may not occur, and that the description includes both the case where the event or situation occurs and the case where it does not occur.

[0137] As used herein, the indefinite articles "a" or "an" and their corresponding definite article "the" mean at least one or more than one, unless otherwise specified.

[0138] Abbreviations well known to those skilled in the art may be used (for example, time as "h" or "hrs", gram as "g" or "gm", milliliter as "mL", room temperature as "rt", nanometer as "nm", and similar abbreviations).

[0139] The specific preferred values disclosed for components, raw materials, additives, dimensions, conditions, and similar characteristics, and ranges thereof, are for illustrative purposes only and do not exclude other defined values or other values within defined ranges. The systems, kits, and methods of the present disclosure can include any value or any combination of any of the values, specific values, more specific values, and preferred values described herein.

[0140] Unless otherwise specified, it is never intended that any method described herein be construed as requiring that its steps be performed in a specific order. Thus, no particular order should be presumed unless the method claims actually recite the order to be followed by the steps or it is specifically stated in the claims or the specification that the steps are to be limited to a particular order.

[0141] It will be apparent to those skilled in the art that various modifications and changes can be made without departing from the spirit or scope of the disclosed embodiments. Since modifications, combinations, sub - combinations, and variations of the disclosed embodiments incorporating the spirit and essence of the embodiments can be envisioned by those skilled in the art, the embodiments of the present disclosure should be construed to include all within the scope of the appended claims and their equivalents.

[0142] Hereinafter, preferred embodiments of the present invention will be described item by item.

[0143] Embodiment 1 A cell - culture matrix for culturing cells in a fixed - bed reactor, the cell - culture matrix comprising a first base material including a first layer having opposing surfaces separated by a layer thickness, a first ordered regular array of openings passing through the first layer, and a first physical structure that is substantially regular and uniform and configured such that cells grow thereon, the first physical structure separating the openings of the first opening array from each other, and A second substrate material comprising a second layer having opposing surfaces separated by a layer thickness, an ordered and regular second array of apertures passing through the second layer, and a second physical structure that is substantially regular and uniform and on which cells are configured to grow, the second physical structure separating the apertures of the second array of apertures from each other comprising wherein the first substrate material and the second substrate material are rolled together to form a roll-shaped cell culture bed, and the first physical structure is different from the second physical structure, whereby the packing densities of the first substrate material and the second substrate material are substantially uniform throughout the roll-shaped cell culture bed Cell culture matrix

[0144] Embodiment 2 The cell culture matrix according to Embodiment 1, wherein the variation in the packing densities of the first substrate material and the second substrate material is less than about 20 times, less than about 10 times, less than about 5 times, or less than about 2 times throughout the roll-shaped cell culture bed

[0145] Embodiment 3 wherein the first substrate material includes a first plurality of fibers defining the first array of apertures, the second substrate material includes a second plurality of fibers defining the second array of apertures, the first physical structure includes a first spacing of the first substrate material, the first spacing being the sum of the fiber diameter of the first plurality of fibers and the aperture diameter of the first array of apertures, and the second physical structure includes a second spacing of the second substrate material, the second spacing being the sum of the fiber diameter of the second plurality of fibers and the aperture diameter of the second array of apertures The cell culture matrix according to Embodiment 1 or Embodiment 2

[0146] Embodiment 4 The cell culture matrix according to Embodiment 3, wherein the second spacing is different from the first spacing

[0147] Embodiment 5 The cell culture matrix according to Embodiment 4, wherein the second interval is within about 100% of the first interval, within about 90% of the first interval, within about 75% of the first interval, within about 50% of the first interval, within about 25% of the first interval, within about 15% of the first interval, within about 10% of the first interval, or within about 5% of the first interval.

[0148] Embodiment 6 The cell culture matrix according to any one of Embodiments 1 to 5, wherein at least one of the first substrate material and the second substrate material includes at least one of a molded polymer lattice, a 3D printed lattice, and a woven mesh.

[0149] Embodiment 7 The cell culture matrix according to any one of Embodiments 1 to 6, wherein at least one of the first substrate material and the second substrate material is a textile substrate.

[0150] Embodiment 8 The cell culture matrix according to any one of Embodiments 1 to 7, wherein at least one of the first substrate material and the second substrate material includes at least one of plain weave, twill weave, Dutch weave, crepe weave, regular mat weave, irregular mat rib weave, irregular weft rib weave, regular weft rib weave, regular warp rib weave, irregular warp rib weave, damask weave, 2 / 2 twill weave, 3 / 3 twill weave, basket weave, point twill weave, royal oxford weave, houndstooth check weave, herringbone weave, or 5 - needle knitting.

[0151] Embodiment 9 The cell culture matrix according to any one of Embodiments 1 to 8, wherein the first substrate material and the second substrate material are arranged as alternating layers of the roll - shaped cell culture bed.

[0152] Embodiment 10 The cell culture matrix according to any one of Embodiments 1 to 9, wherein the first base material and the second base material are in direct physical contact with each other.

[0153] Embodiment 11 The cell culture matrix according to any one of Embodiments 1 to 10, wherein the first base material and the second base material are not separated from each other by a spacer material or a barrier.

[0154] Embodiment 12 A third base material further comprising a third layer having opposing surfaces separated by the thickness of the third layer, a regular and orderly third array of openings passing through the third layer, and a third physical structure that is substantially regular and uniform and configured such that cells grow thereon, the third physical structure separating the openings of the third opening array from each other. The first base material, the second base material, and the third base material are rolled together to form the roll-shaped cell culture bed. The cell culture matrix according to any one of Embodiments 1 to 11.

[0155] Embodiment 13 In the roll-shaped cell culture bed, there is no spacer material or barrier between subsequent base material layers of the roll-shaped cell culture bed. The cell culture matrix according to any one of Embodiments 1 to 12.

[0156] Embodiment 14 The first and second base materials are different from each other in at least one physical dimension. The cell culture matrix according to any one of Embodiments 1 to 13.

[0157] Embodiment 15 The at least one physical dimension is at least one of the diameter of the opening, the thickness of the physical structure, the pattern of the physical structure, the weaving method, and the spacing between the physical structures on both sides of the opening. The cell culture matrix according to Embodiment 1.

[0158] Embodiment 16 The cell culture matrix according to any one of Embodiments 1 to 15, wherein a change in the flow rate of the fluid flowing through the cell culture matrix is uniform across the width of the cell culture matrix, and the width is in a direction perpendicular to the direction of the fluid flow.

[0159] Embodiment 17 The cell culture matrix according to any one of Embodiments 1 to 16, wherein the plurality of base material layers includes at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrroles, and polypropylene oxide.

[0160] Embodiment 18 The cell culture matrix according to any one of Embodiments 1 to 17, wherein the plurality of fibers includes 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.

[0161] Embodiment 19 The cell culture matrix according to any one of Embodiments 1 to 18, wherein the opening includes 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.

[0162] Embodiment 20 A bioreactor system for culturing cells, a cell culture vessel including 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 Embodiments 1 to 19, disposed within the reservoir. A bioreactor system including the same.

[0163] Embodiment 21 The bioreactor system according to embodiment 20, wherein the bioreactor system is configured for perfusion flow through the cell culture matrix during cell culture.

[0164] Embodiment 22 The bioreactor system according to embodiment 20 or 21, wherein the bioreactor system is configured to recover live cells from the cell culture matrix in the reservoir.

[0165] Embodiment 23 A cell culture matrix for culturing cells in a fixed bed reactor, the cell culture matrix comprising: A first base material including a first layer having opposing surfaces separated by a layer thickness, a first array of openings passing through the first layer, and a first physical structure including a first fiber array configured to grow cells thereon and separate the openings of the first array of openings from each other; A second base material including a second layer having opposing surfaces separated by a layer thickness, a second array of openings passing through the second layer, and a second physical structure including a second fiber array configured to grow cells thereon and separate the openings of the second array of openings from each other; Comprising: The first base material and the second base material are rolled together to form a roll-shaped cell culture bed, and The first physical structure is different from the second physical structure. Cell culture matrix.

[0166] Embodiment 24 The cell culture matrix according to embodiment 23, wherein the first physical structure is different from the second physical structure, so that the packing densities of the first base material and the second base material are substantially uniform throughout the roll-shaped cell culture bed.

[0167] Embodiment 25 The cell culture matrix according to Embodiment 23 or Embodiment 24, wherein the first opening array is an orderly and regular first array.

[0168] Embodiment 26 The bioreactor system according to any one of Embodiments 23 to 25, wherein the second opening array is an orderly and regular second array.

[0169] Embodiment 27 The bioreactor system according to any one of Embodiments 23 to 26, wherein the first physical structure and the second physical structure are substantially regular and uniform.

[0170] Embodiment 28 The bioreactor system according to any one of Embodiments 23 to 27, wherein the first physical structure and the second physical structure include fibers in a non-random arrangement.

[0171] Embodiment 29 A cell culture matrix for culturing cells in a fixed bed reactor, the cell culture matrix comprising: a first layer having opposing surfaces separated by a thickness of the first layer, an orderly and regular array of openings passing through the first layer, and a physical structure that is substantially regular and uniform and configured such that cells grow thereon, the physical structure separating the openings from each other, and a first base material including the first base material having a three-dimensional surface defined by the array of openings and the physical structure, and the three-dimensional surface being configured to prevent nesting of the first layer and a second layer of the base material, the second layer being adjacent to and in direct physical contact with the first layer, a cell culture matrix.

[0172] Embodiment 30 The cell culture matrix according to Embodiment 29, wherein the first base material is a single sheet including the first layer and the second layer.

[0173] Embodiment 31 The cell culture matrix according to Embodiment 30, wherein the single sheet is rolled to form a roll-shaped cell culture bed, and a plurality of roll layers radially arranged in the roll-shaped cell culture bed are formed by the rolling, and the plurality of roll layers include the first layer and the second layer.

[0174] Embodiment 32 The cell culture matrix according to any one of Embodiments 29 to 31, wherein the first base material is a woven fabric base material, and the woven fabric base material includes a plurality of fibers woven together by a predetermined method.

[0175] Embodiment 33 The cell culture matrix according to Embodiment 32, wherein the woven fabric base material includes twill weave, Dutch weave, crepe weave, regular mat weave, irregular mat 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, point twill weave, royal oxford weave, houndstooth check weave, herringbone weave, or 5-needle knitting.

[0176] Embodiment 34 Further including a second base material having an orderly regular array of openings passing through the layer, the openings being separated by the base material having a physical structure that is substantially regular and uniform and configured such that cells grow thereon. The second base material includes the second layer, and the first and second base materials have the same physical structure and the same opening array. The cell culture matrix according to any one of Embodiments 29 to 33.

[0177] Embodiment 35 The cell culture matrix according to Embodiment 34, wherein the first and second base material layers are laminated in an alternating arrangement.

[0178] Embodiment 36 The cell culture matrix according to Embodiment 34, wherein the first base material and the second base material are rolled to form a roll-shaped cell culture bed, and by being rolled, a plurality of roll layers radially arranged in the roll-shaped cell culture bed are formed, and the plurality of roll layers include the first layer and the second layer.

[0179] Embodiment 37 Further including a second base material having a regular and orderly array of openings passing through the layer, the openings being separated by the base material having a physical structure that is substantially regular and uniform and configured such that cells grow thereon. The second base material includes the second layer, and the first and second base materials are different from each other in at least one physical dimension, and the first base material and the second base material are arranged as alternating layers of the cell culture matrix. The cell culture matrix according to any one of Embodiments 29 to 33.

[0180] Embodiment 38 The cell culture matrix according to any one of Embodiments 34 to 37, wherein the second base material includes twill weave, Dutch weave, crepe weave, regular mat weave, irregular mat rib weave, irregular weft rib weave, regular weft rib weave, regular warp rib weave, irregular warp rib weave, damask weave, 2 / 2 twill weave, 3 / 3 twill weave, basket weave, point twill weave, royal oxford weave, houndstooth check weave, herringbone weave, 5 - needle knitting, micro - mesh, or mesh netting.

[0181] Embodiment 39 The cell culture matrix according to embodiment 37 or embodiment 38, wherein at least one of 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 between the physical structures on both sides of the opening.

[0182] Embodiment 40 The cell culture matrix according to any one of embodiments 34 to 39, wherein the first substrate material and the second substrate material include at least one of a molded polymer lattice, a 3D printed lattice, and a woven mesh.

[0183] Embodiment 41 The cell culture matrix according to any one of embodiments 29 to 40, wherein a degree of relative rotation between the first substrate material and the second substrate material is random.

[0184] Embodiment 42 The cell culture matrix according to any one of embodiments 29 to 41, wherein a change in a flow rate of a fluid flowing through the cell culture matrix is uniform across a width of the cell culture matrix, and the width is a direction perpendicular to a direction of the fluid flow.

[0185] Embodiment 43 The cell culture matrix according to any one of embodiments 31 to 42, wherein the roll-shaped layers are not separated by a spacer material or a barrier, or are in physical contact with each other.

[0186] Embodiment 44 The cell culture matrix according to any one of embodiments 29 to 43, wherein the first substrate material includes at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrroles, and polypropylene oxide.

[0187] Embodiment 45 The cell culture matrix according to any one of embodiments 32 to 44, wherein the plurality of fibers includes first fibers having a first fiber diameter of from about 30 μm to about 1000 μm, from about 30 μm to about 600 μm, from about 30 μm to about 400 μm, from about 100 μm to about 325 μm, or from about 150 μm to about 275 μm.

[0188] Embodiment 46 The cell culture matrix according to any one of embodiments 29 to 45, wherein the opening has a diameter of from about 100 μm to about 1000 μm, from about 200 μm to about 900 μm, or from about 225 μm to about 800 μm.

[0189] Embodiment 47 When the first substrate material is rolled into a roll-shaped substrate matrix including a roll-shaped layer of the first substrate material, the physical structure and array of the openings are configured such that the variation in the packing density of the roll-shaped layer is less than about 20-fold, less than about 10-fold, less than about 5-fold, or less than about 2-fold across the entire roll-shaped substrate matrix. The cell culture matrix according to any one of embodiments 29 to 46.

[0190] Embodiment 48 A bioreactor system for culturing cells, the system comprising a cell culture vessel including 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 embodiments 29 to 47, disposed within the reservoir. A bioreactor system comprising.

[0191] Embodiment 49 The bioreactor system according to embodiment 48, wherein the bioreactor system is configured for perfusion flow through the cell culture matrix during cell culture.

[0192] Embodiment 50 The bioreactor system according to Embodiment 48 or Embodiment 49, wherein the bioreactor system is configured to recover live cells from the cell culture matrix in the reservoir.

Explanation of Signs

[0193] 100 Cell culture substrate 102 First plurality of fibers 104 Second plurality of fibers 106 Plurality of openings 108 First surface 110 Second surface 200 Cell culture system 202 Bioreactor container 204 Cell culture chamber 206 Cell culture matrix 208 Substrate layer 210 Inlet 212 Outlet 300, 400 Sample cell 600, 600’ Stack of substrate layers 602, 604, 602’, 604’ At least two layers 700 Fiber / cylinder 702 Water 900 Woven mesh 902, 904 Cylinder array

Claims

1. A cell culture matrix for culturing cells in a fixed bed reactor, the cell culture matrix comprising a first substrate material including a first layer having opposing surfaces separated by a layer thickness, a first ordered regular array of openings passing through the first layer, and a first physical structure that is substantially regular and uniform and configured such that cells grow thereon, the first physical structure separating the openings of the first opening array from each other, and a second substrate material including a second layer having opposing surfaces separated by a layer thickness, a second ordered regular array of openings passing through the second layer, and a second physical structure that is substantially regular and uniform and configured such that cells grow thereon, the second physical structure separating the openings of the second opening array from each other wherein the first substrate material and the second substrate material are rolled together to form a roll-shaped cell culture bed, and the first physical structure is different from the second physical structure, whereby the packing densities of the first substrate material and the second substrate material are substantially uniform throughout the roll-shaped cell culture bed cell culture matrix.

2. The first substrate material includes a first plurality of fibers defining the first opening array, the second substrate material includes a second plurality of fibers defining the second opening array, the first physical structure includes a first spacing of the first substrate material, the first spacing being the sum of the fiber diameter of the first plurality of fibers and the opening diameter of the first opening array, and the second physical structure includes a second spacing of the second substrate material, the second spacing being the sum of the fiber diameter of the second plurality of fibers and the opening diameter of the second opening array The cell culture matrix according to claim 1.

3. The cell culture matrix according to claim 2, wherein the second spacing is different from the first spacing.

4. The cell culture matrix according to claim 1, wherein at least one of the first substrate material and the second substrate material includes at least one of a molded polymer lattice, a 3D printed lattice, and a woven mesh.

5. The cell culture matrix according to claim 1, wherein the first substrate material and the second substrate material are arranged as alternating layers of the roll-shaped cell culture bed.

6. A third substrate material including a third layer having opposing surfaces separated by the thickness of the third layer, an ordered, regular third array of openings passing through the third layer, and a third physical structure that is substantially regular and uniform and on which cells are configured to grow, the third physical structure separating the openings of the third opening array from each other, wherein the first substrate material, the second substrate material, and the third substrate material are rolled together to form the roll-shaped cell culture bed. The cell culture matrix according to claim 1.

7. The cell culture matrix according to claim 1, wherein no spacer material or barrier is present between subsequent substrate layers of the roll-shaped cell culture bed.

8. The first and second substrate materials are different from each other in at least one physical dimension. The cell culture matrix according to claim 1.

9. The cell culture matrix according to claim 8, wherein the at least one physical dimension is at least one of the diameter of the opening, the thickness of the physical structure, the pattern of the physical structure, the weaving pattern, and the spacing between the physical structures on both sides of the opening.

10. A cell culture matrix for culturing cells in a fixed bed reactor, the cell culture matrix comprising: a first substrate material including a first layer having opposing surfaces separated by the thickness of the first layer, an ordered, regular array of openings passing through the first layer, and a physical structure that is substantially regular and uniform and on which cells are configured to grow, the physical structure separating the openings from each other including, wherein the first substrate material has a three-dimensional surface defined by the array of openings and the physical structure, and the three-dimensional surface is configured to prevent nesting of the first layer and a second layer of the substrate material, the second layer being adjacent to and in direct physical contact with the first layer. Cell culture matrix.

11. The cell culture matrix according to claim 10, wherein the first substrate material is a single sheet including the first layer and the second layer.

12. The single sheet is rolled to form a roll-shaped cell culture bed, and by being rolled, a plurality of roll layers radially arranged in the roll-shaped cell culture bed are formed, and the plurality of roll layers include the first layer and the second layer. The cell culture matrix according to claim 11.

13. Further comprising a second substrate material having a neat and regular array of openings passing through the layer, the openings being separated by the substrate material having a physical structure that is substantially regular and uniform and configured such that cells grow thereon, the second substrate material includes the second layer, and the first and second substrate materials have the same physical structure and the same opening array. The cell culture matrix according to claim 10.

14. Further comprising a second substrate material having a neat and regular array of openings passing through the layer, the openings being separated by the substrate material having a physical structure that is substantially regular and uniform and configured such that cells grow thereon, the second substrate material includes the second layer, and the first and second substrate materials are different 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. The cell culture matrix according to claim 10.

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