Fixed-bed cell culture reactor for matrix alignment and sample collection

Abstract

A fixed-bed bioreactor for culturing cells on a cell culture substrate is provided. The fixed-bed bioreactor comprises a cell culture vessel having a vessel body defining at least one internal chamber, an inlet fluidly connected to the chamber, and an outlet fluidly connected to the chamber. The vessel body has a first end, a second end, and a longitudinal axis extending in a direction from the first end to the second end. A guide rod is disposed within the internal chamber and extends parallel to the longitudinal axis of the vessel body. The guide rod is configured to hold the cell culture substrate in place within the internal chamber.

Description

Description of Related Applications

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 354,401, filed on June 22, 2022, the content of which is relied upon and incorporated herein in its entirety.

Technical Field

[0002] The present disclosure broadly relates to cell culture bioreactors and substrates for culturing cells. The present disclosure particularly relates to bioreactors incorporating such substrates that enable defined filling and / or sampling of cell culture substrates and substrates.

Background Art

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

[0004] Most of the cells used in bioprocesses are anchorage-dependent, which means that the 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 forms such as T-flasks, Petri dishes, cell factories, cell stacking containers, roller bottles, and HYPERStack® containers. These methods can have significant drawbacks, including difficulty in achieving cell densities high enough to enable the execution of therapy or large-scale production of cells.

[0005] Alternative methods have been proposed to increase the volumetric density of cultured cells. These include microcarrier culture performed in a stirred tank. In this technique, cells attached to the surface of the microcarriers are exposed to a certain shear stress, which has a significant impact on growth and culture performance. Another example of a high-density cell culture system is a hollow fiber bioreactor, where cells can form large three-dimensional aggregates when growing within the interstitial fiber spaces. However, cell growth and performance are significantly inhibited by nutrient deficiency. To mitigate this problem, these bioreactors are made small and are not suitable for large-scale manufacturing.

[0006] Another example of a high-density culture system for anchorage-dependent cells is a packed bed bioreactor system. In this type of bioreactor, a cell substrate is used to provide a surface to which adherent cells can attach. The medium is perfused along the surface or through a semi-porous substrate to provide the nutrients and oxygen necessary for cell growth. For example, a packed bed bioreactor system containing a packed bed of a support or matrix system for cell uptake has already been disclosed in Patent Documents 1 to 3. The packed bed matrix is usually made of a polymer non-woven ultra-fine fiber or porous particles as a substrate. Such a bioreactor functions as a recirculation flow-type bioreactor. One of the major problems with such bioreactors is the non-uniformity of cell distribution inside 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 seeding process. In addition, due to the random fiber packing, the cell capture efficiency and flow resistance across the cross-section of the packed bed are not uniform. For example, the medium flows faster in regions with a low cell packing density and slower in regions with a high resistance due to a large number of captured cells. This results in a channeling effect where nutrients and oxygen are more efficiently delivered to regions with a low volumetric cell density, and regions with a high cell density are maintained under sub-optimal culture conditions.

[0007] Another significant drawback of the packed bed systems disclosed in the prior art is the inability to efficiently harvest viable cells at the end of the cultivation process. When the final product is cells, or when the bioreactor is used as part of a "seed train" (where a cell population is grown in one vessel and then transferred to another vessel for further population growth), cell harvesting is important. Patent Document 4 discloses a bioreactor design for improving the efficiency of cell recovery from a packed bed during the cell harvesting process. The design is based on loosening the packed bed matrix and rocking or agitating the packed bed particles to cause the porous matrix to collide, thus removing the cells. However, this approach requires a lot of time and labor and significantly damages the cells, thus reducing the overall cell viability.

[0008] In some existing bioreactor solutions, small pieces of a cell substrate material consisting of randomly oriented fibers in a non-woven arrangement are used. These pieces are filled into a container to form a packed bed. However, like similar solutions on the market, this type of packed bed substrate also has drawbacks. Specifically, the non-uniform packing of the substrate pieces creates visible channels in the packed bed, resulting in a preferential and non-uniform flow of the medium and distribution of nutrients through the packed bed. In studies of such systems, it has been pointed out that there is a "systematic non-uniform distribution of cells increasing from the top to the bottom of the fixed bed" and that "nutrient gradients... lead to limitations in cell growth and production", all of which "can impair transfection efficiency due to the non-uniform distribution of cells" (Non-Patent Document 1). In studies, agitation of the packed bed may improve dispersion, but there will be other drawbacks (i.e., "the agitation required to improve dispersion during inoculation and transfection will induce an increase in shear stress, which in turn will reduce cell viability", the same document). Another study mentioned that the non-uniform distribution of cells makes it difficult to monitor cell populations using a biomass sensor ("... when cells are distributed non-uniformly, the biomass signal from the cells on the upper carrier may not represent the general view of the entire bioreactor", Non-Patent Document 2).

[0009] In addition, due to the random arrangement of fibers within the substrate sheet and the variation in the filling of small pieces between one packed bed and another packed bed of a bioreactor such as those described above, the substrate varies from culture to culture, making it difficult for customers to predict cell culture performance. Furthermore, since cells are considered to be confined by the packed bed by a randomly packed substrate having a random structure itself, it becomes very difficult or impossible to efficiently harvest the cells.

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] The production of viral vectors for early-stage clinical trials is possible with existing platforms, but to reach late-stage commercial production scales, a platform that can produce large quantities of high-quality products is required.

[0013] In addition, it is desirable to be able to monitor bioreactors used for culturing cells, producing AAVs, or creating seed trains to promote cell growth for biochemical production. When using adherent cell reactors, samples of the growth medium do not contain cells or contain cells to at least a useful extent for monitoring the state of culture on the adherent substrate.

[0014] There is a need for cell culture matrices, systems, and methods that allow users to monitor the state of the cell culture process by examining the cells on the substrate during and / or after the cell culture process, including having a uniform cell distribution, easily achievable and increasing harvest yields, enabling cell culture in a high-density format, and aseptically monitoring the substrate.

Means for Solving the Problems

[0015] According to embodiments of the present disclosure, there is disclosed a cell culture substrate that enables sampling of all or part of the substrate to monitor the state or health of cell culture. Embodiments include a multi-layer fixed-bed cell culture matrix in which one or more layers are specially designed to enable such sampling. Embodiments also include a fixed-bed bioreactor equipped with such a cell culture substrate and / or matrix, including a bioreactor vessel that allows the cell culture substrate to be arranged in a defined array and orientation.

[0016] In order to be able to predict the number and distribution and health of cells within a cell bioreactor bed, in one method, a portion of the substrate is removed in the middle of the cell culture or cell growth process. By sampling during this process, information about the implementation of the cell culture can be used to evaluate the quality and performance of the process. The cell number can be predicted from the sample, and growth can be monitored by sampling at different times. This information can be used to develop and optimize the performance of specific biological processes such as seed trains or virus vector production. In production, contaminated or out-of-specification runs can be terminated, reducing the cost of running the process until an unsatisfactory result is obtained. Growth media and lost production time are significant costs for typical biological processes. Embodiments of the present disclosure remove all or a portion of a fixed-bed cell culture substrate from a housing such that a user can access the bed without breaking the bed or the container of the bioreactor. Thereby, any or a selected portion of the fixed bed can be evaluated. The bed can also be evaluated after the cell culture process or after harvesting of the desired components for "post-mortem" analysis of the cell culture.

[0017] A fixed-bed bioreactor for culturing cells on a cell culture substrate is provided. The fixed-bed bioreactor includes a container body that defines at least one internal chamber, an inlet fluidly connected to the chamber, and a cell culture container having an outlet fluidly connected to the chamber. The container body has a first end, a second end, and a longitudinal axis extending in a direction from the first end to the second end. A guide rod is disposed within the internal chamber and extends parallel to the longitudinal axis of the container body. The guide rod is configured to hold the cell culture substrate in place within the internal chamber.

Brief Description of the Drawings

[0018]

FIG. 1A

FIG. 1B

FIG. 1C

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FIG. 2B

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FIG. 6A

FIG. 6B

FIG. 7A

FIG. 7B

FIG. 7C

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FIG. 9A

FIG. 9B

FIG. 9C

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FIG. 13

Mode for Carrying Out the Invention

[0019] Various embodiments of the present disclosure will be described in detail, if any, with reference to the drawings. References to various embodiments are not intended to limit the scope of the invention, which is limited only by the appended claims. In addition, any examples described herein are not limiting and merely describe some of the many possible embodiments of the invention recited in the claims.

[0020] Embodiments of the present disclosure include a cell culture substrate and a cell culture bioreactor incorporating such a substrate that enables sampling of the substrate or a portion of the substrate to monitor cell culture.

[0021] In conventional large-scale cell culture bioreactors, different types of packed-bed bioreactors have been used. Typically, these packed beds contain a porous matrix to maintain adherent or suspension cells and support growth and proliferation. The packed-bed matrix provides a high surface area to volume ratio, and thus, the cell density can be higher than in other systems. However, the packed bed often functions as a depth filter, in which case the cells are physically trapped or entrapped in the fibers of the matrix. Therefore, due to the linear flow of the cell inoculum material through the packed bed, the cells are exposed to a non-uniform distribution within the packed bed, resulting in variations in cell density across the depth or width of the packed bed. For example, the cell density will be higher in the inlet region of the bioreactor and significantly lower near the outlet of the bioreactor. Such non-uniform distribution of cells within the packed bed significantly hinders the scalability and predictability of such bioreactors in bioprocess manufacturing, and can even result in a decrease in efficiency with respect to cell growth or virus vector production per unit surface area or volume of the packed bed.

[0022] Another problem encountered in packed-bed bioreactors disclosed in the prior art is the channeling effect. The local fiber density at any given cross-section of the packed bed is not uniform due to the randomness of the packed nonwoven fibers. The medium flows faster in regions of low fiber density (high bed permeability) and much slower in regions of high fiber density (lower bed permeability). The non-uniform medium perfusion across the packed bed results in the channeling effect, which manifests as significant gradients of nutrients and metabolites that adversely affect the overall cell culture and the performance of the bioreactor. Cells located in regions of low medium perfusion will starve and, in many cases, die due to nutrient deprivation or metabolite toxicity. Cell harvesting is yet another problem encountered when bioreactors filled with nonwoven fiber scaffolds are used. Cells released at the end of the cell culture process are captured inside the packed bed due to its function as a depth filter, and the cell recovery rate is very low. This severely limits the use of such bioreactors in bioprocesses where live cells are the product. Therefore, the non-uniformity results in regions with different exposures to flow and shear, effectively reducing the available cell culture area, causing non-uniform cultures, and hindering transfection efficiency and cell release.

[0023] To address these and other problems of existing cell culture solutions, embodiments of the present disclosure provide cell growth substrates, matrices of such substrates, and / or packed bed systems using such substrates that enable efficient and high-yield cell culture of adherent-dependent cells and the production of cell products (e.g., proteins, antibodies, virus particles). Embodiments include a porous cell culture matrix fabricated from a regular ordered array of porous substrate materials that enables uniform cell seeding and medium / nutrient perfusion, as well as efficient cell harvesting. Embodiments also provide scalable cell culture solutions for substrates and bioreactors on which cells can be seeded and grown and / or cell products can be harvested, from process development scale to full production size scale, without sacrificing the uniform performance of the embodiments. For example, in some embodiments, the bioreactor can be easily scaled up from process development scale to production scale with equivalent virus genomes (VG / cm 2 ) per unit surface area of the substrate. The harvest stability and scalability of the embodiments described herein enable use in an efficient seed train for growing cell populations at multiple scales on the same cell substrate. In addition, the embodiments described herein provide a cell culture temperament 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 described herein can produce from 10 16 to 10 18 virus genomes (VG) per batch.

[0024] In one embodiment, a matrix is provided that has a structurally defined surface area for adherent cells to attach and proliferate, has good mechanical strength, and forms a highly uniform number of interconnected fluid networks when assembled within a packed bed or other bioreactor. In certain embodiments, a mechanically stable and non-degradable woven fabric mesh can be used as a substrate to assist in adherent cell production. The cell culture matrix disclosed herein supports the attachment and proliferation of anchorage-dependent cells in a high volume density format. Uniform cell seeding of such a matrix, as well as efficient harvesting of cells or other products of the bioreactor, can be achieved. In addition, embodiments of the present disclosure provide a uniform cell distribution during the seeding process, assist cell culture to achieve a confluent monolayer or multilayer of adherent cells on the disclosed matrix, limit nutrient diffusion, and avoid the formation of large and / or uncontrollable 3D cell aggregates with increased metabolite concentrations. Therefore, in that matrix, there are no diffusion limitations during bioreactor operation. In addition, the matrix enables easy and efficient cell harvesting from the bioreactor. The structurally defined matrix of one or more embodiments enables complete cell recovery and consistent cell harvesting from the packed bed of the bioreactor.

[0025] According to some embodiments, a cell culture method is also provided that uses a bioreactor having a matrix for producing a therapeutic protein, antibody, viral vaccine, or viral vector in a bioprocess.

[0026] In contrast to existing cell culture substrates used in cell culture bioreactors (i.e., randomly ordered fibrous nonwoven substrates), embodiments of the present disclosure include cell culture substrates having a distinct ordered structure. The distinct ordered structure results in consistent and predictable cell culture outcomes. In addition, the substrate has an open porous structure that prevents cell entrapment and allows a uniform flow through the packed bed. This configuration can improve cell seeding, nutrient supply, cell growth, and cell harvesting. According to one or more particular embodiments, it has a thin sheet-like structure with a first and a second surface separated by a relatively small thickness, such that the thickness of the sheet is small relative to the width and / or length of the first and second surfaces of the substrate, and a matrix is formed of the substrate material. In addition, a plurality of pores or openings are formed through the thickness of the substrate. The substrate material between the openings is sized and shaped to attach cells to the surface of the substrate material as if it were a substantially two-dimensional (2D) surface, while allowing appropriate fluid flow around the substrate material and through the openings. In some embodiments, the substrate is a polymeric material and can be formed as a molded polymeric sheet; a polymeric sheet with openings through its thickness; a number 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 matrix has a high surface area to volume ratio for culturing anchorage-dependent cells. According to various embodiments, the matrix can be arranged or packed within the bioreactor in the particular manner described herein for uniform cell seeding and growth, uniform media perfusion, and efficient cell harvesting.

[0027] Embodiments of the present disclosure can achieve a practical-sized viral vector platform that can produce viral genomes at a scale of up to or exceeding about 10 14 per batch, up to or exceeding about 10 15 per batch, up to or exceeding about 10 16 per batch, up to or exceeding about 10 17 per batch, up to or exceeding about 10 16 per batch, up to or exceeding about 10 viral genomes per viral genome, or up to or exceeding about 10 viral genomes per viral genome. In some embodiments, the production is about 1015 from about 10 18 or more viral genomes. For example, in some embodiments, the viral genome yield is about 10 15 to about 10 16 viral genomes per batch, or about 10 16 to about 10 19 viral genomes per batch, or about 10 16 to about 10 18 viral genomes per batch, or about 10 17 to about 10 19 viral genomes per batch, or about 10 18 to about 10 19 viral genomes per batch, or about 10 18 or more viral genomes per batch.

[0028] In addition, the embodiments disclosed herein enable not only the attachment and growth of cells to the cell culture substrate, but also the harvesting of the cultured cells in a viable state. The inability to harvest viable cells is a significant drawback in current platforms, which presents difficulties in constructing and maintaining a sufficient number of cells for production capacity. According to aspects of the embodiments of the present disclosure, it is possible to harvest viable cells from the cell culture substrate, including 80% to 100% viability, or about 85% to about 99% viability, or about 90% to about 99% viability. 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 harvested cells are viable. The cells may be released from the cell culture substrate, for example, using trypsin, TrypLE, or Accutase®.

[0029] Figures 1A and 1B show, respectively, 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 made from a first plurality of fibers 102 extending in a first direction and a second plurality of fibers 104 extending in a second direction. The woven fibers of the substrate 100 form a plurality of openings 106, which can be defined by one or more widths or diameters (e.g., D1, D2). The size and shape of the openings 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 may be characterized as a macroscale, two-dimensional sheet or layer. However, upon closer inspection of the woven mesh, a three-dimensional structure becomes apparent due to the undulations of the intersecting fibers of the mesh. Thus, as shown in FIG. 1C, the thickness T of the woven mesh 100 will 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 intending to be bound by theory, the three-dimensional structure of the substrate 100 provides a large surface area for culturing adherent cells, and conveniently, the structural rigidity of the mesh is thought to be able to provide a consistent and predictable cell culture matrix structure that allows for uniform fluid flow.

[0030] In FIG. 1B, the openings 106 have a diameter D1 defined as the distance between fibers 102 that are opposite each other, and a distance D2 defined as the distance between fibers 104 that are opposite each other. D1 and D2 may or may not be equal, depending on the weave shape. If D1 and D2 are not equal, the larger one can be referred to as the outer diameter, and the smaller one can be referred to as the inner diameter. In some embodiments, the diameter of the opening may refer to the widest part of the opening. Unless otherwise specified, the diameter of the opening, as used herein, will refer to the distance between parallel fibers on opposite sides of the opening.

[0031] Predetermined fibers of the plurality of fibers 102 have a thickness t1, and predetermined fibers of the plurality of fibers 104 have a thickness t2. In the case of fibers with a circular cross-section, as shown in FIG. 1A, or in the case of 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 plurality of fibers 102 have the same thickness t1, and all of the plurality of fibers 104 have the same thickness t2. In addition, t1 and t2 may be equal. However, in one or more embodiments, t1 and t2 are not equal, such as when the plurality of fibers 102 are different from the plurality of fibers 104. In addition, each of the plurality of fibers 102 and the plurality of fibers 104 may contain 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 relative to the size of the cells cultured thereon, so that the fibers provide a planar approximation 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 order of the diameter of the cells). Due to the three-dimensional characteristics of the fabric mesh, as shown in FIGS. 1A to 1C, the 2D surface area of the fibers available for cell adhesion and proliferation exceeds the surface area for adhesion on an equivalent flat 2D surface.

[0032] In one or more embodiments, the fibers can have a diameter in the range of from about 50 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. On a microscopic scale, due to the scale of the fibers compared to cells (e.g., the diameter of the fibers is larger than that of cells), the surface of a single fiber presents an approximation of a 2D surface for adherent cells to attach and proliferate. The fibers can be woven into a mesh having openings ranging from about 100 μm × 100 μm to about 1000 μm × 1000 μm. In some embodiments, the openings can have a diameter of from about 50 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 and are not intended to limit the possible feature sizes of the mesh for 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 cell culture matrix is made from a number of adjacent mesh layers (e.g., a laminate of individual layers or a rolled mesh layer).

[0033] Factors such as the diameter of the fibers, the diameter of the openings, and the weaving method / pattern will determine the surface area available for cell attachment and growth. In addition, when the cell culture matrix includes a laminate, roll, or other arrangement of overlapping substrates, the packing density of the cell culture matrix will affect the surface area of the packed bed matrix. The packing density can be replaced by the packing thickness of the substrate material (e.g., the space required for a layer of the substrate). For example, if a laminate of the cell culture matrix has a specific height, it can be said that each layer of the laminate has a packing thickness determined by dividing the total height of the laminate by the number of layers in the laminate. The packing thickness varies depending on the diameter of the fibers and the weaving method, but can also vary based on the alignment of adjacent layers in the laminate. For example, due to the three-dimensional characteristics of the knitted layer, there is a specific amount of interlocking or overlap that adjacent layers can accommodate based on their alignment with each other. In a first alignment, adjacent layers can fit tightly together, but in a second alignment, there may be no overlap, such as when the lowest point of the upper layer is in direct contact with the highest point of the lower layer. For certain applications, it may be desirable to provide a cell culture matrix with a lower packing density of the layers (e.g., when higher permeability is a priority) or a higher packing density of the layers (e.g., when maximizing the surface area of the substrate is a priority). According to one or more embodiments, the packing thickness can be from about 50 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.

[0034] Due to the structural factors described above, the surface area of the cell culture matrix can be determined regardless of whether it is a single layer of the cell culture substrate or a cell culture matrix having multiple layers of the substrate. For example, in a specific embodiment, a single layer of a circular woven mesh substrate with a diameter of 6 cm has an area of about 68 cm 2It may have an effective surface area. The "effective surface area" as used herein is the total surface area of the fibers in the portion of the substrate material available for cell attachment and growth. Unless otherwise specified, references to "surface area" refer to this effective surface area. According to one or more embodiments, a single woven mesh substrate layer with a diameter of 6 cm has 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 given by way of example only, and some embodiments may have different effective surface areas. The cell culture matrix can also be characterized in terms of porosity, as described in the examples herein.

[0035] The substrate mesh can be made from single or multifilaments of polymeric materials suitable for cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. The mesh substrate may have different patterns or weaves, including, for example, knitting, warp knitting, or weaving (e.g., plain weave, twill weave, dutch weave, five needle weave).

[0036] The interfacial chemistry of the mesh filaments will need to be altered to provide the desired cell adhesion properties. Such alterations can be made by chemical treatment of the polymeric 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 that exhibits cell adhesion properties, such as, for example, collagen or Matrigel®. Alternatively, cell adhesion properties can be imparted to the surface of the filament fibers of the mesh during a treatment process with various types of plasma, 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.

[0037] By using a structurally defined culture matrix with sufficient rigidity, a high uniformity of flow resistance is achieved across the matrix or packed bed. According to various embodiments, the matrix can be installed in a monolayer or multilayer configuration. This flexibility eliminates diffusion limitations and enables uniform delivery of nutrients and oxygen to the cells attached to the matrix. In addition, the open matrix has no cell uptake regions in the packed bed structure, allowing for complete cell harvest with high viability at the end of the culture. The matrix also provides packing uniformity for the packed bed and enables direct scale-up from a process development unit to a large-scale industrial bioprocess unit. The ability to directly harvest cells from the packed bed eliminates the need to resuspend the matrix in a stirred or mechanically shaken vessel (which would add complexity and could impart harmful shear stress to the cells). Furthermore, the high packing density of the cell culture matrix results in high bioprocess productivity at an industrially manageable volume.

[0038] As used herein, "structurally defined" means that the structure of the substrate conforms to a predetermined design and is not random. Therefore, a structurally defined substrate can be a textile design, 3D printed, molded, or formed by several other techniques known in the art that cause its structure to follow a predetermined planned structure.

[0039] FIG. 2A shows an embodiment of a matrix having a multilayer substrate 200, and FIG. 2B is a plan view of the same multilayer substrate 200. The multilayer substrate 200 includes a first mesh substrate layer 202 and a second mesh substrate layer 204. Despite the overlap of the first and second mesh substrate layers 202 and 204, the mesh shape (e.g., the ratio of the aperture diameter to the fiber diameter) is such that the apertures of the first and second mesh substrate layers 202 and 204 overlap as shown by the aperture 206 without filaments in FIG. 2B, providing a path for fluid to flow through the entire thickness of the multilayer substrate 200.

[0040] FIG. 3 shows a cross-sectional view of the multilayer substrate 200 along line B-B of FIG. 2B. Arrow 208 indicates a possible fluid flow path that passes through the opening of the second substrate layer 204 and then bypasses the filaments of the first substrate layer 202. The shape of the mesh substrate layer is designed to allow for an efficient and uniform flow through one or more substrate layers. In addition, the structure of the matrix 200 is capable of accommodating fluid flow through the matrix in multiple directions. For example, as shown in FIG. 3, the direction of the bulk fluid flow (as indicated by arrow 208) is perpendicular to the major surfaces of the first and second substrate layers 202 and 204. However, the matrix can also be oriented with respect to the fluid flow such that the sides of the substrate layers are parallel to the bulk flow direction. FIG. 4 shows a cross-sectional view of the multilayer substrate 200 along line C-C of FIG. 3, and due to the structure of the matrix 200, fluid flow (arrow 210) can pass through the flow paths in the multilayer substrate 200. In addition to the fluid flow being perpendicular or parallel to the first and second surfaces of the mesh layer, the matrix can be arranged such that a number of substrate pieces are at intermediate angles and even randomly arranged with respect to the fluid flow. This flexibility in direction is made possible by the substantially isotropic flow behavior of the woven substrate. In contrast, substrates for adherent cells in existing bioreactors do not exhibit this behavior, and instead, their packed beds tend to create preferential flow paths and have anisotropic permeable substrate materials. The flexibility of the matrices of the present disclosure allows for use in a variety of applications and bioreactor or vessel designs while enabling better and more uniform permeability throughout the bioreactor vessel.

[0041] As described herein, according to one or more embodiments, the cell culture substrate can be used within a bioreactor vessel. For example, the substrate can be used within a packed bed bioreactor structure or other structures within a three-dimensional culture chamber. However, the embodiments are not limited to three-dimensional culture spaces, and the substrate can be used in what is considered a two-dimensional culture surface structure where one or more layers of the substrate are flat, such as within a flat-bottom culture dish, to provide a culture substrate for cells. The vessel can be a disposable vessel that can be discarded after use due to concerns about contamination.

[0042] According to one or more embodiments, a cell culture system is provided in which a cell culture matrix is used within a culture chamber of a bioreactor vessel. FIG. 5 shows an example of a cell culture system 300 that includes a bioreactor vessel 302 having a cell culture chamber 304 inside the bioreactor vessel 302. Inside the cell culture chamber 304 is a cell culture matrix 306 made from a stack of substrate layers 308. The substrate layers 308 are stacked such that the first or second surface of one substrate layer faces the first or second surface of an adjacent substrate layer. The bioreactor vessel 302 has an inlet 310 at one end for media, cells, and / or nutrients to enter the culture chamber 304, and an outlet 312 at the opposite end for removing media, cells, or cell products from the culture chamber 304. By enabling the substrate layers to be stacked in this way, the system can be easily expanded without adversely affecting cell attachment and growth for efficient fluid flow through the defined structure and stacked substrates. The vessel 302 may generally be described as having an inlet 310 and an outlet 312, but in some embodiments, one or both of the inlet 310 and the outlet 312 may be used to flow media, cells, or other contents into and out of the culture chamber 304. For example, the inlet 310 may be used to flow media or cells into the culture chamber 304 during cell seeding, perfusion, or culture phases, but may also be used to remove media, cells, or cell products through the inlet 310 during the harvest phase. Therefore, the terms “inlet” and “outlet” are not intended to limit the function of those openings.

[0043] In one or more embodiments, the flow resistance and bulk density of the packed bed can be controlled by alternately arranging substrate layers of different shapes. Specifically, the mesh size and shape (e.g., fiber diameter, aperture diameter, and / or aperture shape) define the resistance of the fluid flow in the packed bed format. By alternately arranging meshes of different sizes and shapes, the flow resistance can be controlled or altered at one or more specific portions of the bioreactor. This enables better uniformity of liquid perfusion within the packed bed. For example, 10 layers of mesh A (Table 1) followed by 10 layers of mesh B (Table 1) followed by 10 layers of mesh C (Table 1) can be stacked to achieve the desired packed bed characteristics. As another example, the packed bed can start with 10 layers of mesh B, followed by 50 layers of mesh C, and then continue with 10 layers of mesh B. Such repeating patterns can continue until the bioreactor is completely filled with mesh. These are merely examples and are used for illustrative purposes without intending to limit to the possible combinations. In fact, various combinations of meshes of different sizes are possible to obtain different profiles of bulk density and flow resistance of the cell growth surface. For example, by alternately arranging meshes of different sizes, a packed bed column can be assembled having zones of different volume cell densities (e.g., a series of zones creating a pattern of low / high / low / high density, etc.).

[0044] In FIG. 5, the bulk flow direction is from the inlet 310 to the outlet 312, and in this example, the first and second major surfaces of the substrate layer 308 are perpendicular to the bulk flow direction.

[0045] The cell culture matrix can be arranged in a number of structures 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 a defined cell culture space within the culture chamber. The multiple layers of substrate can thus be stacked to a predetermined height. As described above, the substrate layers may be arranged such that the first and second faces of one or more layers are perpendicular to the bulk flow direction of the medium passing through the defined culture space within the culture chamber, or the first and second faces of one or more layers may be parallel to the bulk flow direction. In one or more embodiments, the cell culture matrix includes one or more substrate layers 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 may have first and second faces that are parallel or perpendicular to the bulk flow direction, or at an angle therebetween.

[0046] In one or more embodiments, the cell culture system includes a plurality of individual pieces of cell culture substrate in a packed bed structure, and the length and / or width of the pieces of substrate are small relative to the culture chamber. As used herein, a piece of substrate is considered to have a small length and / or width relative to the culture chamber if the length and / or width of the piece of substrate is about 50% or less of the length and / or width of the culture space. Thus, the cell culture system may include a plurality of pieces of substrate filled into 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 alignment, such as when the pieces are oriented in a substantially similar orientation (e.g., horizontal, vertical, or at an angle between 0° and 90° with respect to the bulk flow direction).

[0047] As used herein, the "predetermined culture space" refers to the space within the culture chamber that is occupied by the cell culture matrix and in which cell seeding and / or culturing is to be performed. The defined culture space can fill substantially all of the culture chamber or may occupy a portion of the space within the culture chamber. As used herein, the "bulk flow direction" is defined as the direction of the bulk mass flow of the fluid or medium within or on top of the cell culture matrix during cell culturing and / or during the inflow or outflow of the medium into or out of the culture chamber.

[0048] In one or more embodiments, the cell culture matrix is fixed within the culture chamber by a fixing mechanism. The fixing mechanism may fix a portion 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 portion of the cell culture matrix to a member extending through the culture chamber, such as a member extending parallel to the longitudinal axis of the culture chamber or a member extending perpendicular to the longitudinal axis thereof. However, in one or more other embodiments, the cell culture matrix may be housed within the culture chamber without being fixed and attached to the wall of the chamber or bioreactor vessel. For example, the matrix may be housed such that it is held within a predetermined area of the bioreactor vessel by the boundaries of the culture chamber or other structural members within the chamber, without being firmly fixed to those boundaries or structural members.

[0049] By using a cell culture matrix according to embodiments of the present disclosure, such as a matrix including a fabric or mesh substrate, a roller bottle container is provided with an increased surface area available for adherent cells to attach, proliferate, and function. Specifically, by using a fabric mesh substrate of a single fiber polymer material within a roller bottle, the surface area can be increased from about 2.4 times to about 4.8 times, or up to about 10 times, the surface area of a standard roller bottle. As described herein, each single fiber strand of the mesh substrate can present itself as a 2D surface for adherent cells to attach to. In addition, multiple layers of the mesh can be placed within the roller bottle to increase the total surface area available to about 2 to 20 times the surface area of a standard roller bottle. Therefore, existing roller bottle equipment and processes, including cell seeding, medium exchange, and cell harvesting, can be modified by adding the improved cell culture matrix disclosed herein with minimal impact on existing operating infrastructure and processing steps.

[0050] The bioreactor vessel comprises one or more outlets that can be attached to inlet and / or outlet means as required. Through the one or more outlets, liquid, medium, or cells can be supplied to or removed from the chamber. A single port in the vessel can serve both inlet and outlet functions, or multiple ports can be provided for dedicated inlets and outlets.

[0051] The packed bed cell culture matrix of one or more embodiments can consist of a woven cell culture mesh substrate, without any other form of cell culture substrate disposed within or dispersed throughout the cell culture matrix. That is, the woven cell culture mesh substrate of embodiments of the present disclosure is an effective cell culture substrate that does not require the types of irregular non-woven substrates used in existing solutions. This enables a cell culture system with a simple design and structure while providing the other advantages described herein related to flow uniformity, harvestability, etc. for high-density cell culture substrates.

[0052] As described herein, the provided cell culture substrates and bioreactor systems present a number of 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 for in vivo and in vitro gene therapy applications. Uniform cell seeding and distribution maximizes the viral vector yield per vessel, and its design enables the harvesting of viable cells, which can be useful for seed trains consisting of multiple expansion periods using the same platform. In addition, embodiments herein are scalable from process development scale to production scale, thereby ultimately saving development time and cost. The methods and systems disclosed herein also enable the automation and control of the cell culture process to maximize vector yield and improve reproducibility. Finally, the number of vessels required to reach a production level scale of viral vectors (e.g., 10 16 to 10 18 AAV VG per batch) can be significantly reduced compared to other cell culture solutions.

[0053] Embodiments are not limited to the rotation of the vessel about a central longitudinal axis. For example, the vessel can rotate about an axis that is not centered with respect to the vessel. In addition, the axis of rotation can be a horizontal or vertical axis.

[0054] The present disclosure describes a method of cutting and perforating a layer of a cell culture substrate, including a polymer mesh substrate, to create a substrate and a removable sample piece. The present disclosure also describes methods and apparatus for aseptically removing a sample from a bioreactor. By taking samples during the cell culture process, information regarding its implementation can be used to evaluate the quality and performance of the culture process. The cell count can be inferred from the sample, and growth can be monitored by taking samples at different times or at different locations within the bioreactor. This information can be used to develop and optimize the parameters of specific biological processes such as seed trains and viral vector production. In production, contaminated or out-of-specification processes can be aborted, reducing the cost of implementing the process to completion without satisfactory results. Growth media and lost production time are significant costs for typical biological processes.

[0055] In an embodiment herein, the sample portion of the substrate is separable from the remainder of the cell culture substrate with a low force, ideally manually, to take a sample without disturbing the main mesh body during the sampling process. For example, with a relatively small (e.g., manually applied) force, the sample portion can be separated from the remainder of the substrate by the tension between the sample portion to which the force is applied and the remainder of the substrate. To keep the removal force low, a separation boundary can be provided between the sample portion and the remainder of the substrate. This separation boundary can be formed by other cutting means such as scoring, perforating, laser cutting, or punching, and can be used to create a layer of the substrate that includes a separable piece of the substrate that can be removed from the fixed bed.

[0056] In some embodiments, a woven polymer mesh substrate having woven fibers that define an ordered array of pores or apertures is used. Since each of the fibers in the mesh is very strong, it is desirable that there be no fibers extending between the removable sample and the body of the mesh to facilitate removal of the sample with a small force. It is also desirable that the mesh layer be robust when handled during the manufacturing and assembly processes used to make the mesh laminate bioreactor floor. To accomplish this, some of the fibers can be cut in such a way as to leave the woven portion of the mesh that connects the sample to the mesh body, as shown in FIG. 6A. Due to the relative rigidity of the fibers, which can be formed from various polymers (including PET) disclosed herein, the woven fibers remain attached even though individual fibers are cut to create a separation boundary for the sample portion. The line in FIG. 6A indicates the separation boundary. FIG. 6B shows the sample portion after it has been removed from the remainder of the cell culture substrate.

[0057] The mesh layers with the sample pieces cut can be removed from the bioreactor by opening the bioreactor housing and pulling them out of the floor with a sterilized instrument, or they can be removed from the reactor by using a sterile sample collection port.

[0058] The diversity of the sample collection pieces can be derived from a single layer. In embodiments using a woven substrate, for most cutting patterns, the weave and the direction of the warp threads when engaging the cutting pattern are thought to maintain structural integrity and allow for easy removal. Since some of the cutting patterns created are not very affected by the orientation of the mesh fibers and the orientation of the mesh need not be precisely controlled, it is convenient to use these patterns in manufacturing.

[0059] Figures 7A-7C show another embodiment in which the sample collection layer includes a number of sample collection portions. The shape of the sample collection portion includes a rectangular end portion inside the sample collection portion that is internal to the outer edge of the sample collection layer, and a tapered end portion at the outer end of the outer edge of the sample collection layer. The tapered end portion allows for easy removal of the sample collection portion through a port in the side wall of the bioreactor. The substrate material may be such that the size of the port in the side wall may be the same as or slightly larger than the narrow end side, and the wide portion of the sample collection portion can be slightly bent or curved when pulled through the opening in the side wall. FIG. 7B shows an enlarged view of an individual sample collection portion after removal, and FIG. 7C shows an example of the relative sizes between the side wall port in the bioreactor and the sample collection portion, although the relative sizes can vary in different embodiments.

[0060] FIG. 8 shows an embodiment in which the sample layer of the substrate has a tether formed into the sample portion, and thus the sample portion can be removed by pulling on the tether. The embodiment includes a method of assembling a bioreactor in which the layer of the substrate is added to the housing of the bioreactor until it reaches the height of the sample collection port. At this point, the sample collection layer is inserted into the bioreactor vessel and the tether is pulled through the port. A sterile container outside the port can be used to perform sterile sample collection.

[0061] FIG. 9A shows a sample layer having six pie-shaped sample collection portions. The number and shape of the sample collection portions can vary. In this case, the separation boundary is laser cut through the fibers of the fabric mesh substrate. The sample layer also has alignment features on the left side of the layer, which can be useful for holding the sample layer in a predetermined position so that the sample collection portions are in a predetermined position to facilitate sample collection. These alignment features can be designed to engage corresponding features inside the side wall of the container. FIG. 9B shows three pie-shaped sample portions stained to show the presence of adherent cells on the substrate. FIG. 9C shows the three pie-shaped sample portions after sample collection following a collection procedure for harvesting cells from the substrate. Comparing FIGS. 9B and 9C shows the effectiveness of the harvesting procedure in this example.

[0062] Figure 10 shows an exploded view of a fixed bed reactor 1000 according to an embodiment of the present disclosure. The reactor 1000 includes a main body 1002 shown in FIG. 10 as a cylindrical container wall surrounding a cell culture space 1003 for accommodating a cell culture substrate 1004. The cell culture substrate 1004 may include one or more porous materials, such as those disclosed herein, in a stacked or wound arrangement. Embodiments of the present disclosure include a fixed bed reactor 1000 that can provide for easy sampling of the cell culture substrate 1004 and / or controlled positioning of the cell culture substrate 1004 within the cell culture space 1003. For example, as described below, the reactor 1000 can serve to maintain the position of the cell culture substrate 1004, such that the cell culture substrate 1004 does not move during operation, or the layers of the cell culture substrate 1004 are arranged within the cell culture space 1003 in a predetermined orientation relative to each other.

[0063] The reactor 1000 has at both ends of the main body 1002 a first end 1006 and a second end 1008 through which a culture medium, cells, and / or cell by-products can be introduced into and / or removed from the cell culture space 1003. The first and second ends 1006, 1008 can be sealed by a lower cap 1010 and an upper cap 1012, respectively, to maintain the hygiene of the cell culture space. Each of the lower and upper caps 1010, 1012 is provided with one or more fluid inlets and / or outlets 1011, 1013 for the fluid flow through the reactor 1000 during operation. An inlet distribution plate 1014 can be used adjacent to the lower cap 1010 to uniformly distribute the fluid across the cross-section of the cell culture space 1003. Similarly, an outlet distribution plate 1016 or a collection plate can be used adjacent to the upper cap 1012 to assist in collecting the fluid from the cell culture space 1003 and directing it towards the outlet of the upper cap 1012. Using the inlet and outlet distribution plates 1014 and 1016 helps to improve the uniformity of the fluid passing through the cell culture space 1003 by promoting a uniform flow of the fluid across the cross-sectional area of the cell culture space 1003 without unevenness at the bottom and top of the cell culture substrate 1004. The inlet and outlet distribution plates 1014 and 1016 are shown in FIG. 10 as separate components from the lower and upper caps 1010 and 1012, but the distribution plates 1014, 1016 could be considered integral with the lower and upper caps 1010, 1012 without any problem.

[0064] The reactor 1000 can be enclosed using various closing mechanisms including one or more of a welded seal, an adhesive, a clamp, and a gasket. For example, FIG. 10 shows a sanitary flange 1020 at the top of the cell culture space 1003. The sanitary flange 1020 engages with a sanitary seal 1022 disposed between the sanitary flange 1020 and the top cap 1012. A sanitary clamp 1024 can firmly engage the top cap 1012 to the sanitary flange 1020. The advantage of using this arrangement is that the sanitary clamp 1024 can be easily removed to provide access to the cell culture space 1003 (e.g., to sample the cell culture during use).

[0065] The reactor 1000 also includes a guide rod 1030. The guide rod 1030 extends through the cell culture space 1003 and is used to align the cell culture substrate 1004 within the cell culture space 1003 and / or to align multiple layers of the cell culture substrate 1004 with each other. For example, as shown in FIG. 11, the cell culture substrate fixed bed 1104 can be adapted to the guide rod 1030 via the substrate alignment feature 1106. The substrate alignment feature 1106 is designed to engage with the rod alignment feature 1136 in such a way that the fixed bed and / or the individual substrate layers within the fixed bed remain in the desired position and / or orientation. In embodiments where the fixed bed includes, for example, multiple layers of stacked substrate material, it may be desirable for each individual layer to have a specific orientation relative to the other layers. For example, in the case of a substrate layer having a predetermined structure of rigid fibers arranged in a predetermined direction, it may be desirable to vary the orientation of the fibers of the layer within the fixed bed. In embodiments, this may include rotating multiple layers by a predetermined angle around the central longitudinal axis of the cell culture space 1003. When adjacent layers are rotated relative to each other, the packing density of the layers can be controlled by controlling the amount of nesting that occurs between adjacent layers. This also affects the porosity and / or pore shape of the packed bed, which in turn affects the fluid flow through the fixed bed. For example, in some embodiments, each layer can be rotated by approximately 45° relative to one or both of the adjacent layers in the laminate to improve fluid flow and cell culture performance. By way of example, the substrate alignment feature 1106 can be a cutout in each layer of the fixed bed, where the position of the substrate alignment feature 1106 is rotated and shifted around the layer so that the layers are relatively rotated when the multi-layer substrate alignment features 1106 are aligned. Next, the corresponding rod alignment feature 1136 holds the substrate alignment feature 1106 in alignment, which in turn maintains the individual layers in their desired orientation.

[0066] As shown in FIG. 12, the guide rod 1230 can be attached to the structures (such as the distribution plates 1214 and 1216) at both ends of the cell culture substrate 1203 so that the guide rod 1230 holds the cell culture substrate 1203 therein, and the entire fixed bed can be taken out from the rest of the fixed bed reactor that houses the wall 1200 of the reactor. Thereby, for example, it becomes possible to easily take out and sample the fixed bed without disturbing the rest of the fixed bed or disturbing the alignment of the substrate layer. FIG. 13 shows an example of an embodiment in which the sample substrate layer 1232 housed in the fixed bed has one or more perforations 1234 so that a part of the substrate 1232 can be easily removed. It is considered that an additional guide rod 1231 or other structural members may be provided to reinforce the structural integrity of the removable substrate core. The embodiment is not limited to the specific arrangement of the guide rod, distribution plate, and / or sample mesh shown in FIGS. 12 and 13, but rather shows an example of various embodiments for sampling a sample from a reactor.

[0067] Definition "Total synthesis" or "complete synthesis" refers to cell culture articles such as microcarriers or the surface of culture vessels that are completely made from synthetic feedstock materials and do not contain any animal-derived or animal-source materials. The disclosed fully synthetic cell culture articles have no risk of xenogeneic contamination.

[0068] Terms such as "comprising" mean to include without limitation, that is, to be inclusive and not exclusive.

[0069] "User" refers to a person who uses the systems, methods, articles, or kits disclosed herein, including a person who cultures cells to harvest cells or cell products, or a person who uses cells or cell products cultured and / or harvested according to the embodiments described herein.

[0070] In describing embodiments of the present disclosure, for example, amounts of components in a composition, concentrations, volumes, process temperatures, process times required, yields, flow rates, pressures, viscosities, and similar values, as well as ranges thereof, or dimensions of components, and similar values, as well as ranges thereof, the term "about" is, for example, by typical measurement and handling procedures used in the preparation of materials, compositions, composites, concentrates, components, articles of manufacture, or use formulations; by inadvertent errors in these procedures; by differences in the manufacture, source, or purity of starting materials or components used to carry out the method; and by similar considerations. The term "about" also encompasses amounts that vary due to the aging of a composition or formulation having a particular initial concentration or mixture, and amounts that vary due to the mixing or processing of a composition or formulation having a particular initial concentration or mixture.

[0071] "Optional" or "optionally" means that the event or circumstance described thereafter may or may not occur, and that the description includes examples where the event or circumstance occurs and examples where it does not occur.

[0072] Nouns, unless otherwise specified, include at least one, or one or more, objects.

[0073] Abbreviations well known to those skilled in the art may be used (e.g., "h" or "hrs" for hours, "g" or "gm" for grams, "mL" for milliliters, and "rt" for room temperature, "nm" for nanometers, and similar abbreviations).

[0074] The specific values and preferred values, as well as ranges thereof, disclosed for components, ingredients, additives, dimensions, conditions, and similar attributes are for illustrative purposes only and do not exclude other predetermined values or other values within a predetermined range. The systems, kits, and methods of the present disclosure can include any value or combination of the values, specific values, more specific values, and preferred values described herein, including apparent or potential intermediate values and ranges.

[0075] Unless otherwise specified, none of the methods described herein are intended to be construed as requiring that the steps be performed in any particular order. Accordingly, where a method claim does not actually recite an order to be followed by its steps, or where not otherwise specifically stated in the claim or description that the steps are to be limited to a particular order, no particular order is intended to be implied.

[0076] It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments without departing from the spirit or scope of the disclosed embodiments. Since modifications, combinations, sub - combinations and variations of the disclosed embodiments that include the spirit and substance of the disclosed embodiments will occur to those skilled in the art, the disclosed embodiments are to be construed as including all within the scope of the appended claims and their equivalents.

[0077] Hereinafter, preferred embodiments of the present invention will be described item by item.

[0078] Embodiment 1 In a fixed - bed bioreactor for culturing cells on a cell - culture substrate, a cell - culture vessel having a container body defining at least one internal chamber, an inlet fluid - connected to the chamber, and an outlet fluid - connected to the chamber, wherein the container body has a first end, a second end, and a longitudinal axis extending from the first end in the direction of the second end, the cell - culture vessel, and a guide rod disposed within the internal chamber and extending parallel to the longitudinal axis of the container body, comprising, the guide rod being configured to hold the cell - culture substrate in place within the internal chamber, the fixed - bed bioreactor.

[0079] Embodiment 2 The fixed - bed bioreactor according to Embodiment 1, wherein the internal chamber is configured to hold a multi - layer cell - culture substrate.

[0080] Embodiment 3 The fixed-bed bioreactor according to Embodiment 1, further comprising a cell culture substrate disposed in the internal groove.

[0081] Embodiment 4 The fixed-bed bioreactor according to Embodiment 3, wherein the cell culture substrate is a multi-layer cell culture substrate.

[0082] Embodiment 5 The fixed-bed bioreactor according to Embodiment 4, wherein the guide rod is configured to maintain rotational alignment of an individual layer of the multi-layer cell culture substrate with respect to another individual layer of the multi-layer cell culture substrate.

[0083] Embodiment 6 The fixed-bed bioreactor according to any one of Embodiments 1 to 5, wherein the guide rod has an outer surface defining a cross-sectional profile of the guide rod configured to engage with the cell culture substrate.

[0084] Embodiment 7 The fixed-bed bioreactor according to Embodiment 6, wherein the cell culture substrate includes a cutout configured to engage with or at least partially surround the cross-sectional profile of the guide rod.

[0085] Embodiment 8 An inlet distribution plate disposed between the inlet and the internal groove, and An outlet distribution plate disposed between the outlet and the internal groove, The fixed-bed bioreactor according to any one of Embodiments 1 to 7, further comprising the same.

[0086] Embodiment 9 The fixed-bed bioreactor according to Embodiment 8, wherein the guide rod is attached to the inlet distribution plate.

[0087] Embodiment 10 The second end of the container body is at an end of the internal groove opposite to the inlet distribution plate, The fixed-bed bioreactor according to embodiment 9, wherein the guide rod and the inlet distribution plate are removable from the container body through a second end of the container body.

[0088] Embodiment 11 The fixed-bed bioreactor according to embodiment 9 or 10, wherein the guide rod is attached to the outlet distribution plate.

[0089] Embodiment 12 The fixed-bed bioreactor according to any one of embodiments 9 to 11, wherein the cell culture substrate is at least partially retained by the inlet distribution plate and the guide rod and is removable from the container body.

[0090] Embodiment 13 The fixed-bed bioreactor according to any one of embodiments 1 to 12, wherein the cell culture substrate has a structurally defined surface for culturing cells thereon, and the structurally defined surface defines a regular ordered array of openings penetrating the thickness of the cell culture substrate.

[0091] Embodiment 14 At least a part of the cell culture substrate constitutes a sample substrate, and the sample substrate is defined by a separation boundary between the sample substrate and the rest of the cell culture substrate. The fixed-bed bioreactor according to any one of embodiments 1 to 13, wherein the separation boundary is made to separate the sample substrate from the rest of the cell culture substrate.

[0092] Embodiment 15 The fixed-bed bioreactor according to embodiment 14, wherein the separation boundary includes at least one of a score, a cut therein or therethrough, or a locally thin portion in the cell culture substrate.

[0093] Embodiment 16 The fixed-bed bioreactor according to any one of embodiments 13 to 15, wherein the structurally defined surface is made of one or more fibers.

[0094] Embodiment 17 The fixed-bed bioreactor according to Embodiment 16, wherein one or more fibers of the layer are woven with each other in a regular arrangement.

[0095] Embodiment 18 The fixed-bed bioreactor according to any one of Embodiments 1 to 17, wherein the tank is defined by a length and a width, the length extending from a first end of the tank adjacent to the inlet to a second end of the tank adjacent to the outlet, and the cell culture matrix having a width substantially extending in the width of the tank.

[0096] Embodiment 19 The fixed-bed bioreactor according to any one of Embodiments 1 to 18, wherein the fixed-bed bioreactor is configured to aseptically remove a sample portion from the cell culture container.

Description of Reference Numerals

[0097] 100 Cell culture substrate 102 First plurality of fibers 104 Second plurality of fibers 106, 206 Opening 108 First surface 110 Second surface 200 Multilayer substrate, matrix 202 First substrate layer 204 Second substrate layer 300 Cell culture system 302 Bioreactor container 304 Cell culture chamber 306 Cell culture matrix 308 Substrate layer 310 Inlet 312 Outlet 1000 Fixed-bed reactor 1002 Main body 1003 Cell culture space 1004, 1203 Cell culture substrate 1006 First end 1008 Second end 1010 Lower cap 1011 Fluid inlet 1012 Upper cap 1013 Fluid outlet 1014 Inlet distribution plate 1016 Outlet distribution plate 1020 Sanitary flange 1022 Sanitary seal 1024 Sanitary clamp 1030, 1230, 1231 Guide rod 1136 Rod alignment feature 1200 Reactor wall 1214, 1216 Distribution plate 1232 Sample matrix layer 1234 Perforation

Claims

1. In a fixed-bed bioreactor for culturing cells on a cell culture substrate, a cell culture vessel having a vessel body defining at least one internal tank configured to hold a multi-layer cell culture substrate, an inlet fluidly connected to the tank, and an outlet fluidly connected to the tank, wherein the vessel body has a first end, a second end, and a longitudinal axis extending from the first end towards the second end, a cell culture substrate disposed within the internal tank, and a guide rod disposed within the internal tank and extending parallel to the longitudinal axis of the vessel body, comprising: the guide rod being configured to hold the cell culture substrate in place within the internal tank, the cell culture substrate being a multi-layer cell culture substrate, and the guide rod being configured to maintain rotational alignment of individual layers of the multi-layer cell culture substrate relative to other individual layers of the multi-layer cell culture substrate, a fixed-bed bioreactor.

2. The fixed-bed bioreactor according to claim 1, wherein the guide rod has an outer surface defining a cross-sectional profile of the guide rod configured to engage with the cell culture substrate.

3. The fixed-bed bioreactor according to claim 2, wherein the cell culture substrate includes a cutout configured to engage with or at least partially surround the cross-sectional profile of the guide rod.

4. an inlet distribution plate disposed between the inlet and the internal tank, and an outlet distribution plate disposed between the outlet and the internal tank, further comprising the fixed-bed bioreactor according to claim 1.

5. The fixed-bed bioreactor according to claim 4, wherein the guide rod is attached to the inlet distribution plate.

6. The second end of the vessel body is at an end of the internal tank opposite to the inlet distribution plate, the fixed-bed bioreactor according to claim 5, wherein the guide rod and the inlet distribution plate are removable from the vessel body through the second end of the vessel body.

7. The fixed-bed bioreactor according to claim 5, wherein the guide rod is attached to the outlet distribution plate.

8. The fixed-bed bioreactor according to claim 5, wherein the cell culture substrate is at least partially retained by the inlet distribution plate and the guide rod and is removable from the vessel body.

9. The fixed-bed bioreactor according to claim 1, wherein the cell culture substrate has a structurally defined surface for culturing cells thereon, and the structurally defined surface defines a regular ordered array of openings penetrating the thickness of the cell culture substrate.

10. At least a part of the cell culture substrate constitutes a sample substrate, and the sample substrate is defined by a separation boundary between the sample substrate and the rest of the cell culture substrate. The fixed-bed bioreactor according to claim 1, wherein the separation boundary is made to separate the sample substrate from the rest of the cell culture substrate.

11. The fixed-bed bioreactor according to claim 10, wherein the separation boundary includes at least one of a score, a cut therein or therethrough, or a locally thin portion in the cell culture substrate.

12. The fixed-bed bioreactor according to claim 9, wherein the structurally defined surface is made of one or more fibers.

13. The fixed-bed bioreactor according to claim 12, wherein one or more fibers of the layer are woven with each other in a regular arrangement.

14. The fixed-bed bioreactor according to claim 1, wherein the trough is defined by a length and a width, the length extending from a first end of the trough adjacent to the inlet to a second end of the trough adjacent to the outlet, and the cell culture matrix has a width substantially extending over the width of the trough.

15. The cell culture substrate has a structurally defined surface for culturing cells thereon, and the structurally defined surface is made of a plurality of fibers defining a regular ordered array of openings penetrating the thickness of the cell culture substrate. Individual layers of the multilayer cell culture substrate include the cutouts. The cutouts of each individual layer of the multilayer cell culture substrate are aligned with the cutouts of all other individual layers of the multilayer cell culture substrate, and the orientation of the plurality of fibers in one or more layers of the multilayer cell culture substrate is different from the orientation of the plurality of fibers in other layers of the multilayer cell culture substrate. The fixed-bed bioreactor according to claim 3, wherein the orientation of the plurality of fibers in one or more layers of the multilayer cell culture substrate is rotated at a predetermined angle around the longitudinal axis of the container body.

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