Fixed bed cell culture reactor vessel for substrate alignment and sampling

Abstract

A fixed bed bioreactor for culturing cells on a cell culture substrate is provided. A fixed bed bioreactor includes a cell culture vessel having a vessel body defining at least one internal reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir. 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 in the inner reservoir and extends parallel to a longitudinal axis of the container body. The guide rod is configured to hold the cell culture substrate secured in the internal reservoir.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority under 35 U.S.C. §119 to U.S. Provisional Application Serial No. 63 / 354,401, filed on June 22, 2022, the contents of which are relied upon and incorporated herein by reference in their entirety. Technical Field

[0003] The present disclosure relates generally to cell culture bioreactors and substrates for culturing cells. In particular, the present disclosure relates to cell culture substrates and bioreactors incorporating such substrates that enable defined packing and / or sampling of the substrates. Background Art

[0004] In the bioprocessing industry, large-scale cell culture is performed for the purpose of producing hormones, enzymes, antibodies, vaccines, and cell therapies. The cell and gene therapy market is growing rapidly, with promising treatments entering clinical trials and moving quickly toward commercialization. However, a single cell therapy dose may require billions of cells or trillions of viruses. Therefore, being able to provide large quantities of cell products in a short period of time is critical to clinical success.

[0005] Most cells used in bioprocessing are anchorage dependent, meaning that cells need a surface to attach to in order to grow and function. Traditionally, the culture of adherent cells is performed on two-dimensional (2D) cell attachment surfaces that are integrated into one of a variety of container formats, such as: T-flasks, culture dishes, cell factories, cell stack containers, roller bottles, and These approaches can have significant drawbacks, including the difficulty in achieving cell densities high enough to be useful for therapeutic or large-scale production of cells.

[0006] Alternative methods have been suggested to increase the volume density of cultured cells. These include microcarrier cultures performed in stirred tanks. In this scheme, cells attached to the surface of the microcarriers are subjected to constant shear stress, resulting in a significant effect on proliferation and culture performance. Another example of a high-density cell culture system is a hollow fiber bioreactor, in which cells can form large three-dimensional aggregates as they proliferate in the spaces between the fiber spaces. However, cell growth and performance are significantly inhibited due to a lack of nutrients. To alleviate this problem, these bioreactors are made small and unsuitable for large-scale production.

[0007] Another example of a high-density culture system for anchoring dependent cells is a packed bed bioreactor system. In this type of bioreactor, a cell substrate is used to provide an attachment surface for attaching cells. The medium is perfused along the surface or through a semi-porous substrate to provide nutrients and oxygen required for cell growth. For example, previous U.S. Patents No. 4,833,083, No. 5,501,971 and No. 5,510,262 have disclosed packed bed bioreactor systems containing a support or matrix system to capture cells. The packed bed matrix is ​​usually made of porous particles as a substrate or non-woven microfibers of a polymer. This type of bioreactor functions as a recirculating flow-through bioreactor. One of the obvious problems is that the cell distribution in the packed bed of this type of bioreactor is not uniform. For example, the packed bed functions as a depth filter, and cells are mainly captured at the inlet area, resulting in a gradient of cell distribution during the inoculation step. In addition, due to the random fiber packing, the flow resistance of the cross section of the packed bed and the cell capture efficiency are not uniform. For example, the medium flows quickly through areas with low cell packing density, while slowly flowing through areas with higher resistance due to a higher number of trapped cells. This creates a channeling effect, where nutrients and oxygen are more efficiently delivered to areas with lower volume cell density, while areas with higher cell density are maintained under non-optimal culture conditions.

[0008] Another significant drawback of the packed bed systems disclosed in the prior art is that intact living cells cannot be efficiently harvested at the end of the culture process. If the final product is cells or if the bioreactor is used as part of an "inoculation sequence" (wherein a cell population is grown in one container and then transferred to another container to allow the population to grow further), then the harvesting of cells is critical. U.S. Patent No. 9,273,278 discloses a bioreactor design that improves the efficiency of recovering cells from the packed bed during the cell harvesting step. It is based on loose packed bed matrix and vibration or stirring of packed bed particles to achieve porous matrix collision and thereby separate cells. However, this solution is laborious and may result in significant cell damage, thereby reducing overall cell viability.

[0009] Some existing bioreactor solutions use small strips of cell substrate materials consisting of randomly oriented fibers arranged in a nonwoven. These strips are loaded into a container to produce a packed bed. However, like similar solutions on the market, this type of packed bed substrate has defects. Specifically, the non-uniform loading of the substrate strips produces visually visible channels in the packed bed, resulting in preferential and non-uniform medium flow and nutrient distribution of the packed bed. Studies on such systems have noted "systematic heterogeneous distribution of cells, their number increases from the top to the bottom of the fixed bed" and "nutrient gradients ... lead to limiting cell growth and production", which all lead to "uneven distribution of cells that may affect transfection efficiency". (Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells (rational plasmid design and bioprocess optimization, to improve the productivity of recombinant adeno-associated virus (AAV) in mammalian cells), Journal of Biotechnology, November 2016, pp. 290-297). Studies have noted that agitation of packed beds can improve distribution, but can have other drawbacks (i.e., "agitation necessary for better dispersion during inoculation and transfection induces increased shear stress, which in turn leads to reduced cell viability," supra). Another study noted that uneven distribution of cells makes it difficult to monitor the cell population using biomass sensors ("…if cells are unevenly distributed, the biomass signal of cells on the top carriers may not show the full picture of the entire bioreactor." Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale, Human Gene Therapy, Vol. 26, No. 8, 2015).

[0010] Furthermore, due to the random arrangement of fibers in the substrate strips and variations in strip packing from one packed bed to another (similar to those discussed above), it may be difficult for the consumer to predict cell culture performance as the substrate changes between cultures. Furthermore, random packing substrates (which themselves have a random structure) make it very difficult or impossible to efficiently harvest cells as the cells are believed to be captured by the packed bed.

[0011] Although viral vectors can be manufactured using existing platforms for early clinical trials, platforms that can produce high-quality products in larger quantities are needed to reach late-stage commercial manufacturing scale.

[0012] In addition, it would be desirable to be able to monitor bioreactors used for cell culture or production of AAV or to generate inoculum sequences to aid in cell expansion for biochemical production. When using attached cell reactors, samples of growth media do not contain cells, or at least not to a usable degree for monitoring the status of cultures on attached substrates.

[0013] There is a need for cell culture substrates, systems and methods that enable cell culture in high density formats with uniform cell distribution and ease of access and increased harvest yields, while also allowing the user to monitor the status of the cell culture process by inspecting the cells on the substrate during and / or after the cell culture process, including monitoring the sterility of the substrate. Summary of the invention

[0014] According to embodiments of the present disclosure, disclosed cell culture substrates enable sampling of the entire substrate or a portion of the substrate to monitor the state or health of the cell culture. Embodiments include multi-layer fixed bed cell culture substrates having one or more layers specifically designed to enable such sampling. Embodiments also include fixed bed bioreactors having such cell culture substrates and / or matrices, including enabling the arrangement of the cell culture substrate in a specified arrangement or orientation.

[0015] In order to be able to estimate the number of cells in the attached cell bioreactor bed and their distribution and health, a method uses a portion of the substrate to be removed in the middle of the cell culture or cell expansion process. By sampling during the process, information about the cell culture operation can be used to evaluate the quality and performance of the process. Cell counts can be estimated from the samples, and growth can be monitored by sampling at different times. This information can be used to develop and optimize the performance of specific bioprocesses (such as inoculation sequences and viral vector production). In production, batches that are contaminated or do not meet specifications will be terminated, thereby reducing the cost of running the process to the end without satisfactory results. For typical bioprocesses, growth media and lost production time represent significant costs. The embodiments of the present disclosure enable the removal of all or part of the fixed bed cell culture substrate from the housing, so that the user has access to the bed without destroying the bed or bioreactor container. This enables the evaluation of any portion or selected portion of the fixed bed. The bed can also be reached after the cell culture process or after the harvest of the desired components is completed for "post-mortem" analysis of cell culture.

[0016] A fixed bed bioreactor for culturing cells on a cell culture substrate is provided. The fixed bed bioreactor includes a cell culture container having: a container body defining at least one internal reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir. 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 arranged in the internal reservoir and extends parallel to the longitudinal axis of the container body. The guide rod is configured to keep the cell culture substrate fixed in the internal reservoir. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A A perspective view showing a three-dimensional model of a cell culture substrate according to an embodiment of the present disclosure.

[0018] Figure 1B yes Figure 1A Two-dimensional plan view of the substrate.

[0019] Figure 1C yes Figure 1B Cross section of the substrate along line AA.

[0020] Figure 2A A perspective view showing a multi-layered cell culture substrate according to an embodiment.

[0021] Figure 2B A plan view showing a multilayer cell culture substrate according to an embodiment.

[0022] Figure 3 Display according to the embodiment Figure 2B Cross-sectional view of the multilayer cell culture substrate along line BB.

[0023] Figure 4 Display according to the embodiment Figure 3 Cross-sectional view of the multilayered cell culture substrate along line CC.

[0024] Figure 5 A schematic diagram showing a cell culture system according to an embodiment.

[0025] Fig. 6A A plan view of a cell culture substrate sampling layer having a separation boundary defining a substrate sampling portion is shown according to one or more embodiments.

[0026] Figure 6B A schematic diagram showing a sampled portion after separating the sampled portion from the remainder of the substrate sampling layer according to one or more embodiments. Figure 6B Plan view of the sampling layer and sampling part of the cell culture substrate.

[0027] Fig. 7AA plan view of a cell culture substrate sampling layer having a plurality of substrate sampling portions with tapered ends is shown according to one or more embodiments.

[0028] Figure 7B Show from Figure 8 Individual substrate sampling portions of B.

[0029] Figure 7C show Figure 8 B includes individual substrate sampling portions and a port through which the substrate sampling portion can be extracted from the fixed bed cell culture substrate.

[0030] Figure 8 is a plan view of a substrate sampling layer with a tethered sampling portion according to some embodiments.

[0031] Fig.9A is a photograph of a substrate sampling layer of a woven PET mesh according to some embodiments.

[0032] Fig. 9B is a photograph of a crystal violet stained sampling section of a woven PET mesh prior to harvesting attached cells according to some embodiments.

[0033] Fig. 9C is a photograph of a crystal violet stained sample portion of a woven PET mesh substrate after harvesting attached cells according to some embodiments.

[0034] Fig.10 is an exploded view of a fixed bed reactor for cell culture according to an embodiment.

[0035] Fig.11 Shown is a cell culture bed and a guide rod with a keyhole feature according to an embodiment.

[0036] Fig.12 is a drawing of a fixed bed reactor vessel with a removable substrate for sampling according to an embodiment.

[0037] Fig.13 yes Fig.12 Close-up of section 1213 of . DETAILED DESCRIPTION

[0038] Various embodiments of the present disclosure are described in detail below with reference to the accompanying drawings (if any). Reference to various embodiments does not limit the scope of the invention, which is limited only by the scope of the appended claims. In addition, any examples listed in this specification are not limiting and only list some of the many possible embodiments of the claimed invention.

[0039] Embodiments of the present disclosure include cell culture substrates and cell culture bioreactors incorporating such substrates, enabling sampling of the substrate or a portion of the substrate to monitor cell culture.

[0040] In conventional large-scale cell culture bioreactors, different types of packed bed bioreactors are used. Generally speaking, these packed beds contain porous matrices to retain attached or suspended cells and to support growth and proliferation. The packed bed matrix provides a high surface area-to-volume ratio, so the cell density will be higher than other systems. However, packed beds often act as depth filters, in which cells are physically captured or entangled in the fibers of the matrix. Thus, due to the linear flow of the cell inoculum through the packed bed, the cells are subjected to heterogeneous distribution in the packed bed, resulting in changes in cell density across the depth or width of the packed bed. For example, the cell density may be higher in the inlet area of ​​the bioreactor and significantly lower at the outlet closer to the bioreactor. The non-uniform distribution of cells in this packed bed significantly hinders the scalability and predictability of such bioreactors in bioprocessing manufacturing, and may even result in a decrease in the growth efficiency of cells or viral vector production per unit surface area or volume of the packed bed.

[0041] Another problem encountered by the packed bed bioreactor disclosed in the prior art is the channel effect. Due to the random nature of the packed nonwoven fibers, the local fiber density at any given cross section of the packed bed is uneven. The medium flows quickly in areas with low fiber density (high bed permeability), while it flows much slower in areas with high fiber density (lower bed permeability). As a result, the non-uniform medium perfusion on the packed bed produces a channel effect, which manifests itself as a significant gradient of nutrients and metabolites, which has a negative impact on overall cell culture and bioreactor performance. Cells located in low medium perfusion areas will starve and die very commonly due to lack of nutrients or metabolite poisoning. Cell harvesting is another problem encountered when using a bioreactor filled with a nonwoven fibrous scaffold. Since the packed bed functions as a depth filter, the cells released at the end of the cell culture process are captured in the packed bed, and cell recovery is very low. This significantly limits the application of such bioreactors in bioprocessing where living cells are products. Thus, non-homogeneity results in areas with different fluid and shear exposure, effectively reducing the available cell culture area, leading to non-uniform cultures, and interfering with transfection efficiency and cell release.

[0042] To address these and other problems with existing cell culture protocols, embodiments of the present disclosure provide cell growth substrates, matrices of such substrates, and / or packed bed systems using such substrates, enabling efficient and high-yield cell culture for the production of anchor-dependent cells and cell products (e.g., proteins, antibodies, viral particles). Embodiments include porous cell culture substrates made from an ordered and regular array of porous substrate materials, which enable uniform cell seeding and media / nutrient perfusion, as well as efficient cell harvesting. Embodiments also enable scalable cell culture solutions with substrates and bioreactors, capable of seeding and growing cells and / or harvesting cell products, from process development specifications to full production size specifications, without sacrificing the uniform performance of the embodiments. For example, in some embodiments, a bioreactor can be easily scaled from a process development specification to a production specification, with a comparable viral genome per unit substrate surface area (VG / cm 2 ). The harvestability and scalability of the embodiments herein enable them to be used for efficient seeding sequences for growing cell populations on multiple scales on the same cell substrate. In addition, the embodiments herein provide cell culture substrates with high surface areas, which, in combination with other features described herein, enable high-yield cell culture solutions. For example, in some embodiments, the cell culture substrates and / or bioreactors discussed herein can produce 10 16 Up to 10 18 A viral genome (VG).

[0043] In one embodiment, the provided matrix has a surface area with a limited structure for attaching (adherent) cells to attach (attach) and proliferate, which has good mechanical strength and forms a highly uniform and diverse interconnected fluid network when assembled into a packed bed or other bioreactor. In a specific embodiment, a mechanically stable non-degradable woven mesh can be used as a substrate to support the production of attached cells. The cell culture matrix disclosed herein supports the attachment and proliferation of anchorage-dependent cells in the form of high volume density. Such a matrix can achieve uniform cell inoculation, as well as efficient harvesting of other products of cells or bioreactors. In addition, the embodiments of the present disclosure support cell culture to provide uniform cell distribution during the inoculation step and achieve a confluent monolayer or multilayer of attached cells on the disclosed matrix, and can avoid the formation of large and / or uncontrollable 3D cell aggregates with limited nutrient diffusion and increased metabolite concentrations. Thus, the matrix eliminates diffusion restrictions during the operation of the bioreactor. In addition, the matrix can be harvested from the bioreactor simply and efficiently. The structurally limited matrix of one or more embodiments realizes complete cell recovery and consistent cell harvesting from the packed bed of the bioreactor.

[0044] According to some embodiments, a method for cell culture using a bioreactor having a matrix is ​​also provided for bioprocessing production of therapeutic proteins, antibodies, viral vaccines or viral vectors.

[0045] Different from the existing cell culture substrate (that is, the nonwoven substrate of disordered fiber) for cell culture bioreactor, the embodiment of the present disclosure includes the cell culture substrate with limited and ordered structure.The limited and ordered structure realizes consistent and predictable cell culture results.In addition, the substrate has an open porous structure, which prevents cell capture and realizes uniform flow through the packed bed.This structure realizes improved cell inoculation, nutrient delivery, cell growth and cell harvesting.According to one or more specific embodiments, the matrix is ​​formed by substrate material, and the substrate material has a thin sheet structure, with the first and second sides separated by a smaller thickness, so that the sheet thickness is small relative to the width and / or length of the first and second sides of the substrate.In addition, a plurality of holes or openings passing through the substrate thickness are formed.The size and geometrical morphology of the substrate material between the openings allow cells to attach to the surface of the substrate material, as if it is an approximate two-dimensional (2D) surface, while also allowing around the substrate material and passing the sufficient fluid flow of the opening. In some embodiments, the substrate is: a polymer-based material and can be formed into a molded polymer sheet; a polymer sheet with openings punched through the thickness; a plurality of filaments fused into a mesh layer; a 3D printed substrate; or a plurality of filaments woven into a mesh layer. The physical structure of the substrate has a high surface-to-volume ratio for culturing anchorage-dependent cells. According to various embodiments, the substrate can be arranged or loaded into a bioreactor in some of the ways discussed herein for uniform cell seeding and growth, uniform media perfusion, and efficient cell harvesting.

[0046] Embodiments of the present disclosure can achieve a practical-sized viral vector platform that can produce viral genomes in batches of greater than about 10 14 Viral genomes; each batch is greater than about 10 15 Viral genomes; each batch is greater than about 10 16 Viral genomes; each batch is greater than about 10 17 viral genomes; or up to or greater than about 10 per batch 16 In some embodiments, each batch produces about 10 15 to about 10 18 For example, in some embodiments, the viral genome yield can be about 10 per batch. 15 to about 10 16 viral genomes; or about 10 per batch 16 to about 10 19viral genomes; or about 10 per batch 16 -10 18 viral genomes; or about 10 per batch 17 to about 10 19 viral genomes; or about 10 per batch 18 to about 10 19 viral genomes; or about 10 per batch 18 or more viral genomes.

[0047] In addition, the embodiments disclosed herein can not only realize cell attachment and growth of cell culture substrates, but also can realize the survival harvest of cultured cells. It is an obvious defect of the current platform that viable cells cannot be harvested, and this causes difficulty in establishing and maintaining a sufficient number of cells to realize production capacity. According to the aspects of the embodiments of the present disclosure, viable cells can be harvested from cell culture substrates, including 80% to 100% survival rate, or about 85% to about 99% survival rate, or about 90% to about 99% survival rate. For example, for the cells harvested, it is at least 80% survival rate, at least 85% survival rate, at least 90% survival rate, at least 91% survival rate, at least 92% survival rate, at least 93% survival rate, at least 94% survival rate, at least 95% survival rate, at least 96% survival rate, at least 97% survival rate, at least 98% survival rate, or at least 99% survival rate. For example, trypsin, TrypLE or Accutase can be used to release cells from cell culture substrates.

[0048] Figure 1A and 1B A three-dimensional (3D) stereogram 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 are shown, respectively. The cell culture substrate 100 is a woven mesh layer made of a first group of multiple fibers 102 along a first direction and a second group of multiple fibers 104 along 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 weave type (e.g., the number, shape, and size of the threads; the angles between intersecting threads, etc.). The woven mesh can be characterized as a macroscopic two-dimensional sheet or layer. However, a close examination of the woven mesh reveals a three-dimensional structure caused by the rise and fall of the intersecting fibers of the mesh. Therefore, as Figure 1CAs shown, the thickness T of the woven mesh 100 can be thicker than the thickness of the single fiber (e.g., t1). As used herein, the thickness T is the maximum thickness between the first side 108 and the second side 110 of the woven mesh. Without wishing to be bound by theory, it is believed that the three-dimensional structure of the substrate 100 is advantageous because it provides a large surface area to culture attached cells, and the structural rigidity of the mesh can provide a consistent and predictable cell culture matrix structure that achieves uniform fluid flow.

[0049] exist Figure 1B In the embodiment of the present invention, the opening 106 has a diameter D1 (defined as the distance between the fibers 102 relative to each other) and a diameter D2 (defined as the distance between the fibers 104 relative to each other). Depending on the braiding geometry, D1 and D2 may be equal or unequal. For the case where D1 and D2 are unequal, the larger one may be referred to as the major diameter, and the smaller one may be referred to as the minor diameter. In some embodiments, the diameter of the opening may be referred to as the widest part of the opening. Unless otherwise stated, the opening diameter as used herein may represent the distance between the parallel fibers on the opposite sides of the opening.

[0050] A given fiber of the plurality of fibers 102 has a thickness t1, and a given fiber of the plurality of fibers 104 has a thickness t2. Figure 1A In the case of a circular cross-section or other three-dimensional cross-section fiber as shown, thickness t1 and t2 are the maximum diameters or thicknesses of the fiber cross-section. According to some embodiments, the plurality of fibers 102 all have the same thickness t1, and the plurality of fibers 104 all have the same thickness t2. In addition, t1 and t2 may be equal. However, in one or more embodiments, t1 and t2 are unequal, such as when the plurality of fibers 102 are different from the plurality of fibers 104. In addition, the plurality of fibers 102 and the plurality of fibers 104 may each contain two or more fibers of different thicknesses (e.g., t 1a ,t 1b etc., and t 2a ,t 2b 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 an approximately flat surface relative to the cell angle, which can achieve better cell attachment and growth compared to some other solutions where the fiber size is small (e.g., sized to the cell diameter). Due to the three-dimensional nature of the woven mesh, such as Figures 1A-1C As shown, the 2D surface area of ​​the fibers available for cell attachment and proliferation exceeds the surface area for attachment on an equivalent flat 2D surface.

[0051] In one or more embodiments, the fiber can have a diameter range of about 50 μm to about 1000 μm, about 100 μm to about 750 μm, about 125 μm to about 600 μm, about 150 μm to about 500 μm, about 200 μm to about 400 μm, about 200 μm to about 300 μm, or about 150 μm to about 300 μm. For the microscopic level, due to the specifications of the fiber compared to the cell (e.g., the fiber diameter is larger than the cell), the surface of the monofilament fiber presents attachment and proliferation for attached cells in an approximately 2D plane. The fiber can be woven into a mesh with an opening range of about 100 μm x 100 μm to about 1000 μm x 1000 μm. In some embodiments, the openings may have diameters of about 50 μm to about 1000 μm, about 100 μm to about 750 μm, about 125 μm to about 600 μm, about 150 μm to about 500 μm, about 200 μm to about 400 μm, or about 200 μm to about 300 μm. These ranges of filament diameters and opening diameters are examples of some embodiments, but are not intended to limit the possible characteristic dimensions of the mesh according to all embodiments. The combination of fiber diameter and opening diameter is selected to provide efficient and uniform fluid flow through the substrate, such as when the cell culture substrate comprises multiple adjacent mesh layers (e.g., stacks of individual layers or rolled mesh layers).

[0052] Factors such as fiber diameter, opening diameter, and weaving type / pattern determine the surface area available for cell attachment and growth. In addition, when the cell culture matrix includes stacking, coiling, or other arrangements of overlapping substrates, the packing density of the cell culture matrix can affect the surface area of ​​the packed bed matrix. The packing density changes with the packing thickness of the substrate material (e.g., the space required for the layer of the substrate). For example, if the stack of the cell culture matrix has a certain height, each layer in the stack can be considered to have a packing thickness, which is determined by dividing the total height of the stack by the number of layers in the stack. The packing thickness changes based on the fiber diameter and weaving, but also changes based on the alignment of adjacent layers in the stack. For example, due to the three-dimensional nature of the woven layer, there is a certain amount of interlocking or overlapping that can be accommodated by the adjacent layers based on their mutual alignment. In the first alignment, the adjacent layers can fit closely together; but in the second alignment, the adjacent layers can have zero 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 substrate with a lower density of layer packing (e.g., when high permeability is a priority) or a higher density of layer packing (e.g., when maximizing substrate surface area is a priority). According to one or more embodiments, the packing thickness can be: about 50 μm to about 1000 μm, about 100 μm to about 750 μm, about 125 μm to about 600 μm, about 150 μm to about 500 μm, about 200 μm to about 400 μm, about 200 μm to about 300 μm.

[0053] The above structural factors can determine the surface area of ​​the cell culture substrate, whether it is the surface area of ​​a single layer of the cell culture substrate or the surface area of ​​the cell culture substrate having multiple layers of the substrate. For example, in a specific embodiment, a single layer of a woven mesh substrate having a circular shape and a diameter of 6 cm can have a surface area of ​​about 68 cm. 2 As used herein, "effective surface area" is the total surface area of ​​fibers in the portion of the substrate material that is 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 having a diameter of 6 cm may have an effective surface area of ​​about 50 cm 2 To about 90cm 2 , about 53cm 2 To about 81cm 2 , about 68cm 2 , about 75cm 2 , or about 81cm 2 These effective surface area ranges are provided for example only, and some embodiments may have different effective surface areas. Cell culture substrates may also be characterized by porosity, as discussed in the Examples herein.

[0054] The substrate mesh can be made from monofilament or multifilament fibers of polymeric materials compatible with cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinyl pyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. The mesh substrate can have different patterns or weaves, including, for example, knitted, warp-knitted, or woven (e.g., plain, twill, Dutch, five-needle knit).

[0055] The surface chemistry of the mesh threads may need to be modified to provide the desired cell attachment properties. Such modifications may be performed by chemical treatment of the polymer material of the mesh or by grafting cell attachment molecules onto the thread surface. Alternatively, the mesh may be coated with a thin layer of a biocompatible hydrogel with demonstrated cell attachment properties, including, for example, collagen or Alternatively, the surfaces of the wire fibers of the mesh may be imparted with cell attachment properties through treatment processes with various types of plasmas, process gases, and / or chemicals known in the industry. However, in one or more embodiments, the mesh is capable of providing an efficient cell growth surface without surface treatment.

[0056] By using a culture matrix with sufficient rigidity that is limited in structure, high flow resistance uniformity on matrix or packed bed can be achieved. According to various embodiments, matrix can be deployed in single layer or multilayer form. This flexibility eliminates diffusion restriction and provides uniform nutrients and oxygen transfer for the cells attached to matrix. In addition, open matrix lacks any cell capture area in the packed bed structure, and complete cell harvesting with high survival rate is achieved at the end of cultivation. Matrix also transmits packing uniformity for packed bed and realizes direct scalability from process development unit to large-scale industrial bioprocessing unit. The ability to directly harvest cells from packed bed eliminates the demand for matrix to be resuspended in stirring or mechanical oscillation container, which can increase complexity and cause cells to suffer harmful shear stress. In addition, the high packing density of cell culture matrix obtains high bioprocessing production capacity that volume can be managed on industrial scale.

[0057] As used herein, "structurally defined" means that the structure of the substrate conforms to a predetermined design and is not random. Thus, a structurally defined substrate may be a woven design, 3D printed, molded, or formed by some other technique known in the art that allows the structure to conform to a predetermined planned structure.

[0058] Figure 2A An embodiment of a substrate having a multi-layer substrate 200 is shown, and Figure 2B2 is a plan view of the same multi-layer substrate 200. The multi-layer substrate 200 includes a first web substrate layer 202 and a second web substrate layer 204. Although the first and second substrate layers 202 and 204 overlap, the web geometry (e.g., the ratio of opening diameter to fiber diameter) is such that the openings of the first and second substrate layers 202 and 204 overlap and provide a path for fluid to flow through the total thickness of the multi-layer substrate 200, as shown in FIG. Figure 2B As shown in the wire-free opening 206 in FIG.

[0059] Figure 3 show Figure 2B BB in FIG. 2 shows a cross-sectional view of a multilayer substrate 200 at line BB in FIG. Arrow 208 shows a possible fluid flow path through an opening in the second substrate layer 204 and then around the filaments in the first substrate layer 202. The geometry of the web substrate layers is designed to allow efficient and uniform flow through one or more substrate layers. In addition, the structure of the substrate 200 can accommodate fluid flow through the substrate in multiple orientations. For example, Figure 3 As shown, the direction of bulk fluid flow (as indicated by arrows 208) is perpendicular to the major side surfaces of the first and second substrate layers 202 and 204. However, the substrates may also be oriented relative to the flow such that the sides of the substrate layers are parallel to the bulk flow direction. Figure 4 Display edge Figure 3 200, and the structure of the matrix 200 allows fluid flow (arrow 210) through the fluid path in the multilayer substrate 200. In addition to the fluid flow perpendicular to or parallel to the first and second sides of the mesh layer, the matrix can also be arranged with multiple substrates at intermediate angles, or even randomly arranged relative to the fluid flow. This orientation flexibility is achieved by the substantially isotropic flow behavior of the woven substrate. In contrast, substrates for attaching cells in existing bioreactors do not exhibit this behavior, and instead, their packed beds tend to produce preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the matrix of the present disclosure enables it to be used in a variety of applications and bioreactor or container designs while achieving better and more uniform permeability throughout the bioreactor container.

[0060] As discussed herein, according to one or more embodiments, the cell culture substrate can be used in a bioreactor container. For example, the substrate can be used in a packed bed bioreactor configuration, or other configurations within a three-dimensional cell culture chamber. However, embodiments are not limited to three-dimensional culture spaces, and it is contemplated that the substrate can be used in situations where it may be considered a two-dimensional culture surface configuration, wherein one or more layers in the substrate are flat, such as in a flat-bottomed culture dish to provide a culture substrate for cells. For contamination considerations, the container can be a disposable container that can be discarded after use.

[0061] According to one or more embodiments, a cell culture system is provided wherein a cell culture matrix is ​​used in a culture chamber of a bioreactor vessel. Figure 5 An example of a cell culture system 300 is shown, which includes a bioreactor container 302 having a cell culture chamber 304 inside the bioreactor container 302. In the cell culture chamber 304 is a cell culture substrate 306 made of a stack of substrate layers 308. The substrate layers 308 are stacked, with the first or second side of the substrate layer facing the first or second side of the adjacent substrate layer. The bioreactor container 300 has an inlet 310 at one end for inputting medium, cells and / or nutrients into the culture chamber 304, and an outlet 312 at the opposite end for removing medium, cells or cell products from the culture chamber 304. Due to the limited structure and efficient fluid flow through the stacked substrate, by allowing the substrate layers to be stacked in this way, the system can be simply scaled without negatively affecting cell attachment and proliferation. Although the container 300 may generally have an inlet 310 and an outlet 312 as described, some embodiments may use one or both of the inlet 310 and the outlet 312 to simultaneously allow medium, cells or other contents to enter and leave the culture chamber 304. For example, the inlet 310 can be used to allow media or cells to flow into the culture chamber 304 during the cell seeding, perfusion or culture phase, but can also be used during the harvest phase to remove one or more of the media, cells, or cell products through the inlet 310. Therefore, the terms "inlet" and "outlet" are not intended to limit the functions of those openings.

[0062] In one or more embodiments, the flow resistance and volume density of the packed bed can be controlled by inserting substrate layers of different geometric morphologies. Specifically, the mesh size and geometry (e.g., fiber diameter, opening diameter and / or opening geometry) define the resistance to fluid flow in the packed bed format. By inserting meshes of different sizes and geometries, the flow resistance can be controlled or changed in one or more specific parts of the bioreactor. This will achieve better uniformity of liquid perfusion in the packed bed. For example, 10 layers of mesh A (Table 1) followed by 10 layers of mesh B (Table 1) and then 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, followed by 10 layers of mesh B. Such a repetitive pattern can be continued until the entire bioreactor is filled with meshes. These are merely examples and are used for illustrative purposes and are not intended to limit possible combinations. In fact, various combinations of meshes of different sizes can be used to obtain different profiles of volume density and flow resistance of the cell growth surface. For example, a packed bed column with zones of varying volumetric cell density (eg, a series of zones producing a low / high / low / high etc. density pattern) can be assembled by inserting meshes of different sizes.

[0063] exist Figure 5 3. In the example, the bulk flow direction is the direction from the inlet 310 to the outlet 312, and in this example, the first and second major sides of the substrate layer 308 are perpendicular to the bulk flow direction.

[0064] Depending on the desired system, the cell culture substrate can be arranged in the culture chamber with a variety of configurations. For example, in one or more embodiments, the system includes one or more layers of substrate, and the width extends on the width of the restricted cell culture space in the culture chamber. The multilayer substrate can be stacked to a predetermined height in this way. As discussed above, the substrate layer can be arranged so that the first and second sides of the one or more layers are perpendicular to the body flow direction of the culture medium through the restricted culture space in the culture chamber, or the first and second sides of the one or more layers can be parallel to the body flow direction. In one or more embodiments, the cell culture substrate includes one or more substrate layers in a first orientation relative to the body flow, and one or more other layers in a second orientation different from the first orientation. For example, various layers can have a first and second side parallel to or perpendicular to the body flow direction (or in some angles between).

[0065] In one or more embodiments, the cell culture system includes a plurality of discrete sheets of a cell culture substrate in a packed bed configuration, wherein the sheet length and / or width of the substrate is small relative to the culture chamber. As used herein, when the sheet length and / or width of the substrate is approximately 50% or less of the length and / or width of the culture space, it is considered that the sheet length and / or width of the substrate is small relative to the culture chamber. Thus, the cell culture system may include multiple substrates filled into the culture space in a desired arrangement. The arrangement of the substrate sheets may be random or semi-random, or may have a predetermined rule or alignment, such as a sheet orientation that is substantially similar (e.g., horizontal, vertical, or an angle between 0° and 90° relative to the body flow direction).

[0066] As used herein, "defined culture space" refers to the space within the culture chamber that is occupied by the cell culture matrix and in which cells are to be inoculated and / or cultured. The defined culture space can fill approximately the entire culture chamber, or can occupy a portion of the space within the culture chamber. As used herein, "bulk flow direction" is defined as the direction in which the bulk mass of a fluid or culture medium flows through or over the cell culture matrix during cell culture and / or during the flow of the culture medium into or out of the culture chamber.

[0067] In one or more embodiments, the cell culture matrix is ​​fixed in 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 around the mechanism 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 an element passing through the culture chamber, such as an element parallel to the longitudinal axis of the culture chamber, or adheres to an element perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture matrix can be accommodated in the culture chamber without being attached to the wall of the chamber or bioreactor container in a fixed manner. For example, the boundary of other structural elements in the culture chamber or the room can be used to hold the matrix, thereby the matrix is ​​maintained in the predetermined area of ​​the bioreactor container without being fixed to those boundaries or structural elements in a fixed manner.

[0068] By using a cell culture matrix (e.g., a matrix comprising a woven or mesh substrate) according to an embodiment of the present disclosure, an increase in the surface area available for attachment of cells to be attached, propagated, and functionalized is provided for a roll-up bottle container. Specifically, a substrate of a woven mesh of a monofilament polymer material is used in a roll-up bottle, and the surface area can be increased by about 2.4 to about 4.8 times, or about 10 times, compared to a standard roll-up bottle. As discussed herein, each single strand of the mesh substrate can present itself as a 2D surface for attachment of cells to be attached. In addition, a multilayer mesh can be arranged in a roll-up bottle, resulting in a total available surface area increased to about 2 to 20 times that of a standard roll-up bottle. Thus, by adding the improved cell culture matrix disclosed herein, existing roll-up bottle equipment and processing (including cell inoculation, medium exchange, and cell harvesting) can be modified while minimizing the impact on existing operating infrastructure and processing steps.

[0069] The bioreactor container optionally includes one or more outlets that can be attached to an inlet and / or outlet device. Through the one or more outlets, liquid, medium or cells can be supplied to the chamber or removed from the chamber. A single port in the container can play the role of both inlet and outlet, or multiple ports can be provided as special inlet and outlet.

[0070] The packed bed cell culture substrate of one or more embodiments can be composed of a woven cell culture mesh substrate, without any other form of cell culture substrate arranged in the cell culture substrate or interspersed with the cell culture substrate. In other words, the woven cell culture mesh substrate of the embodiment of the present disclosure is an effective cell culture substrate, and does not need to be used for the random non-woven substrate type in the existing scheme. This realizes a cell culture system that simplifies the design and construction, while providing a high-density cell culture substrate with other advantages discussed herein, such as flow uniformity, harvestability, etc.

[0071] As discussed herein, the cell culture substrates and bioreactor systems provided provide many advantages. For example, embodiments of the present disclosure can support the production of any variety of viral vectors (e.g., AAV (all serotypes) and lentivirus), and can be used for in vivo and in vitro gene therapy applications. Uniform cell inoculation and distribution maximize the viral vector yield per container, and the design achieves the harvest of viable cells, which can be useful for inoculation sequences consisting of multiple expansion stages using the same platform. In addition, the embodiments herein can be scalable from process development specifications to production specifications, which ultimately saves development time and cost. The methods and systems disclosed herein also achieve automation and control of cell culture processes to maximize vector yields and improve repeatability. Finally, viral vector production level specifications (e.g., 10 per batch) are achieved. 16 Up to 10 18 The number of vessels required for AAV VG can be greatly reduced compared to other cell culture protocols.

[0072] Embodiments are not limited to containers that rotate around a central longitudinal axis. For example, the container can rotate around an axis that is not centered relative to the container. In addition, the axis of rotation can be a horizontal axis or a vertical axis.

[0073] The present disclosure describes substrates and methods for cutting and punching layers of cell culture substrates (including polymer mesh substrates) to produce detachable sampling sheets. The present disclosure also describes methods and equipment for aseptically removing samples from bioreactors. By sampling during the cell culture process, information about operation can be used to evaluate the quality and performance of the culture process. Cell count can be estimated from the sample, and growth can be monitored by sampling at different times or different positions in the bioreactor. This information can be used to develop and optimize parameters for specific biological processes (such as inoculation sequences and viral vector production). In production, processes that are contaminated or not up to specification can be terminated, thereby reducing the cost of running the process to the end without satisfactory results. For typical biological processes, growth media and lost production time represent significant costs.

[0074] In the embodiments herein, the remainder of the substrate sampling part and the cell culture substrate can be separable with a small active force, thereby allowing to complete sampling manually and not disturbing the main mesh body during the sampling process ideally. For example, a relatively low active force (for example, by manually applying) can cause the sampling part to be separated from the remainder of the substrate via the tension force between the sampling part and the remainder of the substrate that applies the active force. In order to keep the low taking-out active force, a separation boundary can be applied between the sampling part and the remainder of the substrate. This separation boundary can be formed by, for example, marking, punching, laser cutting or other cutting methods (for example, die-cutting), and can be used to produce the layer of the substrate of the separable sheet comprising the substrate that can be taken out from the fixed bed.

[0075] Some embodiments use a woven polymer mesh substrate having woven fibers defining an ordered array of pores or openings. Since each fiber in the mesh is very strong, it is desirable that no fibers run between the detachable sample and the main body of the mesh to facilitate low-force removal of the sample. It is also desirable that the mesh layer be strong when handled during the manufacturing and assembly process for producing the mesh stacked bioreactor bed. To this end, some fibers can be cut in a manner that leaves the woven portion of the mesh connecting the sample to the main mesh body, such as Fig. 7A Because the fibers (which may be formed from various polymers disclosed herein, including PET) are relatively strong, the interwoven fibers may remain attached even though the individual fibers are cut to create the separation boundaries of the sampling portions. Fig. 6A The lines in show the separation boundaries. Figure 6B The sampled portion is shown after it has been separated from the cell culture substrate.

[0076] The mesh layers with sampling tab cuts can be removed from the bioreactor by opening the bioreactor housing and pulling them away from the bed with sterile tools, or they can be removed from the reactor using a sterile sampling port.

[0077] Multiple coupons may be obtained from a single layer. In embodiments employing a woven substrate, for most cut patterns, consideration of the winding direction and the interaction of the weave with the cut pattern is required to maintain structural integrity and achieve ease of removal. Some cut patterns are produced that are less sensitive to the orientation of the web fibers, and these patterns are advantageous for use in manufacturing because precise control of the web orientation is not required.

[0078] Figures 7A-7CAnother embodiment is shown in which the sampling layer contains multiple sampling portions. The shape of the sampling portion includes a rectangular end on the inner side of the sampling portion within the perimeter of the sampling layer and a tapered end on the outer end at the perimeter of the sampling layer. The tapered end allows for easy removal of the sampling portion through a port in the side wall of the bioreactor. The substrate material is such that the size of the port in the side wall can be or slightly larger than the narrow end side, and the wider portion of the sampling portion can fold or bend slightly when it is pulled through the opening in the side wall. Figure 7B A magnified view showing a single sampled portion after separation, and Figure 7C Examples of relative sizes between the sidewall port and the sampling portion in the bioreactor are shown, but in various embodiments, the relative sizes may be varied.

[0079] Figure 8 Embodiments are shown where a sampling layer of substrate has a tie-down molded into the sampling portion so that the tie-down can be pulled to remove the sampling portion. Embodiments include methods of assembling a bioreactor where a substrate layer is added to the housing of the bioreactor until the sampling port elevation is reached. At this point, the sampling layer is inserted into the bioreactor vessel and the tie-down is pulled through the port. Sterile sampling can be accomplished using a sterile container on the exterior of the port.

[0080] Fig.9A A sampling layer is shown with six pie-shaped sampling portions. The number and shape of the sampling portions can be varied. In the case here, the separation boundary laser cuts through the fibers of the woven web substrate. The sampling layer also includes an alignment feature on the left side of the layer, which can be used to hold the sampling layer in a predetermined position so that the sampling portion is in a predetermined position for convenient sampling. The alignment feature can be designed to match a corresponding feature on the interior of the container side wall. Fig. 9B Shown are three pie-shaped sample sections stained to reveal the presence of attached cells on the substrate. Fig. 9C Shows three pie-shaped sampling sections from which cells were sampled after the harvesting process to harvest cells from the substrate. Fig. 9B and 9C , showing the effectiveness of the harvesting process in this example.

[0081] Fig.10 An exploded view of a fixed bed reactor vessel 1000 is shown according to an embodiment of the present disclosure. The vessel 1000 includes a body 1002 ( Fig.101004). The cell culture space 1003 for accommodating the cell culture substrate 1004 is shown as a cylindrical container wall. The cell culture substrate 1004 may include one or more porous materials (such as those disclosed herein) in a stacked or rolled arrangement. Embodiments of the present disclosure include a fixed bed reactor container 1000, which can provide easy sampling of the cell culture substrate 1004 and / or for controlled placement of the cell culture substrate 1004 in the cell culture space 1003. For example, as explained below, the container 1000 can help maintain the position of the cell culture substrate 10004 so that the cell culture substrate 1004 is not offset during operation, or so that the layers of the cell culture substrate 1004 are arranged in a predetermined orientation relative to each other in the cell culture space 1003.

[0082] The container 1000 has a first end 1006 and a second end 1008 on either end of the body 1002, through which media, cells and / or cell byproducts 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 bottom end cap 1010 and a top end cap 1012, respectively, to maintain the sanitary state of the cell culture space. The bottom and top ends 1010, 1012 provide one or more fluid inlets and / or outlets 1011, 1013, respectively, for allowing fluid to flow through the bioreactor container 1000 during operation. An inlet distribution plate 1014 can be used adjacent to the bottom end cap 1010 to evenly distribute the fluid over 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 top end cap 1012 to help collect liquid from passing through the cell culture space 1003 and direct it to the outlet in the top end cap 1012. The use of inlet and outlet distribution plates 1014 and 1016 helps improve the uniformity of flow through cell culture space 1003 by promoting a uniform and even flux of fluid over the cross-sectional area of ​​cell culture space 1003 at the bottom and top of cell culture substrate 1004. Fig.10 The inlet and outlet distribution plates 1014 and 1016 are shown as discrete components relative to the bottom and top end caps 1010 and 1012 , but it is contemplated that the distribution plates 1014 , 1016 may be integrated with the end caps 1010 , 1012 .

[0083] Reactor vessel 1000 may be closed using a variety of closure mechanisms, including one or more of weld seals, adhesives, clamps, and gaskets. For example, Fig.10A sanitary flange 1020 is shown at the top of the cell culture space 1003. The sanitary flange 1020 is matched with a sanitary seal 1022 disposed between the sanitary flange 1020 and the top cover 1012. A sanitary clamp 1024 can securely mate the top cover 1012 with the sanitary flange 1020. An 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., for sampling of cell cultures during use).

[0084] The reactor vessel 1000 further comprises guide rods 1030. The guide rods 1030 extend through the cell culture space 1003 and are used for aligning the cell culture substrate 1004 in the cell culture space 1003 and / or for aligning the layers of the cell culture substrate 1004 with each other. Fig.11 As shown, the cell culture substrate fixed bed 1104 can be bonded to the guide rod 1130 via the substrate alignment feature 1106. The substrate alignment feature 1106 is designed to engage with the rod alignment feature 1136 in the following manner: the fixed bed and / or the individual substrate layer in the fixed bed is maintained in the desired position and / or orientation. For example, in an embodiment in which the fixed bed includes a multilayer stacked substrate material, it may be desirable to make each individual layer have a specific orientation relative to other layers. For example, in the case where the substrate layer with a defined structure of rigid fibers is arranged in a predetermined direction, it may be desirable to change the orientation of the fibers in the layer in the fixed bed. In an embodiment, this may include rotating the layer around the central longitudinal axis of the cell culture space 1003 by a predetermined angle. If adjacent layers are rotated relative to each other, this can control the packing density of the layer by controlling the amount of nesting between adjacent layers. This also affects the porosity and / or pore geometry of the packed bed, which affects the flow of fluid through the fixed bed. For example, in some embodiments, each layer can be rotated about 45° relative to one or two adjacent layers in the stack to improve fluid flow and cell culture performance. For example, the substrate alignment features 1106 may be cutouts in each layer of a fixed bed, wherein the positions of the substrate alignment features 1106 are arranged in a rotational manner about the layer, thereby causing relative rotation of the layers when the multiple layers of substrate alignment features 1106 are aligned. Corresponding rod alignment features 1136 then maintain the alignment of the substrate alignment features 1106, which in turn maintains the individual layers in their desired orientations.

[0085] like Fig.12As shown, guide rods 1230 can be attached to structures (e.g., distribution plates 1214, 1216) on either end of the cell culture substrate 1203, such that the guide rods 1230 confine the cell culture substrate 1203 therein and enable removal of the entire fixed bed from the remainder of the fixed bed reactor containing the vessel wall 1200. This enables easy removal and sampling of the fixed bed without disturbing, for example, the remainder of the fixed bed or disturbing the alignment of the substrate layer. Fig.13 An example of an embodiment is shown in which a sample substrate layer 1232 contained in a fixed bed has one or more perforations 1234 that allow for easy removal of a portion of the substrate 1232. It is contemplated that additional guide rods 1231 or other structural elements may be provided to enhance the structural integrity of the removable substrate core. Embodiments are not limited to Fig.12 and 13 The specific arrangements of guide rods, distribution plates and / or sample screens shown are not intended to be construed as examples of various embodiments to accomplish sampling from a reactor vessel.

[0086] definition

[0087] "All-synthetic" or "completely synthetic" refers to a cell culture article that is composed entirely of synthetic-derived materials and does not contain any animal-derived or animal-derived materials, such as microcarriers or culture vessel surfaces. The disclosed all-synthetic cell culture articles eliminate the risk of xenogeneic contamination.

[0088] The terms "include", "including" or similar terms mean including but not limited to, which is inclusive and not exclusive.

[0089] "User" refers to those who use the systems, methods, articles, or kits disclosed herein, including those who culture cells to harvest cells or cell products, or those who use cells or cell products cultured and / or harvested according to the embodiments herein.

[0090] In the embodiments described herein, "about" used to modify, for example, the amount, concentration, volume, processing temperature, processing time, yield, flow rate, pressure, viscosity and similar values ​​and ranges of components in the composition or the size of the component and similar values ​​and ranges thereof refers to changes in the numerical amount that may occur, for example, from the conventional measurement and operating processes used to prepare materials, compositions, complexes, concentrates, component parts, products or use formulations; from accidental errors in these processes; from differences in the manufacture, source or purity of the starting materials or ingredients used to implement the method; and similar factors. The term "about" also includes amounts that differ due to aging of a composition or formulation with a specific initial concentration or mixture, and amounts that differ due to mixing or processing a composition or formulation with a specific initial concentration or mixture.

[0091] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0092] As used herein, the indefinite article "a" or "an" and the corresponding definite article "the" mean at least one, or one or more, unless stated otherwise.

[0093] Abbreviations familiar to those skilled in the art may be used (e.g., "h" or "hr" for hours, "g" or "gm" for grams, "mL" for milliliters, "rt" for room temperature, "nm" for nanometers, and the like).

[0094] The specific and preferred numerical values ​​and ranges disclosed in components, ingredients, additives, dimensions, conditions and the like are only for illustration, and they do not exclude other limited numerical values ​​or other numerical values ​​within the limited range. The systems, kits and methods of the present disclosure may include any numerical value or any combination of numerical values, specific numerical values, more specific numerical values ​​and preferred numerical values ​​described herein, including explicit or implicit intermediate values ​​and ranges.

[0095] Unless otherwise stated, it is not intended that any method described herein be construed as requiring that its steps be performed in a specific order. Therefore, when a method claim does not actually state that its steps follow a certain order or it does not specifically indicate in any other way in the claims or description that the steps are limited to a specific order, it is not intended to imply any particular order.

[0096] It will be apparent to those skilled in the art that various modifications and variations may be made without departing from the spirit or scope of the illustrated embodiments. Because those skilled in the art may conceive of various modifications, combinations, sub-combinations, and variations of the disclosed embodiments that incorporate the spirit and substance of the embodiments, the disclosed embodiments should be considered to include all of the contents within the scope of the appended claims and their equivalents.

Claims

1. A fixed bed bioreactor for culturing cells on a cell culture substrate, the fixed bed bioreactor comprising: A cell culture container having: a container body defining at least one internal reservoir, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir, the container body including a first end, a second end, and a longitudinal axis extending in a direction from the first end to the second end; as well as a guide rod arranged in the inner reservoir and extending parallel to the longitudinal axis of the container body, Therein, the guide rods are configured to hold the cell culture substrate fixed in the internal reservoir.

2. The fixed bed bioreactor according to claim 1, wherein: The internal reservoir is configured to hold the multi-layered cell culture substrate.

3. The fixed bed bioreactor of claim 1, further comprising a cell culture substrate disposed in the internal reservoir.

4. The fixed bed bioreactor according to claim 3, wherein: The cell culture substrate is a multi-layered cell culture substrate.

5. The fixed bed bioreactor according to claim 4, wherein: The guide rods are configured to maintain rotational alignment of individual layers of the multi-layer cell culture substrate with other individual layers of the multi-layer cell culture substrate.

6. A fixed bed bioreactor as claimed in any one of the preceding claims, wherein: The guide rod includes an outer surface defining a guide rod cross-sectional profile configured to engage a cell culture substrate.

7. The fixed bed bioreactor according to claim 6, wherein: The cell culture substrate includes a cutting portion configured to match the cross-sectional profile of the guide rod or at least partially surround the cross-sectional profile of the guide rod.

8. A fixed bed bioreactor according to any one of the preceding claims, further comprising: an inlet distribution plate disposed between the inlet and the internal reservoir; as well as An outlet distribution plate is disposed between the outlet and the internal reservoir.

9. The fixed bed bioreactor according to claim 8, wherein: The guide rods are attached to the inlet distribution plate.

10. The fixed bed bioreactor according to claim 9, wherein: The second end of the container body is at the opposite end of the internal reservoir relative to the inlet distribution plate, Wherein, the guide rod and the inlet distribution plate are removable from the container body through the second end of the container body.

11. The fixed bed bioreactor according to claim 9 or 10, wherein: The guide rods are attached to the outlet distribution plate.

12. The fixed bed bioreactor according to any one of claims 9 to 11, wherein: The cell culture substrate at least partially defined by the inlet distribution plate and the guide rods is removable from the container body.

13. A fixed bed bioreactor as claimed in any one of the preceding claims, wherein: The cell culture substrate comprises a structurally defined surface for culturing cells thereon, the structurally defined surface defining an ordered and regular array of openings through the thickness of the cell culture substrate.

14. A fixed bed bioreactor as claimed in any one of the preceding claims, wherein: at least a portion of the cell culture substrate comprises a sampling substrate defined by a separation boundary between the sampling substrate and the remainder of the cell culture substrate, and The separation boundary is configured to separate the sampling substrate from the rest of the cell culture substrate.

15. The fixed bed bioreactor according to claim 14, wherein: The separation boundary comprises at least one of a perforation in the cell culture substrate, a cut in or through the cell culture substrate, or a localized thinned portion of the cell culture substrate.

16. The fixed bed bioreactor according to any one of claims 13 to 15, wherein: The structured surface includes one or more fibers.

17. The fixed bed bioreactor according to claim 16, wherein: The one or more fibers of the layer are woven together in an ordered arrangement.

18. A fixed bed bioreactor as claimed in any one of the preceding claims, wherein The reservoir is defined by a length and a width, the length extending from a first end of the reservoir adjacent the inlet to a second end of the reservoir adjacent the outlet, the cell culture matrix having a width extending substantially across the width of the reservoir.

19. A fixed bed bioreactor as claimed in any one of the preceding claims, wherein The fixed bed bioreactor is configured for aseptic removal of sampled portions from the cell culture vessel.

Images