Fabric cell culture medium

Abstract

To provide a substrate for culturing cells, and a system and method for culturing cells.SOLUTION: A cell culture matrix is provided that has a substrate with a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and passing through the thickness of the substrate. The plurality of openings allows a flow of at least one of cell culture media, cells, and cell products through the thickness of the substrate, and provides a uniform, efficient, and scalable matrix for cell seeding, proliferation, and culturing. The substrate can be formed from a woven polymer mesh material that provides a high surface area to volume ratio for cells and a good fluid flow through the matrix. Bioreactor systems incorporating the cell culture matrix and related methods are also provided.SELECTED DRAWING: Figure 5

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §120 to U.S. Provisional Patent Application No. 62 / 910,696, filed October 4, 2019, and U.S. Provisional Patent Application No. 62 / 801,325, filed February 5, 2019, the contents of which are relied upon and incorporated by reference in their entireties into this application. [Technical Field]

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to substrates for culturing cells, and systems and methods for culturing cells. In particular, the disclosure relates to cell culture substrates, bioreactor systems incorporating the substrates, and methods for culturing cells using the substrates. [Background technology]

[0003] The bioprocessing industry performs large-scale cell culture for the production of hormones, enzymes, antibodies, vaccines, and cell therapies. The cell and gene therapy market is growing rapidly, moving promising treatments into clinical trials and quickly toward commercialization. However, a single cell therapy can require billions of cells and trillions of viruses. Therefore, the ability to rapidly deliver large quantities of cell products is critical for clinical success.

[0004] The majority of cells used in bioprocessing are anchorage-dependent, meaning that they require a surface to attach to in order to grow and function. Traditionally, adherent cell culture is performed on two-dimensional (2D) cell-attachment surfaces incorporated into one of a number of vessel formats, such as T-flasks, Petri dishes, cell factories, CellStack vessels, roller bottles, and HYPERStack® vessels. These approaches can have significant drawbacks, including difficulty achieving cell densities high enough to enable large-scale manufacturing of therapies or cells.

[0005] Alternative methods have been proposed to increase the volumetric density of cultured cells. These include microcarrier cultures carried out in stirred tanks. In this approach, cells attached to the surface of microcarriers are subjected to constant shear stress, which significantly impacts growth and culture performance. Another example of a high-density cell culture system is the hollow fiber bioreactor, where cells can form large, three-dimensional aggregates as they grow within the spaces between the fibers. However, cell growth and performance are significantly hindered by a lack of nutrients. To alleviate this problem, these bioreactors have been miniaturized and are not suitable for large-scale manufacturing.

[0006] Another example of a high-density culture system for anchorage-dependent cells is the packed-bed bioreactor system. In this type of bioreactor, a cell substrate is used to provide a surface for the attachment of adherent cells. Culture medium is perfused along the surface or through a semi-porous substrate, thereby providing the nutrients and oxygen necessary for cell growth. For example, packed-bed bioreactor systems incorporating a packed bed of a support or matrix system for cell entrapment have already been disclosed in Patent Documents 1-3. The packed-bed matrix is ​​typically made of porous particles or nonwoven polymeric microfibers as a substrate. Such bioreactors function as recirculating flow-through bioreactors. One of the key issues with such bioreactors is the uneven distribution of cells within the packed bed. For example, the packed bed acts as a depth filter, trapping cells primarily in the inlet region, creating a gradient in cell distribution during the seeding step. Furthermore, the random fiber packing results in non-uniform flow resistance and cell entrapment efficiency across the cross section of the packed bed. For example, culture medium flows faster through regions with low cell packing density and slower through regions with high resistance due to the large number of trapped cells. This creates a channeling effect in which nutrients and oxygen are efficiently delivered to areas of low cell volume density, while areas of high cell density are maintained in suboptimal culture conditions.

[0007] Another significant drawback of packed-bed systems disclosed in the prior art is the inability to efficiently harvest intact, viable cells at the end of the culture process. Cell harvesting is important when cells are the final product, or when the bioreactor is used as part of a "seed train," in which a cell population is grown in one vessel and then transferred to another vessel for further population growth. U.S. Patent No. 6,299,499 discloses a bioreactor design to improve the efficiency of cell recovery from the packed bed during the cell harvesting step. This is based on loosening the packed-bed matrix and shaking or agitating the packed-bed particles to cause collisions with the porous matrix, thereby detaching the cells. However, this approach is tedious and can cause significant cell damage, reducing overall cell viability.

[0008] One example of a currently commercially available packed-bed bioreactor is the iCellis® manufactured by Pall Corporation. iCellis uses small strips of cell substrate material consisting of randomly oriented fibers in a nonwoven configuration. These strips are packed into a vessel to create a packed bed. However, like similar solutions on the market, this type of packed-bed substrate has drawbacks. Specifically, uneven packing of the substrate strips creates visible channels within the packed bed, which leads to preferential and uneven media flow and nutrient distribution through the packed bed. Studies of iCellis have described "an overall heterogeneous distribution of cells, with cell numbers increasing from the top to the bottom of the fixed bed," as well as "nutrient gradients, which lead to limited cell growth and production," all of which lead to "unequal cell distribution, which can compromise transfection efficiency" (NPL 1). Studies have also noted that rocking the packed bed can improve dispersion but has other drawbacks (i.e., "rocking, necessary for better dispersion during inoculation and transfection, can induce increased shear stress, which leads to reduced cell viability"; ibid.). Another study, with respect to iCellis, noted that uneven cell distribution makes it difficult to monitor the cell population using biomass sensors ("...if the cells are unevenly distributed, the biomass signal from the cells in the top carrier may not provide a complete picture of the bioreactor"; non-patent document 2).

[0009] Furthermore, the random arrangement of fibers within the substrate strip and the variability in strip packing from one iCellis packed layer to another can make it difficult for customers to predict cell culture performance due to substrate variability from culture to culture. Additionally, the packed iCellis substrate makes efficient cell harvesting extremely difficult or impossible, as cells are believed to become trapped in the packed layer.

[0010] Roller bottles have several advantages, such as ease of handling and the ability to monitor cells on an adherent surface. However, from a manufacturing perspective, a major drawback is the low surface area / volume ratio, even though the roller bottle configuration occupies a large amount of manufacturing floor space. Various approaches have been used to increase the surface area available for adherent cells in roller bottle formats. While some solutions have been implemented in commercial products, there is room for improvement to further increase roller bottle productivity. Traditionally, roller bottles are manufactured as a single structure by a blow molding process. This simplicity of manufacturing makes roller bottles economically viable in the bioprocessing industry. While some roller bottle modifications to increase the surface area available for cell culture can be achieved without changing the manufacturing process, the resulting surface area increase is minimal. Other modifications to roller bottle designs significantly complicate the manufacturing process, making roller bottles less economically viable in the bioprocessing industry. Therefore, it would be desirable to provide roller bottles with increased surface area and bioprocessing productivity while using the same blow molding process to manufacture roller bottles.

[0011] While existing platforms are capable of producing viral vectors for early-stage clinical trials, reaching late-stage commercial manufacturing scale requires platforms capable of producing larger numbers of high-quality products. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent No. 4,833,083 [Patent Document 2] U.S. Patent No. 5,501,971 [Patent Document 3] U.S. Patent No. 5,510,262 [Patent Document 4] U.S. Patent No. 9,273,278 [Non-patent literature]

[0013] [Non-Patent Document 1] Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells. Biotechnol. J. 2016, 11, 290-297 [Non-patent document 2] Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale. Human Gene Therapy, Vol. 26, No. 8, 2015 Summary of the Invention [Problem to be solved by the invention]

[0014] There is a need for cell culture matrices, systems, and methods that allow for the cultivation of cells in a high density format with uniform cell distribution, ease of harvest, and increased yield. [Means for solving the problem]

[0015] According to an embodiment of the present disclosure, a cell culture matrix is ​​provided. The cell culture matrix includes a substrate, the substrate comprising: a first surface ;The first one above surface The second opposite surface ;The first one above surface and the second one above. surfaceand a plurality of openings formed in the substrate and passing through the thickness of the substrate. The plurality of openings are configured to allow the flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate. The substrate can be at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh sheet. The substrate has a regular, ordered structure and provides a surface for cell attachment, growth, and eventual cell release.

[0016] According to one embodiment of the present disclosure, a bioreactor system for cell culture is provided, the system including: a cell culture vessel having at least one reservoir; and a cell culture matrix disposed within the at least one reservoir, the cell culture matrix including a woven substrate having a plurality of interwoven fibers having a surface configured for cell attachment.

[0017] According to one or more embodiments, a cell culture system is provided, the system including: a bioreactor vessel; and a cell culture matrix disposed within the bioreactor vessel and configured to culture cells. The cell culture matrix includes a substrate having: a first side; a second side opposite the first side; a thickness separating the first side and the second side; and a plurality of openings formed in the substrate and passing through the thickness of the substrate, the plurality of openings configured to allow flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate.

[0018] According to one or more embodiments, a bioreactor system for culturing cells is provided. The system includes: a cell culture vessel having a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed within the at least one reservoir. The cell culture matrix has a plurality of woven fabric substrates, each of the woven fabric substrates including a plurality of interwoven fibers having a surface configured for cell attachment. The bioreactor system is configured to flow material through the at least one reservoir in a flow direction from the first end to the second end, and the plurality of woven fabric substrates are stacked so that each woven fabric substrate is substantially parallel to each other woven fabric substrate and substantially perpendicular to the flow direction.

[0019] According to one or more embodiments, a bioreactor system for culturing cells is provided. The system includes: a cell culture vessel having a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed within the at least one reservoir, the cell culture matrix including a plurality of woven fabric substrates, each of the woven fabric substrates having a plurality of interwoven fibers with a surface configured for cell attachment. The bioreactor system is configured to flow material through the at least one reservoir in a flow direction from the first end to the second end, and the plurality of woven fabric substrates are stacked so that each woven fabric substrate is generally parallel to each other woven fabric substrate and generally parallel to the flow direction.

[0020] According to one or more embodiments, a bioreactor system for culturing cells is provided. The system includes: a cell culture vessel having a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed within the at least one reservoir. The cell culture matrix includes a woven substrate comprising a plurality of interwoven fibers having a surface configured for cell attachment, the woven substrate being disposed within the at least one reservoir in a rolled configuration, thereby providing a cylindrical cell culture matrix, with a surface of the woven substrate parallel to a longitudinal axis of the cylindrical cell culture matrix.

[0021] According to another embodiment, a method for culturing cells in a bioreactor is provided. The method includes providing a bioreactor vessel having a cell culture chamber therein and a cell culture matrix disposed in the cell culture chamber. The cell culture matrix is ​​provided for culturing cells on the cell culture matrix. The cell culture matrix includes a substrate having: a first side; a second side opposite the first side; a thickness separating the first side and the second side; and a plurality of openings formed in the substrate and passing through the thickness of the substrate. The method further includes: seeding cells on the cell culture matrix; culturing the cells on the cell culture matrix; and harvesting a product of the culturing of the cells. The plurality of openings in the substrate allow the flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate. [Brief explanation of the drawings]

[0022] [Figure 1A] 1 is a perspective view of a three-dimensional model of a cell culture substrate according to one or more embodiments of the present disclosure. [Figure 1B] 2D plan view of the substrate of FIG. 1A [Figure 1C] 1B is a cross-sectional view of the substrate taken along the line A-A of FIG. [Figure 2A]Examples of cell culture substrates according to some embodiments [Figure 2B] Examples of cell culture substrates according to some embodiments [Figure 2C] Examples of cell culture substrates according to some embodiments [Figure 3A] 1 is a perspective view of a multi-layer cell culture substrate according to one or more embodiments. [Figure 3B] FIG. 1 is a plan view of a multi-layer cell culture substrate according to one or more embodiments. [Figure 4] 3C is a cross-sectional view of the multi-layer cell culture substrate of FIG. 3B along line B-B, according to one or more embodiments. [Figure 5] 5 is a cross-sectional view of the multi-layer cell culture substrate of FIG. 4 along line CC, according to one or more embodiments. [Figure 6] 1 is a schematic diagram of a cell culture system according to one or more embodiments. [Figure 7] 1 is a schematic diagram of a cell culture system according to one or more embodiments. [Figure 8] A rolled cylindrical configuration of the cell culture matrix according to one or more embodiments. [Figure 9] Cell culture system incorporating a cell culture matrix in a rolled cylindrical configuration according to one or more embodiments [Figure 10A] 1 is a schematic diagram of a cell culture system according to one or more embodiments. [Figure 10B] 1 is a detailed schematic diagram of a cell culture system according to one or more embodiments. [Figure 11A] 1 is a process flow chart for culturing cells in a cell culture system, according to one or more embodiments. [Figure 11B] OPERATIONS FOR CONTROLLING PERFUSION FLOW RATE OF A CELL CULTURE SYSTEM IN ACCORDANCE WITH ONE OR MORE EMBODIMENTS [Figure 12] Micrograph of stained HEK293T cells on a cell culture substrate, according to one or more embodiments. [Figure 13A] Graph showing cell growth and proliferation data for HEK293T cells on the substrates of FIG. 12 [Figure 13B] Bar graph showing cell viability from Figures 12 and 13A [Figure 14A]Photograph of a layer of cell culture substrate containing stained HEK293T cells from a bioreactor seeded under static conditions. [Figure 14B] Photograph of a layer of cell culture substrate containing stained HEK293T cells from a bioreactor seeded with the cell tumbling method. [Figure 15] 1 is a schematic cross-sectional view of a cell culture system including a spread cell culture substrate, according to one or more embodiments. [Figure 16] FIG. 1 is a cross-sectional view of a roller bottle-style cell culture vessel including a multi-layer cell culture substrate, according to one or more embodiments. [Figure 17A] Photograph of a cell culture substrate with stained cells after seeding in a roller bottle style cell culture system using low rotation speed during cell seeding, according to one or more embodiments. [Figure 17B] Photograph of a cell culture substrate with stained cells after seeding in a roller bottle style cell culture system using a relatively high rotation speed during cell seeding, according to one or more embodiments. [Figure 18A] Photograph of a disc from a cell culture matrix containing stained cells after seeding and growth but prior to harvesting of the cells, according to one or more embodiments. [Figure 18B] Photograph of the disk from FIG. 18A after cell harvesting, according to one or more embodiments. [Figure 19A] Experimental results of whole cell harvest for two examples according to embodiments of the present disclosure compared to the HYPERflask [Figure 19B] Experimental results of total genome copies per vessel for two examples according to embodiments of the present disclosure compared to HYPERflask [Figure 19C] Experimental results of genome copies per surface area for two examples according to embodiments of the present disclosure compared to HYPERflask [Figure 20A] FIG. 1 is a plan view of a modeled multilayer woven mesh cell culture substrate in a close-packed configuration, according to one or more embodiments of the present disclosure. [Figure 20B]FIG. 20B is a side cross-sectional view of the multilayer woven mesh cell culture substrate of FIG. 20A in accordance with one or more embodiments of the present disclosure. [Figure 21A] FIG. 1 is a top view of a modeled multilayer woven mesh cell culture substrate in a loosely packed configuration, according to one or more embodiments of the present disclosure. [Figure 21B] FIG. 21B is a side cross-sectional view of the multilayer woven mesh cell culture substrate of FIG. 21A in accordance with one or more embodiments of the present disclosure. [Figure 22A] Modeled empty space within the dotted volume shown in Figures 20A and 20B [Figure 22B] Modeled empty space within the dotted volume shown in Figures 21A and 21B [Figure 23] Photographs of various mesh samples A-F from Table 5, according to one or more embodiments of the present disclosure. [Figure 24] Bar graph of transmittance of woven mesh samples A to F from Figure 23 [Figure 25] Results of pressure drop tests using specimens A to C from Figure 23 [Figure 26] Schematic of a seed train process according to one or more embodiments of the present disclosure. [Figure 27A] Flow uniformity model for a bioreactor using a woven mesh substrate, according to one or more embodiments of the present disclosure. [Figure 27B] Close-up of the flow uniformity model in Figure 27A [Figure 28] Bar graph of measured permeability of woven and nonwoven cell culture substrates [Figure 29A] Simulated flow velocity around a piece of nonwoven mesh substrate aligned at 90° to the flow direction [Figure 29B] Simulated flow velocity around a piece of nonwoven mesh substrate aligned at 45° to the flow direction [Figure 30A] Simulated flow velocity around a piece of nonwoven mesh substrate with 1 mm gaps between all neighboring substrate pieces [Figure 30B] Simulated flow velocity around a coarse woven mesh with 1 mm gaps between all neighboring substrate pieces [Figure 31] Schematic of the experimental setup for measuring the residence time distribution of different cell culture substrate samples. [Figure 32] Graph showing the change in dye concentration versus time during residence time distribution measurements for woven and nonwoven cell culture substrates. DETAILED DESCRIPTION OF THE INVENTION

[0023] Various embodiments of the present disclosure will now be described in detail, with reference to the drawings, where present. Reference to various embodiments does not limit the scope of the invention, which is limited only by the appended claims. Moreover, any examples set forth herein are not limiting, but merely illustrate some of the many possible embodiments of the claimed invention.

[0024] SUMMARY OF THE DISCLOSURE Embodiments of the present disclosure relate to: cell culture substrates; and cell culture or bioreactor systems incorporating the substrates; and methods of culturing cells using the substrates and bioreactor systems.

[0025] Various types of packed-bed bioreactors have been used in conventional large-scale cell culture bioreactors. These packed beds typically contain a porous matrix to retain attached or suspended cells and support their growth and proliferation. The packed bed matrix provides a high surface area / volume ratio, thus enabling higher cell densities than other systems. However, packed beds often function as depth filters, with cells becoming physically trapped or trapped within the fibers of the matrix. Thus, linear flow of cell inoculum through the packed bed results in non-uniform cell distribution within the packed bed, leading to variations in cell density across the depth or width of the packed bed. For example, cell density can be high in the inlet region of the bioreactor and significantly lower near the outlet of the bioreactor. This non-uniform distribution of cells within the packed bed significantly hinders the scalability and predictability of such bioreactors in bioprocess manufacturing and may even result in reduced efficiency per unit area or volume of the packed bed for cell growth or viral vector production.

[0026] Another problem encountered with packed-bed bioreactors disclosed in the prior art is the channeling 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 not uniform. Culture medium flows rapidly through regions of low fiber density (high packed-bed permeability) and much more slowly through regions of high fiber density (low packed-bed permeability). The resulting uneven medium perfusion across the packed bed creates a channeling effect, which manifests as significant nutrient and metabolite gradients, adversely affecting overall cell culture and bioreactor performance. Cells located in areas of low medium perfusion often starve and die from nutrient deprivation or metabolite poisoning. Cell harvesting is yet another problem encountered when using bioreactors packed with nonwoven fibrous scaffolds. Because the packed bed acts as a depth filter, cells released at the end of the cell culture process are trapped within the packed bed, resulting in extremely low cell recovery rates. This limits the use of such bioreactors in bioprocesses where live cells are the product. Thus, the non-uniformity results in areas with different exposure to flow and shear, which effectively reduces the usable cell culture area, causing non-uniform cultures and hindering transfection efficiency and cell release.

[0027] To address these and other problems with 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, high-yield cell culture for anchorage-dependent cells and production of cell products (e.g., proteins, antibodies, viral particles). Embodiments include porous cell culture matrices fabricated from an ordered, regular array of porous substrate material that allows for uniform cell seeding and media / nutrient perfusion, as well as efficient cell harvest. Embodiments also enable scalable cell culture solutions from process development scale to full production scale using substrates and bioreactors that allow for cell seeding and growth and / or cell product harvest without sacrificing uniform performance of the embodiments. For example, in some embodiments, bioreactors can be easily scaled from process development scale to production scale, and across this production scale, the number of viral genomes per unit surface area of ​​the substrate (VG / cm) can be increased. 2 ) are equivalent. The embodiments described herein are easily harvested and scalable, allowing them to be used in efficient seed trains for growing cell populations at multiple scales on the same cell substrate. Additionally, the embodiments described herein provide cell culture matrices with large surface areas, which, along with other features described herein, enable high-yield cell culture solutions. In some embodiments, for example, the cell culture substrates and / or bioreactors described herein can grow up to 10 cells per batch. 16 ~10 18 It can produce 10 viral genomes (VG).

[0028] In one embodiment, the matrix provides a structurally defined surface area for cell attachment and growth, has good mechanical strength, and forms a highly uniform, interconnected, and numerous fluidic networks when assembled into a packed-bed or other bioreactor. In certain embodiments, a mechanically stable, non-degradable woven mesh can be used as a substrate to support the production of adherent cells. The cell culture matrices disclosed herein support the attachment and growth of anchorage-dependent cells in a high-volume density format. Uniform cell seeding of such matrices is achievable, as is efficient harvesting of cells or other bioreactor products. Furthermore, embodiments of the present disclosure provide uniform cell distribution during the seeding step to support cell culture to achieve a confluent monolayer or multilayer of adherent cells on the disclosed matrices, while avoiding the formation of large and / or uncontrollable 3D cell aggregates, which can result in nutrient diffusion limitations and elevated metabolite concentrations. Thus, the matrix eliminates diffusion limitations during bioreactor operation. Furthermore, the matrix allows for easy and efficient harvesting of cells from the bioreactor. The structurally defined matrix of one or more embodiments allows for complete cell recovery and consistent cell harvest from the packed bed of the bioreactor.

[0029] According to some embodiments, there is also provided a method of cell culture using a bioreactor with a matrix for bioprocessing production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0030] In contrast to existing cell culture substrates used in cell culture bioreactors (i.e., nonwoven substrates with randomly arranged fibers), embodiments of the present disclosure include cell culture substrates with a defined and ordered structure. This defined and ordered structure allows for consistent and predictable cell culture results. Furthermore, the substrate has an open, porous structure, which prevents cell entrapment and allows for uniform flow through the packed bed. This structure allows for improved cell seeding, nutrient delivery, cell growth, and cell harvest. According to one or more specific embodiments, the matrix is ​​formed of a substrate material with a thin, sheet-like structure, which comprises first, second, and third layers separated by a relatively small thickness. surface and the second surface and therefore the thickness of the sheet is determined by the first and second thicknesses of the substrate. surface The matrix is ​​small relative to the width and / or length of the substrate. Additionally, multiple holes or openings are formed through the thickness of the substrate. The substrate material between the openings is sized and shaped to allow cells to adhere to the surface of the substrate material as if it were a substantially two-dimensional (2D) surface, while allowing adequate fluid flow around and through the openings. In some embodiments, the substrate is a polymer-based material and can be formed as: a molded polymer sheet; a polymer sheet with openings punched through its thickness; multiple filaments fused together into a mesh-like layer; a 3D-printed substrate; or multiple filaments woven into a mesh layer. The physical structure of the matrix has a high surface-to-volume ratio for the cultivation of anchorage-dependent cells. According to various embodiments, the matrix can be disposed or packed into a bioreactor in the specific manner described herein for uniform cell seeding and growth, uniform medium perfusion, and efficient cell harvesting.

[0031] An embodiment of the present disclosure is a method for producing approximately 10 14 More than 10 viral genomes per batch 15 More than 10 viral genomes per batch 16 More than 10 viral genomes per batch 17More than 10 viral genomes, or approximately g10 per batch 16 A practically sized viral vector platform capable of producing viral genomes on the scale of more than 10 viral genomes can be achieved. In some embodiments, production is achieved at a rate of about 10 per batch. 15 ~about 10 18 For example, in some embodiments, the yield of viral genomes is about 10 per batch. 15 ~about 10 16 viral genomes, or approximately 10 per batch 16 ~about 10 19 viral genomes, or approximately 10 per batch 16 ~10 18 viral genomes, or approximately 10 per batch 17 ~about 10 19 viral genomes, or approximately 10 per batch 18 ~about 10 19 viral genomes, or approximately 10 per batch 18 It may be one or more viral genomes.

[0032] Furthermore, embodiments of the present disclosure enable not only cell attachment and growth on cell culture substrates, but also viable harvesting of cultured cells. The inability to harvest viable cells is a significant drawback of current platforms, making it difficult to establish and maintain sufficient numbers of cells for production capacity. According to certain aspects of embodiments of the present disclosure, viable cells can be harvested from cell culture substrates with viability of 80% to 100%, or about 85% to about 99%, or about 90% to about 99%. For example, harvested cells may be at least 80% viable, at least 85% viable, at least 90% viable, at least 91% viable, at least 92% viable, at least 93% viable, at least 94% viable, at least 95% viable, at least 96% viable, at least 97% viable, at least 98% viable, or at least 99% viable. Cells can be released from cell culture substrates using, for example, trypsin, TrypLE, or Accutase.

[0033] 1A and 1B show a three-dimensional (3D) perspective view and a two-dimensional (2D) plan view, respectively, of a cell culture substrate 100 according to one or more embodiments of the present disclosure. The cell culture substrate 100 is a woven mesh layer composed of a first plurality of fibers 102 extending in a first direction and a second plurality of fibers 104 extending in a second direction. The woven fibers of the substrate 100 form a plurality of apertures 106, which may be defined by one or more widths or diameters (e.g., D1, D2). The size and shape of the apertures can vary based on the type of weave (e.g., number, shape, and size of filaments; angle between intersecting filaments, etc.). At the microscale, a woven mesh can be characterized as a two-dimensional sheet or layer. However, closer inspection of the woven mesh reveals a three-dimensional structure due to the intersecting fibers of the mesh. Thus, as shown in FIG. 1C, the thickness T of the woven mesh 100 can be greater than the thickness of a single fiber (e.g., t1). As used herein, thickness T is the maximum thickness of the woven mesh between first side 108 and second side 110. Without wishing to be bound by theory, it is believed that the three-dimensional structure of substrate 100 is advantageous because it provides a large surface area for the cultivation of adherent cells and the structural rigidity of the mesh provides a consistent and predictable cell culture matrix structure that allows for uniform fluid flow.

[0034] In FIG. 1B , aperture 106 has a diameter D1, defined as the distance between opposing fibers 102, and a diameter D2, defined as the distance between opposing fibers 104. D1 and D2 may or may not be equal, depending on the weave geometry. When D1 and D2 are unequal, the larger may be referred to as the major diameter and the smaller may be referred to as the minor diameter. In some embodiments, the diameter of an aperture may refer to the widest portion of the aperture. Unless otherwise specified, aperture diameter as used herein will refer to the distance between parallel fibers on opposite sides of an aperture.

[0035] 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. For fibers having a circular cross-section, such as that shown in FIG. 1A, or other three-dimensional cross-section, thicknesses t1 and t2 are the maximum diameter or thickness 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. Furthermore, t1 and t2 may be equal. However, in one or more embodiments, t1 and t2 may not be equal, such as when the plurality of fibers 102 is different from the plurality of fibers 104. Furthermore, the plurality of fibers 102 and the plurality of fibers 104 may each have two or more different thicknesses (e.g., t 1a , t 1b etc., and t 2a , t 2b The mesh may include fibers of various thicknesses (e.g., 1 / 2, 1 / 4 ...

[0036] In one or more embodiments, the fibers may have a diameter 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. At the microscale level, the scale of the fibers relative to cells (e.g., the fiber diameter is larger than that of cells) allows the surface of the monofilament fibers to present an approximate 2D surface for adherent cells to attach and grow on. The fibers can be woven into a mesh with openings of about 100 μm x 100 μm to about 1000 μm x 1000 μm. In some embodiments, the apertures may have a diameter 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 diameter and aperture diameter are examples of some embodiments and are not intended to limit the size of possible features in meshes according to all embodiments. The combination of fiber diameter and aperture diameter is selected to provide efficient and uniform fluid flow through the substrate, for example, when the cell culture matrix includes multiple adjacent mesh layers (e.g., a stack of individual layers or a single wound mesh layer).

[0037] Factors such as fiber diameter, aperture diameter, and weave type / pattern will determine the surface area available for cell attachment and growth. Furthermore, when a cell culture matrix includes a stack, roll, or other configuration of overlapping substrates, the packing density of the cell culture matrix will affect the surface area of ​​the packed layer matrix. Packing density can vary depending on the packing thickness of the substrate material (e.g., the space required for one layer of substrate). For example, if a stack of cell culture matrices has a certain height, each layer of the stack can be said to have a packing thickness determined by dividing the overall height of the stack by the number of layers in the stack. Packing thickness varies based on fiber diameter and weave, but can also vary based on the alignment of adjacent layers within the stack. For example, due to the three-dimensional nature of woven layers, there is a certain amount of interconnection or overlap that adjacent layers can accommodate based on their alignment with each other. In one alignment, adjacent layers can be closely spaced, while in a second alignment, adjacent layers may have zero overlap, such as when the lowest point of the upper layer is directly in contact with the highest point of the lower layer. In certain applications, it may be desirable to provide a cell culture matrix with a low layer packing density (e.g., when high permeability is a priority) or a high layer packing density (e.g., when maximizing the surface area of ​​the substrate 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; or about 200 μm to about 300 μm.

[0038] The structural factors discussed above can determine the surface area of ​​a cell culture matrix (whether it is a single layer of cell culture substrate or a cell culture matrix with multiple layers of substrate). For example, in one particular embodiment, a single layer of woven mesh substrate with a circular shape and a 6 cm diameter has a surface area of ​​approximately 68 cm. 2As used herein, "effective surface area" 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 6 cm diameter has an effective surface area of: about 50 cm 2 ~about 90cm 2 ; approx. 53cm 2 ~Approx. 81cm 2 ; approx. 68cm 2 ; approx. 75cm 2 ; or approximately 81 cm 2 These ranges of effective surface areas are provided as examples only, and some embodiments may have different effective surface areas. The cell culture matrix can also be characterized for porosity, as described in the Examples herein.

[0039] 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, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. Mesh substrates can have a variety of patterns or weaves, including, for example, knitted, warp knitted, or woven (e.g., plain weave, twill weave, monoweave, five needle weave).

[0040] The surface chemistry of the mesh filaments may need to be modified to provide the desired cell attachment properties. Such modification can be achieved by chemical treatment of the mesh's polymeric material 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 attachment properties, including, for example, collagen or Matrigel®. Alternatively, the surface of the mesh's filament fibers can be imparted with cell attachment properties through treatment processes using various types of plasmas, process gases, and / or chemicals known in the art. However, in one or more embodiments, the mesh can provide an efficient cell growth surface even without surface treatment.

[0041] 2A-2C show several different examples of woven meshes according to some possible embodiments of the present disclosure. The fiber diameters and opening sizes of these meshes are summarized in Table 1 below, along with the approximate increase in cell culture surface area provided by a single layer of each mesh relative to an equivalent 2D surface. In Table 1, Mesh A refers to the mesh in FIG. 2A, Mesh B refers to the mesh in FIG. 2B, and Mesh C refers to the mesh in FIG. 2C. The three mesh geometries in Table 1 are merely examples, and embodiments of the present disclosure are not limited to these specific examples. Because Mesh C provides the largest surface area, it may be advantageous for achieving high cell attachment and proliferation densities, thereby providing the most efficient substrate for cell culture. However, in some embodiments, to achieve desired cell distribution or flow characteristics, for example, within a culture chamber, it may be advantageous for the cell culture matrix to include a mesh with a smaller surface area, such as Mesh A or Mesh B, or a combination of meshes with different surface areas.

[0042] [Table 1]

[0043] As shown in the table above, the three-dimensional quality of the mesh increases the surface area for cell attachment and growth compared to a flat, 2D surface of comparable size. This increased surface area aids in the scalable performance achieved by embodiments of the present disclosure. For process development and validation studies, small-scale bioreactors are often required to reduce reagent costs and increase experimental throughput. While applicable to such small-scale studies, embodiments of the present disclosure are also scalable to industrial or production scales. For example, 100 layers of Mesh C in the shape of a 2.2 cm diameter circle were packed into a cylindrical packed bed with an inner diameter of 2.2 cm, increasing the total surface area available for cell attachment and growth to approximately 935 cm. 2 To scale up such a bioreactor ten-fold, a similar setup can be used with a cylindrical packed bed of 7 cm inner diameter and 100 beds of the same mesh. In such a case, the total surface area is 9,350 cm. 2 In some embodiments, the available surface area is about 99,000 cm 2 / L or more. Plug-type perfusion in a packed bed results in a flow rate of ml / min / cross-sectional surface area of ​​the packed bed (cm 2 The same flow rate, expressed in units of sieving per square meter, can be used for both small-scale and large-scale versions of the bioreactor. The larger surface area allows for higher seeding and cell growth densities. According to one or more embodiments, the cell culture substrates described herein can support up to 22,000 cells / cm. 2 For reference, the seeding density of Corning HyperFlask® is 20,000 cells / cm on a two-dimensional surface. 2 That's about it.

[0044] Another benefit of the large surface area and high cell seeding or growth density is that the costs of the embodiments disclosed herein can be comparable to or less than competitive solutions, particularly the cost per cell product (e.g., per cell or per viral genome) can be less than or equal to other packed bed bioreactors.

[0045] In further embodiments of the present disclosure described below, the woven mesh substrate can be packed into a bioreactor in a cylindrical roll format (see Figures 8 and 9). In such embodiments, scalability of the packed-bed bioreactor can be achieved by increasing the overall length of the mesh strips and their length. The amount of mesh used in this cylindrical roll configuration can be varied based on the desired packing density of the packed bed. For example, the cylindrical rolls can be tightly packed with tight rolls or loosely packed with loose rolls. The packing density will often be determined by the surface area of ​​the cell culture substrate required for a given application or scale. In one embodiment, the required length of mesh is determined by the following formula:

[0046]

number

[0047] where L is the total length of mesh needed to pack the bioreactor (i.e., H in FIG. 8), R is the inner radius of the packed-bed culture chamber, r is the radius of the internal support (support 366 in FIG. 9) around which the mesh is wrapped, and t is the thickness of one layer of mesh. In such a configuration, scalability of the bioreactor can be achieved by increasing the diameter or width of the packed-bed cylindrical roll (i.e., W in FIG. 8) and / or increasing the height H of the packed-bed cylindrical roll to provide more substrate surface area for the seeding and growth of adherent cells.

[0048] The use of a structurally defined, sufficiently rigid culture matrix achieves high flow-resistance uniformity across the matrix or packed bed. According to various embodiments, the matrix can be deployed in a single-layer or multi-layer format, flexibly eliminating diffusion limitations and providing uniform delivery of nutrients and oxygen to cells attached to the matrix. Furthermore, the coarse-grained matrix does not contain cell-trapping regions within the packed-bed configuration, allowing for complete cell harvest with high viability at the end of the culture. The matrix also provides packing uniformity in the packed bed, enabling direct scalability from process development units to large-scale industrial bioprocessing units. The ability to directly harvest cells from the packed bed eliminates the need for resuspension of the matrix in a stirring or mechanically shaking vessel, which adds complexity and potentially imposes harmful shear stress on the cells. Furthermore, the high packing density of the cell culture matrix results in high bioprocess productivity at manageable industrial scales.

[0049] Figure 3A shows an embodiment of a matrix having a multi-layer substrate 200, and Figure 3B is a plan view of the same multi-layer substrate 200. The multi-layer substrate 200 includes a first mesh substrate layer 202 and a second mesh substrate layer 204. Despite the overlap between the first substrate layer 202 and the second substrate layer 204, the mesh geometry (e.g., ratio of opening diameter to fiber diameter) is such that the openings in the first substrate layer 202 and the second substrate layer 204 overlap, providing a path for fluid flow through the entire thickness of the multi-layer substrate 200, as shown by the filament-free opening 206 in Figure 3B.

[0050] FIG. 4 shows a cross-sectional view of the multilayer substrate 200 taken along line B-B in FIG. 3B. Arrows 208 indicate possible fluid flow paths through the openings in the second substrate layer 204 and then around the filaments in the first substrate layer 202. The geometry of the mesh substrate layers is designed to allow efficient and uniform flow through one or more substrate layers. Furthermore, the structure of the matrix 200 can accommodate multiple orientations of fluid flow through the matrix. For example, as shown in FIG. 4, the direction of bulk fluid flow (as indicated by arrows 208) is perpendicular to the major surfaces of the first substrate layer 202 and the second substrate layer 204. However, the matrix can also be oriented relative to flow so that the surfaces of the substrate layers are parallel to the bulk flow direction. FIG. 5 shows a cross-sectional view of the multilayer substrate 200 taken along line C-C in FIG. 4, where the structure of the matrix 200 allows fluid flow (arrows 210) through multiple fluid paths within the multilayer substrate 200. In addition to fluid flow being perpendicular or parallel to the first and second sides of the mesh layer, the matrix can be configured with multiple substrate pieces at multiple intermediate angles or in a random arrangement relative to the fluid flow. This orientation flexibility is made possible by the essentially isotropic flow behavior of the woven substrate. In contrast, substrates for adherent cells in existing bioreactors do not exhibit this behavior; instead, their packed layers tend to form preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the matrix of the present disclosure allows its use in a variety of applications and bioreactor or container designs, achieving better and more uniform permeability throughout the bioreactor vessel.

[0051] As described herein, the cell culture substrates can be used within a bioreactor vessel, according to one or more embodiments. For example, the substrates can be used within a packed-bed bioreactor configuration, or, in other configurations, within a three-dimensional culture chamber. However, embodiments are not limited to three-dimensional culture spaces; the use of substrates that can be considered two-dimensional culture surface configurations is also contemplated, where one or more layers of the substrate are placed flat, for example, in a flat-bottom culture dish, to provide a culture substrate for cells. Due to contamination concerns, the vessels can be single-use vessels that can be disposed of after use.

[0052] According to one or more embodiments, a cell culture system is provided that utilizes a cell culture matrix within a culture chamber of a bioreactor vessel. FIG. 6 illustrates an example of a cell culture system 300 that includes a bioreactor vessel 302 having a cell culture chamber 304 therein. Within the cell culture chamber 304 is a cell culture matrix 306 comprised of a stack of substrate layers 308. The substrate layers 308 are stacked such that a first or second side of one substrate layer faces a first or second side of an adjacent substrate layer. The bioreactor vessel 302 has an inlet 310 at one end for introducing 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. This stacking of substrate layers allows the system to be easily scalable without adversely affecting cell attachment and growth due to the defined structure and efficient fluid flow through the stacked substrates. Although vessel 302 may generally be described as having an inlet 310 and an outlet 312, some embodiments may use one or both of inlet 310 and outlet 312 to flow medium, cells, or other contents into and out of culture chamber 304. For example, inlet 310 may be used to flow medium or cells into culture chamber 304 during a cell seeding, perfusion, or culture phase, but may also be used to remove one or more of medium, cells, and cell products through inlet 310 during a harvest phase. The terms "inlet" and "outlet" are not intended to limit the function of these openings.

[0053] In one or more embodiments, the flow resistance and volumetric density of the packed bed can be controlled by interleaving substrate layers of different geometries. In particular, mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the fluid flow resistance of the packed bed format. By interleaving meshes of different sizes and geometries, the flow resistance can be controlled or varied in one or more specific sections of the bioreactor, thereby improving the uniformity of fluid perfusion within the packed bed. For example, 10 layers of mesh A (Table 1) can be stacked, followed by 10 layers of mesh B (Table 1), followed by 10 layers of mesh C (Table 1), to achieve the desired packed bed characteristics. As another example, the packed bed can begin with 10 layers of mesh B, followed by 50 layers of mesh C, followed by 10 layers of mesh B. This repeating pattern can be continued until the entire bioreactor is packed with mesh. These are merely examples and are used for illustrative purposes without intending to limit the possible combinations. In practice, various combinations of different sized meshes are possible to achieve different profiles of cell growth surface volumetric density and flow resistance. For example, by interleaving different sized meshes, a packed bed column can be constructed having multiple zones of varying volumetric cell densities (e.g., a series of zones forming a low / high / low / high, etc. density pattern).

[0054] In Figure 6, the bulk flow direction is from inlet 310 to outlet 312, and in this example, the first and second major surfaces of substrate layer 308 are perpendicular to the bulk flow direction. In contrast, the example shown in Figure 7 is of an embodiment in which system 320 includes a bioreactor vessel 322 and a stack of substrates 328 within culture chamber 324, where culture chamber 324 has first and second sides parallel to the bulk flow direction, and the bulk flow direction corresponds to the direction indicated by the flow lines entering inlet 330 and exiting outlet 332. Thus, matrices of the disclosed embodiments can be employed in either configuration. In each of systems 300 and 320, substrates 308, 328 are sized and shaped to fill the interior space defined by culture chambers 304, 324, thereby filling the culture space within each vessel to maximize the efficiency of the cell growth surface in terms of cell number per unit volume. 7 illustrates multiple inlets 330 and multiple outlets 332, it is contemplated that system 320 may be fed by a single inlet and have a single outlet. However, according to various embodiments herein, a distribution plate may be used to help distribute medium, cells, or nutrients across the cross-section of the packed bed, thereby improving the uniformity of fluid flow through the packed bed. Thus, multiple inlets 330 illustrate how a distribution plate may be provided with multiple holes across the cross-section of the packed bed to create a more uniform flow.

[0055] FIG. 8 illustrates one embodiment of a matrix in which the substrate is formed into a cylindrical roll 350. For example, a sheet of matrix material including a mesh substrate 352 is rolled into a cylinder about a central longitudinal axis y. The cylindrical roll 350 has a width W along a dimension perpendicular to the central longitudinal axis y and a height H along a direction parallel to the central longitudinal axis y. In one or more preferred embodiments, the cylindrical roll 350 is designed to be placed within a bioreactor vessel such that the central longitudinal axis y is parallel to the direction of bulk fluid flow F through the bioreactor or culture chamber containing the cylindrical roll. FIG. 9 illustrates a cell culture system 360 having a bioreactor vessel 362 containing a cell culture matrix 364 in such a cylindrical roll configuration. Like the cylindrical roll 350 of FIG. 8, the cell culture matrix 364 has a central longitudinal axis, which extends into the plane of the paper in FIG. 9. The system 360 further includes a central support member 366 around which the cell culture matrix 364 is positioned. While the central support member 366 can be provided solely for physical support and / or alignment of the cell culture matrix 364, it can also serve other functions according to some embodiments. For example, the central support member 366 can include one or more openings for supplying culture medium to the cell culture matrix 364 along the length H of the matrix. In other embodiments, the central support member 366 can include one or more attachment sites for holding one or more portions of the cell culture matrix 364 to the interior portion of the cylindrical roll. These attachment sites can be hooks, clasps, posts, clamps, or other means for attaching the mesh sheet to the central support member 366.

[0056] FIG. 10A illustrates a cell culture system 400 according to one or more embodiments. The system 400 includes a bioreactor 402 containing a cell culture matrix according to one or more embodiments disclosed herein. The bioreactor 402 can be fluidly connected to a medium conditioning vessel 404, and the system can supply cell culture medium 406 from the medium conditioning vessel 404 to the bioreactor 402. The medium conditioning vessel 404 can include sensors and control components found in typical bioreactors used in the bioprocessing industry for suspension culture, fed-batch culture, or perfusion culture. These include, but are not limited to, DO sensors, pH sensors, an oxygenator / gas sparging unit, a temperature probe, and nutrient and base addition ports. The gas mixture supplied to the sparging unit can be controlled by a gas flow controller for N, O, and CO gases. The medium conditioning vessel 404 also contains an impeller for mixing the medium. All of the medium parameters measured by the above-mentioned sensors can be controlled by a medium conditioning control unit 418, which is in communication with the medium conditioning vessel 404 and can measure and / or adjust the state of the cell culture medium 406 to desired levels. As shown in FIG. 10A, the medium conditioning vessel 404 is provided as a vessel separate from the bioreactor vessel 402. This can be advantageous in that the medium can be conditioned away from where the cells are being cultured and then the conditioned medium can be delivered to the cell culture space. However, in some embodiments, medium conditioning can be performed within the bioreactor vessel 402.

[0057] Media from the medium 406 conditioning vessel 404 is delivered to the bioreactor 402 via an inlet 408, which may include an injection port for seeding a cell inoculum to initiate cell cultivation. The bioreactor vessel 402 may also include one or more outlets 410 through which the cell culture media 406 exits the vessel 402. Additionally, cells or cell products may exit through the outlet 410. One or more sensors 412 may be provided in line to analyze the contents of the stream exiting the bioreactor 402. In some embodiments, the system 400 includes a flow control unit 414 for controlling the flow into the bioreactor 402. For example, the flow control unit 414 can receive a signal from one or more sensors 412 (e.g., an O sensor) and, based on the signal, adjust the flow into the bioreactor 402 by sending a signal to a pump 416 (e.g., a peristaltic pump) upstream of the inlet 408 to the bioreactor 402. Thus, based on one or a combination of factors measured by sensor 412, pump 416 can control flow to bioreactor 402 to achieve desired cell culture conditions.

[0058] The medium perfusion rate is controlled by a signal processing unit 414, which collects and compares sensor signals from sensors located in the medium conditioning vessel 404 and at the outlet 410 of the packed-bed bioreactor. Due to the characteristics of the medium perfusion flow through the packed-bed bioreactor 402, nutrient, pH, and oxygen gradients develop along the packed bed. The bioreactor perfusion flow rate can be automatically controlled by a flow control unit 414 operably connected to a peristaltic pump 416 according to the flowchart of FIG. 11.

[0059] One or more embodiments of the present disclosure provide a cell inoculation step that differs from conventional methods. In conventional methods, a packed bed of a conventional matrix is ​​filled with culture medium, and the concentrated inoculum is injected into a medium circulation loop. The cell suspension is pumped through the bioreactor at a high flow rate to reduce the non-uniformity of cell seeding due to entrapment on the conventional packed bed matrix. In such conventional methods, pumping of cells at a high flow rate through the circulation loop continues, perhaps for several hours, until the majority of the cells are entrapped in the packed bed bioreactor. However, due to the non-uniform nature of depth filtration in conventional packed bed bioreactors, cells are distributed non-uniformly within the packed bed, resulting in a high cell density in the inlet region of the bioreactor and a low cell density in the outlet region of the bioreactor.

[0060] In contrast, according to an embodiment of the present disclosure, a volume of cell inoculum equal to the void volume of the bioreactor's culture chamber is injected directly into the packed bed through the cell inoculum injection port at the inlet 408 of the bioreactor 402 ( FIG. 10A ). The uniform and continuous fluid pathways present in the cell culture matrices described herein then uniformly distribute the cell suspension within the packed bed. To prevent cell settling due to gravity during the initial seeding phase, perfusion of the medium can begin immediately after inoculum injection. The perfusion flow rate is maintained below a preprogrammed threshold to balance gravity and prevent cells from being washed out of the packed-bed bioreactor. Thus, during the initial cell attachment phase, cells are gently rolled within the packed bed, achieving uniform cell distribution and attachment to the available substrate surface.

[0061] FIG. 10B shows a more detailed schematic diagram of a cell culture system 420 according to one or more embodiments. The basic structure of system 420 is similar to system 400 of FIG. 10A, with a packed-bed bioreactor 422 including a vessel containing a packed bed of cell culture material, such as a woven PET mesh, and a separate medium conditioning vessel 424. However, in contrast to system 400, system 420 shows the details of the system, including sensors, user interfaces and controls, and various inlets and outlets for medium and cells. According to some embodiments, medium conditioning vessel 424 is controlled by a controller 426 to provide the appropriate temperature, pH, O2, and nutrients. In some embodiments, bioreactor 422 can also be controlled by controller 426, while in other embodiments, bioreactor 422 is contained within a separate perfusion circuit 428, where a pump is used to control the flow of medium through perfusion circuit 428 based on detection of O2 at or near the outlet of bioreactor 422.

[0062] The systems of Figures 10A and 10B can be operated according to process steps according to one or more embodiments. As shown in Figure 11A, these process steps can include process preparation (S1), cell seeding and attachment (S2a, S2b), cell growth (S3), transfection (S4a, S4b), viral vector production (S5a, S5b), and harvesting (S6a, S6b).

[0063] FIG. 11 shows an example of a method 450 for controlling flow in a perfusion bioreactor system, such as the system 400 of FIG. 10A or 10B. According to method 450, in step S21, certain parameters of the system 400 are predetermined by bioreactor optimization runs. From these optimization runs, values ​​for pH1, pO1, [glucose]1, pH2, pO2, [glucose]2, and maximum flow rate can be determined. The values ​​of pH1, pO1, and [glucose]1 are measured in the cell culture chamber of the bioreactor 402 in step S22, and pH2, pO2, and [glucose]2 are measured by sensor 412 at the outlet of the bioreactor vessel 402 in step S23. Based on these values ​​in S22 and S23, the perfusion pump control unit makes a decision in S24 to maintain or adjust the perfusion flow rate. For example, if pH2 ≧ pH 2min , pO2 ≥ pO 2min , and [glucose]2 ≥ [glucose] 2min If at least one of the following conditions is met, the perfusion flow rate of the cell culture medium to the cell culture chamber may be continued at the current flow rate (S25). If the current flow rate is equal to or less than a predetermined maximum flow rate of the cell culture system, the perfusion flow rate is increased (S27). Furthermore, if the current flow rate is greater than a predetermined maximum flow rate of the cell culture system, the controller of the cell culture system: (1) increases the pH 2min , pO 2min , and [glucose] 2min At least one of: (2) pH, pO, and [glucose]; and (3) bioreactor vessel height can be reassessed (S26).

[0064] The cell culture matrix can be arranged in a number of configurations within the culture chamber, depending on the desired system. For example, in one or more embodiments, the system includes one or more layers of substrate having a width that spans the width of a predetermined cell culture space within the culture chamber. Multiple layers of substrate can be stacked in this manner to a predetermined height. As described above, the substrate layers can be arranged such that first and second sides of one or more layers are perpendicular to the bulk flow direction of culture medium through the predetermined culture space within the culture chamber, or the first and second sides of one or more layers can 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 relative to the bulk flow direction and one or more other layers in a second orientation different from the first orientation. For example, the various layers can have first and second sides that are parallel or perpendicular to the bulk flow direction, or at some angle therebetween.

[0065] In one or more embodiments, the cell culture system includes multiple separate pieces of cell culture substrate in a packed bed configuration, where the length and width of the pieces of substrate are small compared to the culture chamber. As used herein, a "piece of substrate" is considered to have a length and / or width that is small compared 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 multiple pieces of substrate packed 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 multiple pieces oriented in a generally similar orientation (e.g., horizontally, vertically, or at an angle between 0° and 90° relative to the bulk flow direction).

[0066] As used herein, "defined culture space" refers to the space within a culture chamber occupied by a cell culture matrix where cells will be seeded and / or cultured. The defined culture space can occupy substantially the entire culture chamber or may occupy a portion of the space within the culture chamber. As used herein, "bulk flow direction" is defined as the direction of bulk mass flow of fluid or culture medium through or over the cell culture matrix during cell culturing and / or during inflow or outflow of culture medium to or from the culture chamber.

[0067] In one or more embodiments, the cell culture matrix is ​​secured within the culture chamber by a securing mechanism. The securing mechanism can secure a portion of the cell culture matrix to a wall of the culture chamber surrounding the matrix or to a chamber wall at one end of the culture chamber. In some embodiments, the securing mechanism attaches 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 to a member extending perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture matrix can be contained within the culture chamber without being fixedly attached to a wall of the chamber or bioreactor vessel. For example, the matrix can be contained by a boundary or other structural member of the culture chamber, thereby retaining the matrix within a predetermined area of ​​the bioreactor vessel without securing the matrix to these boundary or structural members.

[0068] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. The culture chamber can contain a cell culture matrix and substrate according to one or more of the embodiments described herein. In the roller bottle configuration, the bioreactor vessel can be operably attached to a means for moving the bioreactor vessel about a central longitudinal axis of the vessel. For example, the bioreactor vessel can be rotated about the central longitudinal axis. This rotation can be continuous (e.g., continuous in one direction) or discontinuous (e.g., intermittent rotation in a single direction or alternating directions, or oscillating back and forth). In operation, the rotation of the bioreactor vessel causes movement of cells and / or fluids within the chamber. This movement can be considered relative to the walls of the chamber. For example, as the bioreactor vessel rotates about its central longitudinal axis, gravity can continue to direct fluids, culture medium, and / or unattached cells toward the bottom of the chamber. However, in one or more embodiments, the cell culture matrix is ​​essentially fixed relative to the vessel and therefore rotates with the vessel. In one or more other embodiments, the cell culture matrix can be unattached and can move freely relative to the vessel as desired as the vessel rotates. The movement of the vessel can allow the cells to attach to the cell culture matrix while exposing the cells to cell culture medium or liquid and oxygen or other gases within the culture chamber.

[0069] The use of cell culture matrices according to embodiments of the present disclosure, such as matrices comprising a woven or mesh substrate, provides roller bottle containers with increased surface area available for attachment, growth, and functionalization of adherent cells. In particular, the use of a woven mesh substrate of monofilament polymer material within a roller bottle can increase the surface area by about 2.4 to about 4.8 times, or even about 10 times, compared to standard roller bottles. As described herein, each monofilament strand of the mesh substrate can present itself as a 2D surface for adherent cells to attach. Furthermore, multiple layers of mesh can be placed within a roller bottle, increasing the total available surface area by about 2 to 20 times compared to standard roller bottles. Thus, existing roller bottle equipment and processes, including cell seeding, medium changes, and cell harvesting, can be modified with the addition of the improved cell culture matrices disclosed herein with minimal impact on existing operational infrastructure and processing steps.

[0070] The bioreactor vessel optionally includes one or more outlets that can be attached to the inlet and / or outlet means through which fluid, medium, or cells can be supplied to or removed from the chamber. A single port on the vessel may function as both an inlet and an outlet, or multiple ports may be dedicated to inlet and outlet functions.

[0071] In one or more embodiments, the packed-bed cell culture matrix can be comprised of a woven cell culture mesh substrate, without any other form of cell culture substrate disposed or interspersed within the cell culture matrix. That is, the woven cell culture mesh substrate of the disclosed embodiments is an effective cell culture substrate that eliminates the need for the irregular, nonwoven substrates used in existing solutions. This allows for a cell culture system with simplified design and construction, while providing the high-density cell culture substrate with the other advantages described herein, such as flow uniformity and ease of harvesting.

[0072] As described herein, the cell culture substrates and bioreactor systems provided herein offer numerous advantages. For example, embodiments of the present disclosure can support the production of any of a number of viral vectors, such as AAV (all serotypes) and lentivirus, and are applicable to in vivo and ex vivo gene therapy applications. Uniform cell seeding and distribution maximizes viral vector yield per vessel, and these designs also allow for viable cell harvest, which may be useful for seed trains consisting of multiple growth periods using the same platform. Furthermore, embodiments herein are scalable from process development scale to production scale, ultimately saving development time and costs. The disclosed methods and systems also enable automation and control of the cell culture process, thereby maximizing vector yield and improving reproducibility. Finally, the number of vessels (e.g., 10 per batch) required to reach production-level scale for viral vectors can be reduced. 16 ~10 18 The number of AAV vectors required can be significantly reduced compared to other cell culture solutions.

[0073] Embodiments are not limited to containers that rotate about a central longitudinal axis. For example, the container may rotate about an axis that is off-center relative to the container. Furthermore, the axis of rotation may be horizontal or vertical. [Example]

[0074] To demonstrate the effectiveness of the disclosed cell culture matrices, cell culture systems, and related methods, cell seeding and culture studies were conducted according to the following examples.

[0075] Example 1 In Example 1, a cell culture matrix with a polyethylene terephthalate (PET) woven mesh substrate (see Figure 12) was tested under static cell culture conditions. The PET mesh was washed with ethanol and plasma-treated in oxygen RF plasma. Gelatin was adsorbed onto the surface of the mesh filaments to promote cell attachment. Disk-shaped pieces of the mesh were placed in Corning® ultra-low attachment (ULA) 6-well plates. HEK293T cells were seeded at different seeding densities (50K / cm). 2 , 75K / cm 2 , 100K / cm 2 ) onto the mesh discs, and cell culture was carried out for 3 days. Cells on the filament surface were stained with a fluorescent green cell tracker dye. Figure 12 shows the results of visualizing cells on the filament surface in this manner. The size of the mesh filaments relative to the size of the cells allows the monofilament fibers to effectively act as a two-dimensional surface for cell attachment and proliferation. Cell proliferation was measured by harvesting cells from the mesh and counting them using a Beckman Coulter Vi-Cell® cell counter. The results demonstrated good cell attachment and proliferation on the cell culture matrix under static cell culture conditions. For example, Figure 13A shows the total cell counts per well for each seeded mesh at 24, 48, and 72 hours. In addition to cell counts, cell viability is shown in Figure 13B, demonstrating extremely high viability across multiple seeding densities.

[0076] Example 2 In Example 2, cells were cultured in a packed-bed bioreactor system such as that shown in FIG. 6 according to an embodiment of the present disclosure. The packed bed was cylindrical and made of a stack of multiple circular or disc-shaped cell culture devices. Specifically, in Example 2, the packed bed had a height of approximately 25 mm and contained 100 woven PET mesh substrates, each with a diameter of approximately 20 mm. The mesh used corresponded to Mesh C in Table 1. The total two-dimensional surface area available for cell attachment was approximately 760 cm.2 The bioreactor was inoculated with 8 ml of a HEK293T cell suspension (2 million cells / ml) directly into the packed bed. Perfusion of medium was initiated immediately after the introduction of the cell suspension, with the perfusion flow rate set at 3 ml / min. Perfusion at this rate was continued for 24 hours, after which the flow rate was reduced to 1 ml / min. The perfusion flow rate was then adjusted to maintain pO2 ≥ 50% saturation and pH ≥ 7 at the bioreactor outlet. After 2–3 days, the cells were stained with crystal violet, and the bioreactor was disassembled to confirm the uniformity of cell attachment within the matrix. Figures 14A and 14B show three disks of packed bed matrix with attached HEK293T cells stained with crystal violet dye. Figure 14A shows the results from a bioreactor seeded under static conditions. Based on the staining variability, uneven cell attachment was observed after 3 days of culture. Specifically, the bottom of the packed bed (corresponding to the bottom disk in the image of FIG. 14A) had a higher concentration of cells, while the top of the packed bed (corresponding to the top disk in the image of FIG. 14A) had fewer cells. FIG. 14B shows results from a bioreactor seeded with a seeding method according to a preferred embodiment, in which cells were continuously rolled within the packed bed during the initial attachment phase. As a result, after two days of cell culture, a uniform cell distribution is observed in all parts of the packed bed, as evidenced by consistent staining of cells in multiple disks from the top to the bottom of the reactor (and from the top to the bottom of the image of FIG. 14B). This indicates that a uniform cell distribution was achieved when the bioreactor was continuously perfused during the cell seeding phase.

[0077] Example 3 In Example 3, cells were cultured in a packed-bed bioreactor system and transfection of HEK293T cells was performed for adeno-associated virus (AAV) production in the bioreactor. The same bioreactor setup as in Example 2 was used in Example 3 (see, e.g., Figure 6). The packed bed contained 100 disks of PET mesh (Mesh C in Table 1). Each disk had a diameter of approximately 20 mm, and the packed bed height was approximately 25 mm, resulting in a total two-dimensional surface area available for cell attachment and growth of approximately 760 cm. 2 The cell temperature was 100°C (50°F). To inoculate the bioreactor, 8 ml of a HEK293T cell suspension (2 million cells / ml) was injected directly into the packed bed. A medium reservoir containing approximately 50 ml of medium was fluidly connected to the bioreactor vessel. For 72 hours, the cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) ATCC® medium containing 10% FBS and 6 mM L-glutamine. When the pH of the medium in the reservoir dropped below 7, the medium was replaced with a fresh medium supply. The perfusion flow rate was adjusted accordingly to maintain a pO2 ≥ 50% saturation and a pH ≥ 7 at the bioreactor outlet. After 72 hours, the cell culture medium was replaced with 50 ml of Corning DMEM (15-018) containing 10% FBS and 6 mM L-glutamine, and transfection reagent was added to a final concentration of 2 μg per ml of AAV2 and PEIpro (1:2 ratio). If the pH in the storage bottle dropped below 7 during the following 72 hours, the cell culture medium was replaced with a fresh supply. The perfusion flow rate was adjusted accordingly to maintain a pO2 ≥ 50% saturation and a pH ≥ 7 at the bioreactor outlet. Cells were harvested using 5X TrypLE. Transfection efficiency was analyzed using a fluorescent flow cytometer, and viral particle and genome titers were analyzed by ELISA and PCR assays. The cell culture results are shown in Table 2. Here, "VP" stands for "viral protein" and "GC" stands for "genome copy."

[0078] [Table 2]

[0079] Example 4 In Example 4, one embodiment of a roller bottle cell culture system was tested. Corning roller bottles #430195 were used, with a surface area of ​​490 cm. 2 To prevent cells from adhering to tissue culture-treated surfaces, roller bottles were treated with a 0.5% solution of BSA for a minimum of 16 hours and washed with water before each experiment. Cells were grown on a standard 2D surface (T-flask) and harvested using a standard protocol, in which cells were released from the surface using a trypsin / EDTA solution, which was then inactivated by the addition of complete medium containing fetal bovine serum (FBS). The cells were then counted using a cell counter, and the cells were harvested at approximately 5 x 10 cells in a total volume of 200 mL. 4 pieces / cm 2Cells were seeded into roller bottles with and without cell culture mesh at a concentration of 0.01 to 0.01.00. As shown in Figures 15 and 16, for roller bottles 500 containing cell culture mesh 502, the mesh was rolled into a tight cylindrical roll 503 for insertion through the bottle's mouth 501. After insertion into the bottle, the cell culture mesh was partially unrolled and unrolled toward the wall of the roller bottle (as indicated by arrow 504). The length of the cell culture mesh was long enough so that after unrolling in the roller bottle, a double layer of cell culture mesh was placed around the inside circumference of the roller bottle, as shown in Figures 15 and 16. Cells were allowed to attach to the surface at various rotation speeds (0.5 to 4 rpm) for 16+ hours in an incubator at 37°C with 5% CO2 and 95% relative humidity. After cell attachment, the speed was reduced to approximately 1 rpm for typical growth of cells in roller bottles. Medium measurements were performed periodically to determine when medium replacement was necessary. Visualization of cells attached to the mesh surface was performed in roller bottles using crystal violet in a solution containing methanol and paraformaldehyde. After staining of the cells, the mesh was removed from the roller bottle and imaged.

[0080] 17A and 17B show the mesh removed from the roller bottle of Example 4, demonstrating the presence of stained HEK293 cells attached to the double-layer mesh within the roller bottle. The total surface area available for attachment of adherent cells was 2450 cm for roller bottles according to certain embodiments of the present disclosure. 2 On the other hand, roller bottles without mesh substrates have a viscosity of 490 cm 2Figure 17A shows crystal violet staining of cells attached to a PET mesh (corresponding to mesh C in Table 1) that self-aligned into two layers in a roller bottle. Cells were seeded at a roller bottle speed of 0.5 rpm. Note that cells were primarily seeded on the outer layer of the mesh facing the bottle wall. Figure 17B shows crystal violet staining of cells attached to a PET mesh (corresponding to mesh C in Table 1) that self-aligned into two layers in a roller bottle. Cells were seeded at a roller bottle speed of 4 rpm. Note that cells were uniformly seeded on both mesh layers. As can be seen from Figures 17A and 17B, the uniformity of cell seeding depended on the roller bottle rotation speed during the seeding step. In contrast to the standard roller bottle seeding protocol, a high rotation speed was required to uniformly seed cells over the available attachment surface of the mesh.

[0081] Embodiments of the present disclosure have advantages over existing platforms for cell culture and viral vector production. It should be noted that embodiments of the present disclosure can be used to produce numerous types of cells and cell by-products, including, for example, adherent or semi-adherent cells, human embryonic kidney (HEK) cells (e.g., HEK23) (including transfected cells), lentiviruses (stem cells, CAR-T), and viral vectors such as adeno-associated viruses (AAV). While these are examples of some common uses for the bioreactors or cell culture substrates disclosed herein, they are not intended to be limitations on the uses or applications of the embodiments of the present disclosure, and one of skill in the art will understand that these embodiments can be adapted for other uses.

[0082] Example 5 As discussed above, one advantage of embodiments of the present disclosure is the uniformity of flow through the cell culture substrate. Without wishing to be bound by theory, it is believed that the regular or uniform structure of the cell culture substrate provides a consistent, uniform body through which media can flow. In contrast, existing platforms primarily rely on irregular or random substrates, such as felted or nonwoven fibrous materials. The uniform nature of the substrate of the present disclosure can be illustrated by examining the uniform and consistent cell seeding achieved in this substrate. For example, Figure 18A shows three disks (1801, 1802, 1803) of substrate material from Example 5 according to some embodiments of the present disclosure. The disks in Figure 18A are of the woven PET mesh material described herein and each have a diameter of approximately 60 mm. The surface area for a bioreactor packed with layers of 10 to 300 similar disks is approximately 678 to 20,300 cm. 2 In this example, cell culture was performed using a stack of 100 discs. The first disc 1801 was the top disc of a stack of such discs in a bioreactor, the second disc 1802 was the middle disc of the stack, and the third disc 1803 was the bottom disc of the stack. Despite being placed at different locations within the stack, the staining in FIG. 18A shows very consistent cell attachment.

[0083] In the experiment that generated the images in Figures 18A and 18B, the bioreactor was pre-filled with cell culture medium, and the system was pre-conditioned overnight to achieve a steady state of pH 7.2, DO 100%, and 37°C. The entire bioreactor system was filled with 400 ml of ATCC DMEM medium + 10% FBS + 6 mM L-glutamine. HEK293T cells in 30 ml of suspension (5 million cells / mL) were inoculated directly into the packed bed through a three-way port. For the first 48 hours, the pre-conditioned medium was perfused through the bioreactor at a rate of 30 mL / min to ensure uniform cell distribution, attachment, and initial growth within the packed bed. After 48 hours of culture, 200 ml of fresh complete ATCC DMEM medium was added to the system to maintain a glucose level above 1 g / L. The perfusion flow rate was automatically adjusted to maintain a DO at the bioreactor outlet. external ≥45% medium saturation was maintained. 72 hours after inoculation, the culture medium was replaced with 500 ml of Corning DMEM (15-018) + 10% FBS + 6 mM L-glutamine and perfused for 2 hours. The transfection mixture (a 1:2 complex of plasmid DNA and PEI; 0.8 μg total DNA per million cells) was added to a final concentration of 2 μg total DNA per ml of medium 24 hours after transfection, and the culture medium was replaced with 500 ml of fresh complete Corning DMEM (15-018) medium to replenish consumed nutrients. The perfusion flow rate was automatically adjusted to generate DO at the bioreactor outlet. external The saturation was maintained at ≥45%. Glucose levels were monitored during the following 48 hours of culture and supplied by medium addition or replacement as needed to maintain levels above 0.3 g / L. 72 hours after transfection, cells were washed with DPBS and harvested using 1X Accutase solution. Transfection efficiency was analyzed by fluorescent flow cytometry, and viral particle and viral genome titers were analyzed by ELISA and qPCR assays.

[0084] Crystal violet staining was used to highlight the uniform cell growth across the surface of the discs in Figure 18A. Despite the first disc 1801, second disc 1802, and third disc 1803 being spread across the entire cell culture matrix stack, cell growth is consistent across all three discs. The image in Figure 18A was taken after 72 hours of culture, before the cells were harvested from the substrate. Figure 18B shows the same three discs (1801', 1802', and 1803') after the cells were harvested. As indicated by the relative lack of crystal violet staining in Figure 18B, the cells were harvested uniformly across the surface of each disc and across the three discs in the cell culture matrix stack. Based on the analysis, more than 95% of the cells were recovered from the bioreactor. 6780 cm 2 The cell culture results for AAV production on these substrate laminates / vessels of 60 mm diameter with a total surface area of ​​1000 μm are shown in Table 3 below, which shows the yield of transfected cells, transfection efficiency, and yield per cm. 2 The number of viral genomes per 1000 cells is shown. Again, the uniform structure and uniform flow characteristics of the substrate are believed to contribute to this efficient and uniform growth and ease of harvesting.

[0085] [Table 3]

[0086] Table 4 below shows the above results in the context of multiple experiments involving multiple bioreactor vessels of different diameters (29 mm and 60 mm). Data is presented for the smaller (e.g., 29 mm diameter, 1600 cm surface area) 2 ) and the larger one (e.g., diameter 60 mm, surface area 6780 cm 2 ) container and / or packed bed matrix.

[0087] [Table 4]

[0088] As discussed above, embodiments of the present disclosure can provide packed-bed cell culture matrices and / or bioreactors that allow for high-density cell culture in a relatively small practical footprint. For example, the 60 mm cell culture matrix in the examples in Tables 3 and 4 above has a cell density of approximately 6870 cm. 2 For reference, the Corning HYPERflask® has a surface area of ​​approximately 1720 cm 2 The 60 mm diameter cell culture matrices from Tables 3 and 4 can be housed in smaller bioreactors than the HYPERflask, yet still allow for higher cell numbers at harvest and higher total genome copies (GC) or viral genomes (VG) per vessel. Figure 19 shows these numbers for two bioreactor vessels containing the 60 mm diameter substrates from Tables 3 and 4 compared to the HYPERflask, while simultaneously allowing for a 1 cm 2 The GC per cm is also shown. 2 Although the GC per liter is lower than the HYPERflask, it makes up for it with a larger surface area.

[0089] Example 6 To further explore the flow uniformity or permeability of the substrates of the present disclosure, modeling was used to understand the porosity of the three-dimensional cell culture matrix. Sheets of woven PET mesh substrate were modeled in densely packed and loosely packed configurations. These represent the upper and lower limits of packing density of the substrate stack for the particular mesh sheet being modeled. In particular, Figure 20A shows a plan view of the densely packed configuration, and Figure 20B shows a plan view of the same stack. cross sectionFigures 21A and 21B show plan and cross-sectional views, respectively, of the loosely packed configuration. For each modeled configuration, a sample cell 600, 602 was defined. The sample cell contained the same volume of mesh material to analyze the porosity per unit volume of the sample cell 600, 602. The modeled volume of open space within each cell is shown in Figure 22A (for the tightly packed laminate) and Figure 22B (for the loosely packed laminate). Porosity as a percentage of open space was approximately 40.8% for the loosely packed cells and 61.4% for the tightly packed cells. Because the modeled laminates in Figures 20A-21B represent the most tightly and loosely packed configurations of a given mesh material, the porosities of 40.8% and 61.4% are upper and lower bounds on porosity for this particular mesh material. Depending on the alignment and actual packing density when using this mesh material, the porosity can fall between these extremes, however, embodiments of the present disclosure are not limited to this porosity range, as variations in mesh size and placement of the substrate within the cell culture vessel may result in different ranges of porosity.

[0090] In addition to the modeled porosity range, porosity was measured using a packed bed of actual PET woven mesh substrate. This measurement was performed using 100 discs, each 22.4 mm in diameter, stacked in random alignment. The total weight of the 100 disc stack was 5.65±0.2 g. The volume of PET material in this stack was calculated based on a PET density of 1.38 g / cm. 3 Assuming that, the following formula:

[0091]

number

[0092] This gives the PET volume V for 5.65 g of PET (for 100 disks of 22.4 mm diameter). PETwas calculated to be 4.1 ml. Next, the PET volume V PET and the total volume V of the laminate, including the volume of the open space within the laminate. total to the following formula:

[0093]

number

[0094] The stack of 100 disks had a stack height of 25±1 mm. Therefore, with a disk diameter of 22.4 mm, V total Therefore, the porosity of the stacked packed bed is calculated by the following formula:

[0095]

number

[0096] Using Equation 4 and the values ​​above, the porosity was calculated to be 58.4%, which is within the range predicted by the model.

[0097] Example 7 Example 7 compared the permeability of various PET woven mesh substrate materials. Table 5 shows the PET mesh samples used in this comparison.

[0098] [Table 5]

[0099] Photographs of mesh samples A-F are shown in Figure 23. Permeability results for each mesh sample A-F are shown in Figure 24. Figure 25 shows the results of pressure drop tests for samples A-C, where pressure drop tests were performed on multiple stacks of different configurations and packing densities for sample A. The dotted lines represent tight and loose packing densities for mesh sample A, with sample A1 being a loosely packed stack compared to A2. Pressure drop in terms of change in pressure per centimeter (Pa) is plotted against Q / A.

[0100] Example 8 As described herein, embodiments of the present disclosure provide cell culture substrates, bioreactor systems, and methods for culturing cells or cell by-products that are scalable and can be used to provide a cell seed train for expanding a cell population. One problem with existing cell culture solutions is that the technology of a given bioreactor system cannot be part of the seed train. Instead, cell populations are typically expanded on various cell culture substrates. This can have a negative impact on the cell population because cells adapt to specific surfaces, and transferring them to different types of surfaces can lead to inefficiencies. Therefore, it is desirable to minimize such transfers between multiple cell culture substrates or technologies. Using the same cell culture substrate throughout a seed train, as can be achieved by embodiments of the present disclosure, improves the efficiency of cell population expansion. Figure 26 illustrates an example of one or more embodiments, in which a woven cell culture substrate of the present application is used as part of a seed train, allowing a relatively small bioreactor to seed a relatively larger one. Specifically, as shown in Figure 26, a seed train can be initiated with a vial of starter cells, which is then cultured in a first vessel (e.g., a T175 flask from Corning), then a second vessel (e.g., a HyperFlask® from Corning), then a process development scale bioreactor system according to embodiments of the invention (effective substrate surface area = approximately 20,000 cm). 2), and a relatively large bioreactor pilot system according to an embodiment of the present invention (effective substrate surface area = approx. 300,000 cm 2 At the end of this seed train, the cells can be seeded into a manufacturing scale bioreactor vessel according to embodiments of the present disclosure, which may have a volume of, for example, about 5,000,000 cm 2 The surface area of ​​the cells is approximately 100 μm. Harvesting and purification steps can then be performed upon completion of cell culture. As shown in Figure 26, harvesting can be achieved by in situ cell lysis using a detergent (such as Triton X-100) or by mechanical lysis, followed by further downstream processing as needed.

[0101] Advantages of using the same cell culture substrate within a seed train (e.g., from the process development level to the pilot level, or even to the production level) include: efficiencies gained by cells being adapted to the same surface during the seed train and production stages; reduced number of manual handling maneuvers during multiple phases of the seed train; more efficient use of the packed bed due to uniform cell distribution and fluid flow as described herein; and flexibility in the use of mechanical or chemical lysis during harvesting of viral vectors.

[0102] Example 9 To understand the potential improvement in virus production yield of the substrates of the present disclosure, the performance of the PET woven mesh substrate was compared to that of the substrate used in iCellis. Table 6 summarizes the total number of virus particles produced using these substrates in a simplified bioreactor.

[0103] [Table 6]

[0104] The volume of substrate material required to produce a specific number of viral particles can be calculated from the results in Table 6. For example, if the goal for manufacturing-scale viral vector production is 3.00E+18 viral particles, as shown in Table 7, the volume of PET woven mesh required is approximately 1 / 7 of the amount of iCellis substrate required.

[0105] [Table 7]

[0106] Example 10 To demonstrate uniform flow through the coarse mesh substrate of the present disclosure, fluid flow rates through a packed-bed bioreactor were modeled. Figure 27A shows modeling results for a vessel 620 with a packed-bed region 622 of woven PET mesh discs, which has a 6 cm diameter and a 10 cm packed-bed height and consists of 357 discs of woven PET mesh substrate. The magnitude of the fluid velocity is shown according to the scale shown. While the flow velocity is high near the inlet 624 and outlet 626, this velocity is consistent throughout the packed-bed region 622, e.g., along the height of the packed bed and across the width of the packed bed. The region indicated by the dotted line 628 is shown enlarged in Figure 27B, which illustrates the relatively constant velocity as the fluid enters the uniform coarse structure of the cell culture matrix. The packed bed in this example has a flow rate of approximately 24,214 cm. 2 For a given uniform flow as shown in the model, the percentage of this surface area exposed to non-uniform flow (defined as a deviation of more than 2.5% from the mean velocity) was 0%.

[0107] To demonstrate the extent to which this uniform flow persists as the vessel size is scaled, further modeling was performed using vessels of increasing width (as well as wider packed beds), similar to Figure 27A. The percentage of non-uniform flow from these larger vessels is summarized in Table 8. As shown, even when the reactor is scaled to a 60 cm diameter, the amount of non-uniform flow remains below approximately ½ percent of the substrate surface area. This demonstrates that the uniform coarse-woven mesh described herein, unlike existing cell culture substrates, can achieve uniform flow throughout the packed bed.

[0108] [Table 8]

[0109] Example 11 To better understand the difference in permeability between the woven mesh substrate of the present disclosure and the irregular nonwoven substrates currently available on the market, experiments were conducted to measure the permeability of these materials. Specifically, PET woven mesh was compared with the nonwoven substrate used in iCellis and similar commercially available nonwoven substrates with a less-ordered configuration. Permeability measurements were performed for flow perpendicular to the woven mesh substrate thickness in a packed bed of stacked disks, flow through a randomly packed bed of nonwoven substrates, and flow through a fixed sheet of nonwoven substrate material. The nonwoven mesh had a fiber diameter of approximately 20 μm, a thickness of approximately 0.18 mm, and a porosity of 91%. The woven mesh substrate had a diameter of approximately 160 μm and an opening diameter of 250 μm.

[0110] Water was used in the test to simulate cell culture medium for mesh permeability. To simulate the flow conditions typically experienced by substrates in a bioreactor, a peristaltic pump was used to maintain a flow rate of 15-50 ml / cm. 2The pressure drop across the sample was small under the test conditions, so a pressure gauge was used to measure the pressure difference across the sample. Due to the different substrate types and loading methods, the substrates were held in slightly different ways.

[0111] To measure the flow across the nonwoven mesh, the sample was cut into a 12 mm diameter disk, and 10 rows of mesh were held between two open cylindrical chambers sealed with O-rings. A pressure gauge was connected directly to the open chambers to measure the pressure drop.

[0112] To measure flow through randomly packed nonwoven mesh, the mesh was cut into 5 mm x 25 mm strips and packed into a cylindrical chamber with a diameter of 29 mm. A total of 3 g of mesh strips was packed to a volume of 30 ml and a packed bed height of approximately 45 mm. On each side of the packed strip, two disks of loosely woven mesh were used to confine the packed bed. An open space approximately 3 mm thick was present on each side of the packed bed, and two 10 mm thick pieces of porous material were used to redistribute the flow at the inlet and outlet.

[0113] To measure the flow through a coarse-woven mesh, the mesh was cut into 29 mm diameter disks to fit into a cylindrical chamber. A total of 170 disks were packed in a single layer, resulting in a packed bed height of 45 mm. The orientation of the fibers in each layer of mesh was not aligned with each other. The flow was transverse to the mesh disk (i.e., perpendicular to the disk surface). An open space approximately 3 mm thick was present on each side of the packed bed, and two 10 mm thick pieces of porous material were used to redistribute the flow at the inlet and outlet.

[0114] The transmittance is given by equation (5):

[0115]

number

[0116] where: Q = flow rate; K = permeability; A = cross-sectional area of ​​the sample or packed bed; dP = pressure drop across the test sample or packed bed; μ = viscosity of water; and dL = total sample thickness or packed bed height.

[0117] The final calculated permeabilities are summarized in Figure 28. These results show that the nonwoven mesh has a permeability of approximately 7.5 × 10, which is approximately 1 / 50 of the permeability across the coarse woven mesh. -12 m 2 The results show that the nonwoven mesh had an extremely low permeability of 100%, which is approximately 100%. When the nonwoven mesh was cut into small strips and randomly packed, the permeability increased significantly, becoming comparable to that of the coarse-woven mesh. This increase in permeability is thought to be the result of most of the flow bypassing the mesh strips due to the channeling effect described above.

[0118] Based on the measured permeabilities, we simulated the flow through and around the nonwoven and coarse-woven meshes. Simulations were performed using the ANSYS Fluent v19.2 software package. For illustrative purposes, two scenarios were studied: the surface of the substrate material was aligned at (i) 90° and (ii) 45° to the flow direction, as shown in Figures 29A and 29B. In both cases, when the spacing between neighboring meshes in the same stage was 5 mm, most of the flow was around the meshes, with only approximately 0.02–0.005% of the flow passing through the meshes. This resulted in the formation of a significant dead zone behind the meshes and nonuniform flow through the packed bed. This nonuniformity was more severe when the nonwoven mesh pieces were not perfectly aligned perpendicular to the flow direction.

[0119] In the case of the coarse woven mesh, the coarse structure allowed the flow to pass easily through the mesh, preventing the formation of dead zones behind the coarse mesh layer. The regular structure of the woven mesh also likely contributed to the uniform flow distribution through each stage, which in turn led to more uniform flow throughout the packed bed. A comparison is clearly seen in Figures 30A (nonwoven mesh piece) and 30B (coarse woven mesh substrate), which show a close-up view of the flow near the edge of the substrate material. In Figures 30A and 30B, the gap between neighboring mesh pieces in all six directions was reduced to 1 mm, and only the periodic domain of one such mesh piece was simulated. Figure 30A shows that the nonwoven mesh has extremely low permeability, as most of the flow bypasses the substrate, with very little flow passing through the substrate itself. Only 0.17% of the total mass flow passes through the nonwoven mesh. In contrast, the coarse woven mesh has a much higher permeability, and therefore more flow passes through it, as shown in Figure 30B. The flow shortcutting through the voids is weaker when comparing the color bars for the two cases. For the coarse woven mesh, as much as 10.7% of the total mass flow passes through the substrate, demonstrating that the coarse woven mesh has excellent permeability even when packed with voids.

[0120] As described herein, multiple woven mesh disks can be randomly packed with infinite variations in inter-disk alignment. However, the range of possible alignment can be reduced to two theoretical limits based on packing density (i.e., tightest packing and loosest packing). These two ideal or limiting conditions allow large packed layers to be simplified into small periodic domains. Using this model, we found that the permeability through the substrate varies by approximately 10 times from the tightest packing limit to the loosest packing limit. The experimentally measured permeability data described above fell well within this range and served as a good validation point. The model also showed that the permeability in the flow direction was comparable to the permeability in the transverse direction for all packing conditions. This suggests that the woven mesh of the present disclosure does not change flow direction as much as we have observed in nonwoven substrates, resulting in more uniform flow regardless of substrate orientation. The improved flow uniformity of the substrate of the present disclosure is further demonstrated by residence time distribution (RTD) measurements in the following examples.

[0121] Example 12 Residence time distribution (RTD) is useful for studying flow in vessels. Its theory, measurement, and analysis can be found in the textbook: Levenspiel, O. Chemical Reaction Engineering. 3rd ed. 1999. Wiley, New York. Figure 5 shows a schematic diagram of the setup for measuring RTD. The chamber was cylindrical with a diameter of 29 mm and a total packed bed volume of 36 ml. 3.6 g of nonwoven mesh or 200 layers of loosely woven mesh were filled into the vessel chamber. McCormick Green Food Color diluted 1:2000 was used as the tracer for the measurements. Changes in tracer concentration were monitored using UV-vis with a Flowcell. A flow rate of 22.5 ml / ml was used for all experiments. The chamber was first filled with water. After switching to the green dye, the change in OD was recorded. The results are shown in Figure 32.

[0122] The mean residence time t (Equation (6)) and variance σ (Equation (7)) were calculated using the following formulas:

[0123]

number

[0124]

number

[0125] where F is the normalized concentration in the step tracer response. Table 9 summarizes the mean residence times and deviations calculated from the measurements. The coarse-woven mesh exhibited a shorter mean residence time, likely due to its lower porosity and reduced dead zones. The packed bed of coarse-woven mesh had a porosity of approximately 60%, while the nonwoven mesh had a relatively high porosity of approximately 93%. The significantly larger normalized deviations detected in the packed bed of nonwoven mesh suggest that the nonwoven mesh was less uniform or less than ideal.

[0126] [Table 9]

[0127] The above permeability and residence time experiments have shown that the irregularly-type nonwoven cell culture substrates used in current bioreactors have lower permeability than the substrates of the present disclosure. These nonwoven substrates also have different permeability or flow rates depending on the direction of flow relative to the nonwoven substrate, whereas the substrates of the present disclosure exhibit essentially isotropic flow behavior. Due to the irregular flow and short residence time of the nonwoven substrates, it may take longer for nutrients and transfection reagents to reach cells on the substrate surface or the other side of the substrate layer than with the uniformly woven mesh substrates of the present disclosure. In addition, the permeability of the randomly packed nonwoven substrates is high, suggesting a strong channeling effect and the resulting uneven delivery of cells or nutrients.

[0128] Exemplary Implementation Below are descriptions of various aspects of implementations of the presently disclosed subject matter. Each aspect may include one or more of various features, characteristics, or advantages of the presently disclosed subject matter. These implementations are intended to illustrate some aspects of the presently disclosed subject matter and should not be construed as a comprehensive or exhaustive description of all possible implementations.

[0129] Aspect 1 is directed to a cell culture matrix, the cell culture matrix comprising: a substrate having a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and passing through the thickness of the substrate, the plurality of openings configured to allow flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate.

[0130] Aspect 2 is directed to the cell culture matrix of aspect 1, wherein the substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0131] Aspect 3 is directed to the cell culture matrix of aspect 1 or aspect 2, wherein the substrate comprises at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh sheet.

[0132] Aspect 4 is directed to the cell culture matrix of aspect 3, wherein the substrate comprises the woven mesh comprising one or more fibers.

[0133] Aspect 5 is directed to the cell culture matrix of aspect 4, wherein the one or more fibers have at least one of the following cross-sectional shapes: flat, circular, rectangular, and polygonal.

[0134] Aspect 6 is directed to the cell culture matrix of aspect 4 or aspect 5, wherein the one or more fibers comprise at least one of monofilament and multifilament fibers.

[0135] Aspect 7 is directed to the cell culture matrix of any one of Aspects 4 to 6, wherein the one or more fibers comprise first fibers having a first fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0136] Aspect 8 is directed to the cell culture matrix of Aspect 7, wherein the one or more fibers further comprise second fibers having a second fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0137] Example 9 is directed to the cell culture matrix of example 8, wherein the second fiber diameter is different from the first fiber diameter.

[0138] Aspect 10 is directed to the cell culture matrix according to any one of Aspects 1 to 9, wherein the plurality of openings have an opening diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

[0139] Aspect 11 is directed to the cell culture matrix of Aspect 10, wherein the fiber diameter is about 250 μm to about 300 μm and the opening diameter is about 750 μm to about 800 μm; or the fiber diameter is about 270 μm to about 276 μm and the opening diameter is about 785 μm to about 795 μm.

[0140] Aspect 12 is directed to the cell culture matrix of Aspect 10, wherein the fiber diameter is about 200 μm to about 230 μm and the opening diameter is about 500 μm to about 550 μm; or the fiber diameter is about 215 μm to about 225 μm and the opening diameter is about 515 μm to about 530 μm.

[0141] Aspect 13 is directed to the cell culture matrix of Aspect 10, wherein the fiber diameter is about 125 μm to about 175 μm and the opening diameter is about 225 μm to about 275 μm; or the fiber diameter is about 150 μm to about 165 μm and the opening diameter is about 235 μm to about 255 μm.

[0142] Aspect 14 is directed to the cell culture matrix of any one of Aspects 10 to 13, wherein the ratio of the aperture diameter to the fiber diameter is about 1.0 to about 3.5, about 1.25 to about 3.25, about 1.4 to about 3.0, about 1.5 to about 2.9, about 1.5 to about 2.4, or about 2.4 to about 2.9.

[0143] Aspect 15 is directed to the cell culture matrix of any one of aspects 1 to 14, wherein the plurality of openings comprises openings having a square, rectangular, diamond, rhomboid, circular, or oval shape.

[0144] A sixteenth aspect is directed to the cell culture matrix of any one of the first to fifteenth aspects, wherein the plurality of openings are arranged in a regular pattern.

[0145] Aspect 17 is directed to the cell culture matrix of any one of aspects 1 to 16, wherein the cell culture matrix comprises a single layer substrate.

[0146] Example 18 is directed to the cell culture matrix of any one of Examples 1 to 17, wherein the cell culture matrix comprises a multilayer substrate comprising at least one first substrate layer and at least one second substrate layer, the first substrate layer comprising a first side and a second side opposite the first side, the second substrate layer comprising a third side and a fourth side opposite the third side, and the second side facing the third side.

[0147] Example 19 is directed to the cell culture matrix of Example 18, wherein the multi-layer substrate is configured such that the first substrate layer has a predetermined alignment with the second substrate layer.

[0148] Aspect 20 is directed to the cell culture matrix of aspect 19, wherein the multilayer substrate is configured such that fiber intersections of the first substrate layer face openings of the second substrate layer.

[0149] Example 21 is directed to the cell culture matrix of example 19 or example 20, wherein the openings in the first substrate layer at least partially overlap the openings in the second substrate layer.

[0150] Example 22 is directed to the cell culture matrix of Example 21, wherein the openings of the first and second substrate layers are aligned.

[0151] Example 23 is directed to the cell culture matrix of example 18, wherein the multi-layer substrate is configured such that the first substrate layer has random alignment relative to the second substrate layer.

[0152] Aspect 24 is directed to the cell culture matrix of any one of Aspects 1 to 23, wherein the cell culture matrix comprises a plurality of the substrates, each of the plurality of substrates being randomly oriented relative to the other substrates of the plurality of substrates.

[0153] Aspect 25 is directed to the cell culture matrix of any one of aspects 1 to 23, wherein the cell culture matrix comprises a plurality of the substrates in a layered configuration.

[0154] Aspect 26 is directed to the cell culture matrix of aspect 25, wherein the first side and the second side of one of the substrates are generally parallel to the first side and the second side of the other substrates in the stacked configuration.

[0155] Aspect 27 is directed to the cell culture matrix of any one of Aspects 1 to 23, wherein the substrate is in a cylindrical roll configuration.

[0156] Example 28 is directed to the cell culture matrix of Example 27, wherein the cylindrical roll is configured to partially unwind when placed in the culture chamber, thereby expanding to the shape of the culture chamber within the bioreactor vessel.

[0157] Aspect 29 is directed to the cell culture matrix of aspect 28, wherein the cylindrical roll is configured to be inserted into the culture space while the cylindrical roll is in a contracted state and to expand within the culture space when placed within the culture space.

[0158] Example 30 is directed to the cell culture matrix of any one of Examples 1 to 29, wherein the cell culture matrix comprises a plurality of substrates comprising woven meshes of different geometries, the different geometries differing in at least one of fiber diameter, aperture diameter, or aperture geometry.

[0159] Example 31 is directed to the cell culture matrix of example 30, wherein the woven meshes of different geometries are arranged in a predetermined arrangement based on desired flow characteristics within the bioreactor vessel.

[0160] Aspect 32 is directed to the cell culture matrix of aspect 31, wherein the desired flow characteristics include at least one of uniform perfusion of liquid medium throughout the cell culture matrix and distribution of cell growth throughout the cell culture matrix.

[0161] Example 33 is directed to the cell culture matrix of example 31 or example 32, wherein the woven meshes of different geometries include a first mesh having a first geometry and a second mesh having a second geometry, and the predetermined positioning includes the first mesh being upstream of the second mesh with respect to a desired direction of bulk flow of cell culture medium through the cell culture matrix.

[0162] Example 34 is directed to the cell culture matrix of Example 33, wherein the predetermined arrangement comprises disposing the first mesh stack upstream of the second mesh stack.

[0163] Aspect 35 is directed to the cell culture matrix of aspect 33 or aspect 34, wherein the predetermined arrangement comprises an alternating arrangement of multiple stacks of the first mesh and multiple stacks of the second mesh along the bulk flow direction.

[0164] Aspect 36 is directed to the cell culture matrix of any one of aspects 1 to 35, wherein the cell culture matrix is ​​configured for the culture and / or harvest of at least one of cells, proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0165] Example 37 is directed to the cell culture matrix of any one of Examples 1 to 36, wherein the substrate comprises a functionalized surface, the functionalized surface being physically or chemically modified to improve attachment of the adherent cells to the polymer mesh material.

[0166] Example 38 is directed to the cell culture matrix of any one of Examples 1 to 37, wherein the cell culture matrix comprises a surface configured for adsorption or absorption of components in the culture medium onto the surface of the mesh.

[0167] Example 39 is directed to the cell culture matrix of any one of Examples 1 to 38, wherein the cell culture matrix comprises a coating on a surface of the polymer mesh material, the coating configured to promote attachment of the adherent cells.

[0168] Aspect 40 is directed to the cell culture matrix of aspect 39, wherein the cells adhere to the coating.

[0169] Aspect 41 is directed to the cell culture matrix of aspect 39 or aspect 40, wherein the coating is a biological molecule or a synthetic bioactive molecule configured to promote attachment of cells to the cell culture matrix.

[0170] Aspect 42 is directed to the cell culture matrix of any one of aspects 39 to 41, wherein the coating is at least one of a hydrogel, collagen, Matrigel®, a bioactive molecule or peptide, and a biological protein.

[0171] Example 43 is directed to the cell culture matrix of any one of Examples 39 to 42, wherein the functionalized surface is plasma treated.

[0172] Aspect 44 is directed to the cell culture matrix of any one of Aspects 1 to 43, wherein the cells comprise at least one of adherent cells, suspension cells, and weakly adherent cells that adhere to the woven mesh.

[0173] Embodiment 45 is directed to a bioreactor system comprising: a cell culture vessel having at least one reservoir; and a cell culture matrix disposed within the at least one reservoir, the cell culture matrix comprising a woven substrate having a plurality of interwoven fibers having a surface configured for cell attachment.

[0174] Example 46 is directed to the system of Example 45, wherein the woven substrate comprises a uniform arrangement of the plurality of interwoven fibers.

[0175] Example 47 is directed to the system of example 45 or example 46, wherein the woven substrate comprises a plurality of apertures disposed between the plurality of fibers.

[0176] Example 48 is directed to the system of any one of Examples 45-47, wherein the plurality of fibers comprises polymer fibers.

[0177] Example 49 is directed to the system of example 48, wherein the polymer fibers comprise at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0178] Example 50 is directed to the system of any one of Examples 45-49, wherein the cell culture matrix comprises a plurality of woven substrates.

[0179] Aspect 51 is directed to the system described in Aspect 50, wherein each of the plurality of substrates has: a first side; a second side opposite the first side; and a thickness separating the first side and the second side, and the plurality of openings pass through the thickness of the substrate.

[0180] Aspect 52 is directed to a system described in aspect 50 or aspect 51, wherein the multiple substrates are arranged adjacent to one another such that one of the first side and the second side of one of the substrates is adjacent to the other of the first side and the second side of an adjacent substrate.

[0181] Example 53 is directed to the system of any one of Examples 50-52, wherein at least a portion of the plurality of substrates are not separated by a spacer material or barrier.

[0182] Example 54 is directed to the system of any one of Examples 50-53, wherein at least a portion of the substrates are in physical contact with one another.

[0183] Aspect 55 is directed to a system described in any one of aspects 45 to 54, wherein the cell culture container comprises at least one port, and the at least one port is configured to supply or remove material from the at least one reservoir through the at least one port.

[0184] Aspect 56 is directed to the system described in Aspect 55, wherein the at least one port includes at least one inlet for supplying material to the at least one reservoir and at least one outlet for removing material from the at least one reservoir.

[0185] Aspect 57 is directed to the system of aspect 56, wherein the materials include at least one of a medium, cells, and cell products.

[0186] Aspect 58 is directed to a cell culture system comprising: a bioreactor vessel; and a cell culture matrix disposed within the bioreactor vessel and configured for culturing cells, the cell culture matrix comprising: a substrate having a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and passing through the thickness of the substrate, the plurality of openings configured to allow flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate.

[0187] Example 59 is directed to the cell culture system of example 58, wherein the substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0188] Example 60 is directed to the cell culture system of example 58 or example 59, wherein the substrate comprises at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh sheet.

[0189] Example 61 is directed to the cell culture system of example 60, wherein the substrate comprises a woven mesh comprising one or more fibers.

[0190] Example 62 is directed to the cell culture system of Example 61, wherein the one or more fibers have at least one of the following cross-sectional shapes: flat, circular, rectangular, and polygonal.

[0191] Example 63 is directed to the cell culture system of example 61 or example 62, wherein the one or more fibers comprise at least one of monofilament and multifilament fibers.

[0192] Aspect 64 is directed to the cell culture system of any one of Aspects 61 to 63, wherein the one or more fibers comprise first fibers having a first fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0193] Example 65 is directed to the cell culture system of Example 64, wherein the one or more fibers further comprise second fibers having a second fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0194] Example 66 is directed to the cell culture system of example 65, wherein the second fiber diameter is different from the first fiber diameter.

[0195] Aspect 67 is directed to the cell culture system according to any one of Aspects 58 to 64, wherein the plurality of openings have an opening diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

[0196] Aspect 68 is directed to the cell culture system of aspect 67, wherein the fiber diameter is about 250 μm to about 300 μm and the opening diameter is about 750 μm to about 800 μm; or the fiber diameter is about 270 μm to about 276 μm and the opening diameter is about 785 μm to about 795 μm.

[0197] Aspect 69 is directed to the cell culture system of aspect 67, wherein the fiber diameter is about 200 μm to about 230 μm and the opening diameter is about 500 μm to about 550 μm; or the fiber diameter is about 215 μm to about 225 μm and the opening diameter is about 515 μm to about 530 μm.

[0198] Aspect 70 is directed to the cell culture system of aspect 67, wherein the fiber diameter is about 125 μm to about 175 μm and the opening diameter is about 225 μm to about 275 μm; or the fiber diameter is about 150 μm to about 165 μm and the opening diameter is about 235 μm to about 255 μm.

[0199] Aspect 71 is directed to the cell culture system of any one of Aspects 67 to 70, wherein the ratio of the opening diameter to the fiber diameter is about 1.0 to about 3.5, about 1.25 to about 3.25, about 1.4 to about 3.0, about 1.5 to about 2.9, about 1.5 to about 2.4, or about 2.4 to about 2.9.

[0200] Aspect 72 is directed to the cell culture system of any one of aspects 58 to 71, wherein the plurality of openings comprises openings having a square, rectangular, diamond, rhomboid, circular, or oval shape.

[0201] Aspect 73 is directed to the cell culture system of any one of aspects 58 to 72, wherein the plurality of openings are arranged in a regular pattern.

[0202] Example 74 is directed to the cell culture system of any one of Examples 58 to 73, wherein the cell culture matrix comprises a single layer substrate.

[0203] Aspect 75 is directed to the cell culture system of any one of Aspects 58 to 74, wherein the cell culture matrix comprises a multilayer substrate, the multilayer substrate comprising at least one first substrate layer and at least one second substrate layer, the first substrate layer comprising a first side and a second side opposite the first side, the second substrate layer comprising a third side and a fourth side opposite the third side, and the second side facing the third side.

[0204] Example 76 is directed to the cell culture system of example 75, wherein the multi-layer substrate is configured such that the first substrate layer has a predetermined alignment with the second substrate layer.

[0205] Example 77 is directed to the cell culture system of example 76, wherein the multilayer substrate is configured such that intersections of fibers in the first substrate layer face openings in the second substrate layer.

[0206] Example 78 is directed to the cell culture system of example 76 or example 77, wherein the openings in the first substrate layer at least partially overlap with the openings in the second substrate layer.

[0207] Example 79 is directed to the cell culture system of example 78, wherein the openings of the first substrate layer and the second substrate layer are aligned.

[0208] Example 80 is directed to the cell culture system of example 75, wherein the multi-layer substrate is configured such that the first substrate layer has random alignment relative to the second substrate layer.

[0209] Example 81 is directed to the cell culture system of any one of Examples 58 to 80, wherein the cell culture matrix is ​​positioned within the bioreactor vessel such that a bulk flow direction of medium through the bioreactor vessel is parallel or perpendicular to the first side and the second side.

[0210] Example 82 is directed to the cell culture system of any one of Examples 58 to 81, wherein the cell culture matrix comprises a plurality of the substrates randomly packed within the bioreactor vessel.

[0211] Aspect 83 is directed to the cell culture system of any one of aspects 58 to 82, wherein the bioreactor vessel is a packed bed bioreactor.

[0212] Embodiment 84 is directed to a cell culture system according to any one of embodiments 58 to 83, wherein the bioreactor vessel comprises: a culture space disposed within the bioreactor vessel and containing the cell culture matrix; and one or more openings configured to supply or remove fluid from the culture space.

[0213] Aspect 85 is directed to the cell culture system of aspect 84, wherein the one or more openings include an inlet configured to supply a fluid to the interior of the culture space and an outlet configured to allow fluid to be removed from the culture space of the bioreactor vessel.

[0214] Embodiment 86 is directed to the cell culture system of embodiment 85, wherein the bioreactor vessel comprises a first end having the inlet and a second end opposite the first end having the outlet, and the culture space is disposed between the first end and the second end.

[0215] Example 87 is directed to the cell culture system of example 86, wherein the cell culture matrix has a shape corresponding to the shape of the culture space.

[0216] Example 88 is directed to the cell culture system of any one of Examples 58 to 87, wherein the cell culture matrix comprises the polymer mesh material in a cylindrical roll configuration.

[0217] Example 89 is directed to the cell culture system of example 88, wherein a central longitudinal axis of the cylindrical roll is parallel to a direction of flow of the medium.

[0218] Example 90 is directed to the cell culture system of example 88 or example 89, wherein the cylindrical roll is configured to expand to the shape of the culture space in the bioreactor vessel upon unwinding of the cylindrical roll.

[0219] Aspect 91 is directed to a cell culture system described in any one of aspects 88 to 90, wherein the cylindrical roll is configured to be inserted into the culture space while the cylindrical roll is in a contracted state and to expand within the culture space when placed within the culture space.

[0220] Aspect 92 is directed to a cell culture system described in any one of aspects 88 to 91, wherein the cylindrical roll and the culture space are configured so that the polymer mesh material is held in a predetermined position within the culture space by frictional forces between the polymer mesh material and the walls of the culture space.

[0221] Aspect 93 is directed to the cell culture system of aspect 91, wherein the cylindrical roll is configured to be inserted into the culture space through an opening of the bioreactor vessel.

[0222] Aspect 94 is directed to the cell culture system of aspect 93, wherein the opening is one of the inlet and the outlet of the bioreactor vessel.

[0223] Aspect 95 is directed to a cell culture system according to any one of aspects 88 to 94, wherein the bioreactor vessel comprises a substrate support within the culture space, the substrate support being configured to guide, align, or secure the cell culture matrix within the culture space.

[0224] Aspect 96 is directed to the cell culture system of aspect 95, wherein the substrate support comprises a support member extending from one of the first end and the second end toward the other of the first end and the second end, and the cylindrical roll is configured to surround the support member such that the support member is parallel to the central longitudinal axis of the cylindrical roll.

[0225] Example 97 is directed to the cell culture system of any one of Examples 58 to 96, wherein the bioreactor vessel is configured to rotate about a central longitudinal axis of the bioreactor vessel during cell culture.

[0226] Aspect 98 is directed to the cell culture system of aspect 97, wherein the central longitudinal axis is perpendicular to the direction of gravity during cell culture.

[0227] Aspect 99 is directed to the cell culture system of aspect 97 or aspect 98, wherein the cell culture system is configured such that the substrate moves through a cell culture fluid during the rotation of the bioreactor vessel.

[0228] Embodiment 100 is directed to the cell culture system of any one of embodiments 97 to 99, wherein the cell culture system further comprises a rotation means operably coupled to the bioreactor vessel and configured to rotate the bioreactor vessel about the central longitudinal axis.

[0229]

[0033] Aspect 101 is directed to the cell culture system of any one of aspects 58 to 100, wherein the cell culture matrix comprises a plurality of substrates comprising woven meshes of different geometries, the different geometries differing in at least one of fiber diameter, aperture diameter, or aperture geometry.

[0230] Example 102 is directed to the cell culture system of example 101, wherein the woven meshes of different geometries are arranged within the bioreactor vessel in predetermined configurations based on desired flow characteristics within the bioreactor vessel.

[0231] Aspect 103 is directed to the cell culture system of aspect 102, wherein the desired flow characteristics include at least one of uniform perfusion of liquid medium throughout the cell culture matrix and distribution of cell growth throughout the cell culture matrix.

[0232] Example 104 is directed to the cell culture system of example 102 or example 103, wherein the woven meshes of different geometries include a first mesh having a first geometry and a second mesh having a second geometry, and the predetermined location includes the first mesh being upstream of the second mesh with respect to the bulk flow direction.

[0233] Example 105 is directed to the cell culture system of Example 104, wherein the predetermined arrangement comprises placing the first mesh stack upstream of the second mesh stack.

[0234] Example 106 is directed to the cell culture system of example 104 or example 105, wherein the predetermined arrangement comprises an alternating arrangement of multiple stacks of the first mesh and multiple stacks of the second mesh along the bulk flow direction.

[0235] Aspect 107 is directed to the cell culture system of any one of aspects 58 to 106, further comprising means for harvesting said adherent cells or cell by-products.

[0236] Example 108 is directed to the cell culture system of Example 107, wherein the cellular by-products comprise at least one of proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0237]

[0033] Aspect 109 is directed to a cell culture system of any one of aspects 58 to 108, wherein the substrate comprises a functionalized surface, the functionalized surface being physically or chemically modified to improve adhesion of the adherent cells to the polymer mesh material.

[0238]

[0033] Aspect 110 is directed to a cell culture system according to any one of aspects 58 to 109, wherein the cell culture matrix comprises a surface configured for adsorption or absorption of components in the culture medium onto the surface of the mesh.

[0239] Embodiment 111 is directed to a cell culture system described in any one of embodiments 58 to 110, wherein the cell culture matrix comprises a coating on the surface of the polymer mesh material, the coating being configured to promote adhesion of the adherent cells.

[0240] Example 112 is directed to the cell culture system of example 111, wherein the cells adhere to the coating.

[0241] Example 113 is directed to the cell culture system of example 111 or example 112, wherein the coating is a biological molecule or a synthetic bioactive molecule configured to promote cell attachment to the cell culture matrix.

[0242] Aspect 114 is directed to the cell culture system of any one of aspects 111 to 113, wherein the coating is at least one of a hydrogel, collagen, Matrigel, a bioactive molecule or peptide, and a biological protein.

[0243] Example 115 is directed to the cell culture system of any one of Examples 110 to 113, wherein the functionalized surface is plasma treated.

[0244] Example 116 is directed to the cell culture matrix of any one of Examples 58 to 115, wherein the cells comprise at least one of adherent cells, suspension cells, and weakly adherent cells that adhere to the woven mesh.

[0245] Example 117 is directed to the cell culture system of any one of Examples 58 to 116, further comprising a medium conditioning vessel configured to provide medium to the inlet of the bioreactor vessel.

[0246] Embodiment 118 is directed to a bioreactor system comprising: a cell culture vessel comprising a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed within the at least one reservoir, the cell culture matrix comprising a plurality of woven fabric substrates, each of the woven fabric substrates including a plurality of interwoven fibers having a surface configured for attaching cells, the bioreactor system configured to flow material through the at least one reservoir in a flow direction from the first end to the second end, and the plurality of woven fabric substrates are stacked such that each woven fabric substrate is generally parallel to each other of the woven fabric substrates and generally perpendicular to the flow direction.

[0247] Embodiment 119 is directed to the system described in embodiment 118, wherein each of the substrates has: a first side; a second side opposite the first side; and a thickness separating the first side and the second side, and a plurality of openings pass through the thickness of the substrate.

[0248] Aspect 120 is directed to the system of aspect 118 or aspect 119, wherein the substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0249]

[0041] Example 121 is directed to the system of any one of Examples 118-120, wherein the plurality of interwoven fibers comprises first fibers having a first fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0250]

[0041] Example 122 is directed to the system of Example 121, wherein the plurality of interwoven fibers further comprises second fibers having a second fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0251] Example 123 is directed to the system of Example 122, wherein the second fiber diameter is less than or equal to the first fiber diameter.

[0252] Example 124 is directed to the system of any one of Examples 119 to 123, wherein the plurality of openings have an opening diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

[0253] Embodiment 125 is directed to the system of any one of embodiments 119 to 124, wherein the ratio of the aperture diameter to the fiber diameter is about 1.0 to about 3.5, about 1.25 to about 3.25, about 1.4 to about 3.0, about 1.5 to about 2.9, about 1.5 to about 2.4, or about 2.4 to about 2.9.

[0254] Example 126 is directed to the system of any one of Examples 119-125, wherein the plurality of openings are arranged in a regular pattern.

[0255] Embodiment 127 is directed to a system described in any one of embodiments 118 to 126, wherein the cell culture matrix includes a plurality of substrates including woven meshes of different geometries, the different geometries differing in at least one of fiber diameter, opening diameter, or opening geometry.

[0256] Example 128 is directed to the system of Example 127, wherein the woven meshes of different geometries are arranged in a predetermined arrangement based on desired flow characteristics within the bioreactor vessel.

[0257]

[0033] Aspect 129 is directed to the system of any one of aspects 118 to 128, wherein the cell culture matrix is ​​configured for culturing and / or harvesting at least one of cells, proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0258] Embodiment 130 is directed to a system described in any one of embodiments 118 to 129, wherein the substrate has a functionalized surface, and the functionalized surface is physically or chemically modified to improve adhesion of the adherent cells to the polymer mesh material.

[0259] Example 131 is directed to the system of any one of Examples 118-130, wherein the plurality of interwoven fibers are arranged in an ordered, non-random arrangement relative to one another.

[0260] Example 132 is directed to the system of any one of Examples 118-131, wherein at least a portion of the plurality of substrates are not separated by a spacer material or barrier.

[0261] Example 133 is directed to the system of any one of Examples 118-132, wherein at least a portion of the substrates are in physical contact with one another.

[0262] Embodiment 134 is directed to a method of culturing cells in a bioreactor system described in any one of embodiments 118 to 133, the method comprising: seeding cells onto the cell culture matrix; culturing the cells on the cell culture matrix; and harvesting a product of the culturing of the cells, wherein the plurality of openings in the substrate are configured to allow the flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate.

[0263] Example 135 is directed to the method of example 134, wherein the seeding step comprises attaching the cells to the substrate.

[0264] Example 136 is directed to a method according to example 135, wherein the seeding step comprises injecting a cell inoculum directly into the cell culture matrix.

[0265] Example 137 is directed to the method of any one of Examples 134 to 136, further comprising perfusing cell culture medium through the culture chamber after the step of injecting the cell inoculum.

[0266] Embodiment 138 is directed to the method of any one of embodiments 134 to 137, further comprising: providing a medium conditioning vessel fluidly connected to the bioreactor vessel; and supplying the cell culture medium from the medium conditioning vessel to the bioreactor vessel.

[0267] Example 139 is directed to the method of example 138, wherein during or after culturing, at least a portion of the medium is removed from the bioreactor vessel and returned to the medium conditioning vessel.

[0268] Aspect 140 is directed to a method according to any one of aspects 134 to 139, further comprising controlling the flow of the cell culture medium into the cell culture chamber, the cell culture medium comprising at least one of cells, cell culture nutrients, and oxygen.

[0269] Embodiment 141 is directed to a bioreactor system comprising: a cell culture vessel comprising a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed within the at least one reservoir, the cell culture matrix comprising a plurality of woven fabric substrates, each of the woven fabric substrates including a plurality of interwoven fibers having a surface configured for attaching cells, the bioreactor system configured to flow material through the at least one reservoir in a flow direction from the first end to the second end, and the plurality of woven fabric substrates are stacked such that each woven fabric substrate is generally parallel to each other woven fabric substrate and generally parallel to the flow direction.

[0270] Embodiment 142 is directed to the system described in embodiment 141, wherein each of the substrates has: a first side; a second side opposite the first side; and a thickness separating the first side and the second side, and a plurality of openings pass through the thickness of the substrate.

[0271] Aspect 143 is directed to the system of aspect 141 or aspect 142, wherein the substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0272]

[0047] Example 144 is directed to a system of any one of Examples 141 to 143, wherein the plurality of interwoven fibers includes first fibers having a first fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0273]

[0049] Example 145 is directed to the system of Example 144, wherein the plurality of interwoven fibers further comprises second fibers having a second fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0274] Example 146 is directed to the system of Example 145, wherein the second fiber diameter is less than or equal to the first fiber diameter.

[0275] Embodiment 147 is directed to the system of any one of embodiments 142 to 146, wherein the plurality of openings have an opening diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

[0276]

[0047] Aspect 148 is directed to a system according to any one of aspects 142 to 147, wherein the ratio of the aperture diameter to the fiber diameter is from about 1.0 to about 3.5, from about 1.25 to about 3.25, from about 1.4 to about 3.0, from about 1.5 to about 2.9, from about 1.5 to about 2.4, or from about 2.4 to about 2.9.

[0277] Example 149 is directed to the system of any one of Examples 142-148, wherein the plurality of openings are arranged in a regular pattern.

[0278] Embodiment 150 is directed to a system described in any one of embodiments 141 to 149, wherein the cell culture matrix includes a plurality of substrates including woven meshes of different geometries, the different geometries differing in at least one of fiber diameter, opening diameter, or opening geometry.

[0279] Example 151 is directed to the system of Example 150, wherein the woven meshes of different geometries are arranged in a predetermined arrangement based on desired flow characteristics within the bioreactor vessel.

[0280]

[0033] Aspect 152 is directed to the system of any one of aspects 141 to 151, wherein the cell culture matrix is ​​configured for the culture and / or harvesting of at least one of cells, proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0281] Embodiment 153 is directed to a system described in any one of embodiments 141 to 152, wherein the substrate has a functionalized surface, and the functionalized surface is physically or chemically modified to improve adhesion of the adherent cells to the polymer mesh material.

[0282] Example 154 is directed to the system of any one of Examples 141-153, wherein the plurality of interwoven fibers are arranged in an ordered, non-random arrangement relative to one another.

[0283] Example 155 is directed to the system of any one of Examples 141-154, wherein at least a portion of the plurality of substrates are not separated by a spacer material or barrier.

[0284] Example 156 is directed to the system of any one of Examples 141-155, wherein at least a portion of the substrates are in physical contact with one another.

[0285] Embodiment 157 is directed to a method of culturing cells in a bioreactor system described in any one of embodiments 141 to 156, the method comprising: seeding cells onto the cell culture matrix; culturing the cells on the cell culture matrix; and harvesting a product of the culturing of the cells, wherein the plurality of openings in the substrate are configured to allow the flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate.

[0286] Example 158 is directed to the method of example 157, wherein the seeding step comprises attaching the cells to the substrate.

[0287] Example 159 is directed to a method according to example 158, wherein the seeding step comprises injecting a cell inoculant directly into the cell culture matrix.

[0288] Example 160 is directed to the method of any one of Examples 157 to 159, further comprising perfusing cell culture medium through the culture chamber after the step of injecting the cell inoculum.

[0289] Embodiment 161 is directed to the method of any one of embodiments 157 to 160, further comprising: providing a medium conditioning vessel fluidly connected to the bioreactor vessel; and supplying the cell culture medium from the medium conditioning vessel to the bioreactor vessel.

[0290] Example 162 is directed to the method of example 161, wherein during or after culturing, at least a portion of the medium is removed from the bioreactor vessel and returned to the medium conditioning vessel.

[0291] Aspect 163 is directed to a method according to any one of aspects 157 to 162, further comprising controlling the flow of the cell culture medium into the cell culture chamber, the cell culture medium comprising at least one of cells, cell culture nutrients, and oxygen.

[0292] Embodiment 164 is directed to a bioreactor system comprising: a cell culture vessel having a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture matrix disposed within the at least one reservoir, the cell culture matrix including a woven fabric substrate comprising a plurality of interwoven fibers having a surface configured for adhering cells, the woven fabric substrate being disposed within the at least one reservoir in a rolled configuration, thereby providing a cylindrical cell culture matrix, with a surface of the woven fabric substrate parallel to a longitudinal axis of the cylindrical cell culture matrix.

[0293] Embodiment 165 is directed to the system of embodiment 164, wherein the woven fabric substrate is disposed within the at least one reservoir as a cylindrical substrate that at least partially surrounds the central longitudinal axis of the bioreactor vessel.

[0294] Embodiment 166 is directed to the system of embodiment 164 or embodiment 165, wherein the bioreactor system is configured to flow material through the at least one reservoir in a flow direction from the first end to the second end.

[0295] Example 167 is directed to the system of Example 166, wherein the central longitudinal axis of the cylindrical substrate is parallel to the flow direction of the medium.

[0296] Embodiment 168 is directed to a system described in any one of embodiments 164 to 167, wherein the cylindrical substrate comprises a rolled-up woven substrate, and the rolled-up woven substrate is configured to expand and contact the wall of the at least one reservoir by unrolling the rolled-up woven substrate.

[0297] Embodiment 169 is directed to a system described in any one of embodiments 164 to 168, wherein the rolled woven fabric substrate is configured to expand to the internal shape of the at least one reservoir within the cell culture vessel.

[0298] Embodiment 170 is directed to the system described in embodiment 169, wherein the rolled woven fabric substrate is inserted into the culture space while in a contracted roll state and is configured to expand within the reservoir when placed within the reservoir.

[0299] Aspect 171 is directed to a system described in aspect 169 or aspect 170, wherein the rolled woven substrate and the reservoir are configured such that frictional forces between the rolled substrate and the walls of the reservoir hold the rolled substrate in an approximately predetermined position within the reservoir.

[0300] Embodiment 172 is directed to the system of any one of embodiments 169-171, wherein the rolled woven substrate is configured to be inserted into the reservoir through an opening in the cell culture vessel.

[0301] Aspect 173 is directed to the system described in aspect 172, wherein the opening is one of the inlet and the outlet of the cell culture vessel.

[0302] Embodiment 174 is directed to a system described in any one of embodiments 164 to 173, wherein the cell culture vessel comprises a substrate support within the reservoir, the substrate support configured to guide, align, or secure the woven substrate within the culture space.

[0303] Aspect 175 is directed to the system described in Aspect 174, wherein the substrate support comprises a support member extending from one of the first end and the second end toward the other of the first end and the second end, and the rolled woven substrate is configured to surround at least a portion of the circumference of the support member so that the support member is parallel to the central longitudinal axis of the rolled woven substrate.

[0304] Example 176 is directed to the system of any one of Examples 164-175, wherein the central longitudinal axis is perpendicular to the direction of gravity during cell culture.

[0305] Embodiment 177 is directed to a system described in any one of embodiments 164 to 175, wherein at least one of the reservoir and the cell culture matrix is ​​configured to rotate about a central longitudinal axis of the bioreactor vessel during cell culture.

[0306] Embodiment 178 is directed to the system of embodiment 177, wherein the bioreactor system is configured such that the substrate moves through a cell culture fluid during the rotation of the cell culture vessel.

[0307] Embodiment 179 is directed to the cell culture system of embodiment 177 or embodiment 178, wherein the bioreactor system further comprises a rotation means operably coupled to the cell culture vessel and configured to rotate the cell culture vessel about the central longitudinal axis.

[0308]

[0041] Embodiment 180 is directed to a system described in any one of embodiments 164 to 179, wherein the cylindrical cell culture matrix comprises a woven cell culture substrate and does not comprise any other solid material between adjacent surfaces of the cell culture substrate.

[0309] Embodiment 181 is directed to a method of culturing cells in a bioreactor, the method including the steps of: providing a bioreactor vessel comprising: a cell culture chamber within the bioreactor vessel; and a cell culture matrix disposed within the cell culture chamber and configured for culturing cells thereon, the cell culture matrix including a substrate having a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and passing through the thickness of the substrate; seeding cells onto the cell culture matrix; culturing the cells on the cell culture matrix; and harvesting a product of the step of culturing the cells, the plurality of openings in the substrate configured to allow flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate.

[0310] Example 182 is directed to the method of example 181, wherein the substrate comprises at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh sheet.

[0311] Example 183 is directed to the method of example 181 or example 182, wherein the substrate comprises a polymeric material.

[0312] Aspect 184 is directed to the method of aspect 183, wherein the polymer material is at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0313] Example 185 is directed to the method of any one of Examples 181 to 184, wherein the seeding step comprises allowing the cells to adhere to the substrate.

[0314] Example 186 is directed to the method of any one of Examples 181 to 185, wherein the seeding step comprises injecting a cell inoculum directly into the cell culture matrix.

[0315] Example 187 is directed to a method according to example 186, wherein the cell inoculum is injected through a cell inoculum injection port of the bioreactor vessel.

[0316] Example 188 is directed to the method of example 186 or example 187, wherein the volume of the cell inoculum is approximately equal to the void volume of the cell culture chamber.

[0317] Example 189 is directed to the method of any one of Examples 186 to 188, further comprising perfusing cell culture medium through the culture chamber after the step of injecting the cell inoculum.

[0318] Example 190 is directed to the method of any one of Examples 181 to 189, further comprising providing at least one of cell culture medium and oxygen to the cells during culturing.

[0319] Aspect 191 is directed to a method according to aspect 190, wherein the step of supplying the cell culture medium includes a step of flowing the cell culture medium through the cell culture chamber and across the substrate.

[0320] Example 161 is directed to a method according to example 190 or example 191, wherein the step of supplying the cell culture medium comprises: providing a medium conditioning vessel fluidly connected to the bioreactor vessel; and supplying the cell culture medium from the medium conditioning vessel to the bioreactor vessel.

[0321] Example 193 is directed to the method of example 192, wherein during or after culturing, at least a portion of the medium is removed from the bioreactor vessel and returned to the medium conditioning vessel.

[0322] Aspect 194 is directed to a method according to any one of aspects 181 to 193, further comprising controlling the flow of the cell culture medium into the cell culture chamber, the cell culture medium comprising at least one of cells, cell culture nutrients, and oxygen.

[0323] Example 195 is directed to the method of any one of Examples 181 to 194, further comprising analyzing the cell culture medium, the cells, and / or the cell products in or exiting the bioreactor vessel.

[0324] Aspect 196 is directed to a method according to aspect 195, wherein the analyzing step includes measuring at least one of pH1, pO1, [glucose]1, pH2, pO2, [glucose]2, and flow rate, wherein pH1, pO1, and [glucose]1 are measured within the cell culture chamber, and pH2, pO2, and [glucose]2 are measured at the outlet of the cell culture chamber or the bioreactor vessel.

[0325] Aspect 197 is directed to a method according to aspect 195 or aspect 196, wherein the flow of the cell culture medium into the cell culture chamber is controlled based at least in part on the results of the step of analyzing the cell culture medium, the cells, and / or the cell products.

[0326] In embodiment 198, pH2 > pH 2min , pO2 ≥ pO 2min , and [glucose]2 ≥ [glucose] 2min the perfusion flow rate of the cell culture medium into the cell culture chamber is continued at the current flow rate, and pH 2min , pO 2min , and [glucose] 2min is predetermined based on the design of the cell culture system.

[0327] Example 199 is directed to the method of any one of Examples 196-198, wherein the perfusion flow rate is increased if the current flow rate is less than or equal to a predetermined maximum flow rate for the cell culture system.

[0328] Embodiment 200 further comprises: if the current flow rate is greater than the predetermined maximum flow rate of the cell culture system, a controller of the cell culture system: 2min , pO 2min , and [glucose] 2min and reassessing at least one of the pH, pO, and [glucose]; and the bioreactor vessel.

[0329] Embodiment 201 is directed to the method of any one of embodiments 181 to 200, wherein the cells have a viability of greater than about 90% or greater than about 95% after culturing for at least about 24 hours, at least about 48 hours, or at least about 72 hours.

[0330] Example 202 is directed to the method of any one of Examples 181 to 201, wherein the cells comprise at least one of adherent cells attached to the cell culture matrix, suspension cells, and weakly adherent cells.

[0331] Aspect 203 is directed to the method of any one of aspects 181 to 202, wherein the product of the step of culturing the cells comprises at least one of cells, proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0332] Aspect 204 is directed to the method of aspect 203, wherein the product of the step of culturing the cells comprises cells that are at least 80% viable, at least 85% viable, at least 90% viable, at least 91% viable, at least 92% viable, at least 93% viable, at least 94% viable, at least 95% viable, at least 96% viable, at least 97% viable, at least 98% viable, or at least 99% viable.

[0333] Embodiment 205 is directed to a cell culture matrix, the cell culture matrix comprising a woven substrate including a plurality of interwoven fibers and a plurality of openings disposed between the plurality of fibers, each of the fibers having a surface configured to adhere cells.

[0334] Embodiment 206 is directed to the matrix of embodiment 205, wherein the surface of the fibers is configured to releasably adhere cells.

[0335] Example 207 is directed to the matrix of example 205 or example 206, wherein the plurality of fibers comprises polymer fibers.

[0336] Aspect 208 is directed to the matrix of aspect 207, wherein the polymer fibers comprise at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0337] Example 209 is directed to the matrix of any one of Examples 205 to 208, wherein the cell culture matrix further comprises a plurality of woven substrates.

[0338] Embodiment 210 is directed to the matrix of embodiment 209, wherein each of the plurality of substrates has: a first side; a second side opposite the first side; and a thickness separating the first side and the second side, and the plurality of openings pass through the thickness of the substrate.

[0339] Aspect 211 is directed to a matrix described in aspect 209 or aspect 210, wherein the multiple substrates are arranged adjacent to one another such that one of the first side and the second side of one of the substrates is adjacent to the other of the first side and the second side of an adjacent substrate.

[0340] Example 212 is directed to the matrix of any one of Examples 209-211, wherein at least a portion of the plurality of substrates are not separated by a spacer material or barrier.

[0341] Example 213 is directed to the matrix of any one of Examples 205-212, wherein at least a portion of the substrates are in physical contact with one another.

[0342] definition "Wholely synthetic" refers to a cell culture article, such as a microcarrier or culture vessel surface, that is composed entirely of synthetic raw materials and does not contain any animal-derived or animal-sourced materials. The fully synthetic cell culture articles of the present disclosure eliminate the risk of cross-species contamination.

[0343] Terms such as "include" and "includes" mean including but not limited to the subject matter, i.e., inclusive and not exclusive.

[0344] "User" refers to a person who uses a system, method, article, or kit disclosed herein, including a person who cultures cells for harvesting cells or cell products, or who uses cells or cell products cultured and / or harvested according to embodiments herein.

[0345] "About," as used in describing embodiments of the present disclosure to modify values ​​and ranges, such as the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, viscosity, and the like of a component in a composition, or the dimensions of a component, refers to variations in numerical quantities that may arise, for example, from typical measuring and handling procedures used in preparing a material, composition, composite, concentrate, component, article of manufacture, or use formulation; from inadvertent errors in these procedures; from differences in the manufacture, source, or purity of the starting materials or ingredients used in carrying out the method; and from other considerations. The term "about" also encompasses amounts that differ from a particular initial concentration or mixture due to aging of the composition or formulation, and amounts that differ from a particular initial concentration or mixture due to mixing or processing of the composition or formulation.

[0346] "Optional" or "optionally" means: 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 the event or circumstance does not occur.

[0347] The indefinite articles "a" or "an" and their corresponding definite article "the", as used herein, unless otherwise stated, mean at least one, or one or more.

[0348] Abbreviations 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, "rt" for room temperature, "nm" for nanometers, and similar abbreviations).

[0349] Specific preferred values ​​and ranges disclosed for aspects such as components, ingredients, additives, dimensions, conditions, etc. are merely exemplary and do not exclude other defined values ​​or other values ​​within defined ranges. The systems, kits, and methods of the present disclosure can include any value or combination of these values, specific values, more specific values, and preferred values ​​set forth herein, including any intermediate values ​​and intermediate ranges that are stated or implied.

[0350] Unless otherwise specified, it is in no way intended that any method described herein be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps should be followed, or unless it is specifically stated in the claim or description that the steps are limited to a particular order, no particular order is intended to be implied.

[0351] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the embodiments of the present disclosure. Since modifications, combinations, sub-combinations, and variations of these embodiments incorporating the spirit and substance of the embodiments of the present disclosure may occur to those skilled in the art, the embodiments of the present disclosure shall be construed as including all that come within the scope of the appended claims and equivalents thereof.

[0352] Preferred embodiments of the present invention will be described below in detail.

[0353] Embodiment 1 1. A cell culture matrix comprising: The cell culture matrix comprises: a substrate comprising a first side, a second side opposite the first side, a thickness separating the first side and the second side, and a plurality of openings formed in the substrate and passing through the thickness of the substrate; Including, The cell culture matrix, wherein the plurality of openings are configured to allow flow of at least one of cell culture medium, cells, and cell products through the thickness of the substrate.

[0354] Embodiment 2 2. The cell culture matrix of embodiment 1, wherein the substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0355] Embodiment 3 2. The cell culture matrix of embodiment 1, wherein the substrate comprises at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh sheet.

[0356] Embodiment 4 4. The cell culture matrix of embodiment 3, wherein the substrate comprises a woven mesh comprising one or more fibers.

[0357] Embodiment 5 5. The cell culture matrix of embodiment 4, wherein the one or more fibers have a cross-sectional shape that is at least one of flat, circular, rectangular, and polygonal.

[0358] Embodiment 6 5. The cell culture matrix of embodiment 4, wherein the one or more fibers comprise at least one of monofilament and multifilament fibers.

[0359] Embodiment 7 5. The cell culture matrix of embodiment 4, wherein the one or more fibers comprise first fibers having a first fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0360] Embodiment 8 8. The cell culture matrix of embodiment 7, wherein the one or more fibers further comprise second fibers having a second fiber diameter of about 50 μm to about 1000 μm, about 50 μm to about 600 μm, about 50 μm to about 400 μm, about 100 μm to about 325 μm, or about 150 μm to about 275 μm.

[0361] Embodiment 9 9. The cell culture matrix of embodiment 8, wherein the second fiber diameter is different from the first fiber diameter.

[0362] Embodiment 10 2. The cell culture matrix according to embodiment 1, wherein the plurality of openings have an opening diameter of about 100 μm to about 1000 μm, about 200 μm to about 900 μm, or about 225 μm to about 800 μm.

[0363] Embodiment 11 the fiber diameter is about 250 μm to about 300 μm and the aperture diameter is about 750 μm to about 800 μm; or 11. The cell culture matrix of embodiment 10, wherein the fiber diameter is about 270 μm to about 276 μm and the aperture diameter is about 785 μm to about 795 μm.

[0364] Embodiment 12 the fiber diameter is about 200 μm to about 230 μm and the aperture diameter is about 500 μm to about 550 μm; or 11. The cell culture matrix of embodiment 10, wherein the fiber diameter is about 215 μm to about 225 μm and the aperture diameter is about 515 μm to about 530 μm.

[0365] Embodiment 13 the fiber diameter is about 125 μm to about 175 μm and the aperture diameter is about 225 μm to about 275 μm; or 11. The cell culture matrix of embodiment 10, wherein the fiber diameter is about 150 μm to about 165 μm and the aperture diameter is about 235 μm to about 255 μm.

[0366] Embodiment 14 11. The cell culture matrix of embodiment 10, wherein the ratio of the aperture diameter to the fiber diameter is about 1.0 to about 3.5, about 1.25 to about 3.25, about 1.4 to about 3.0, about 1.5 to about 2.9, about 1.5 to about 2.4, or about 2.4 to about 2.9.

[0367] Embodiment 15 2. The cell culture matrix of embodiment 1, wherein the plurality of openings comprises openings having a square, rectangular, diamond, rhomboid, circular, or oval shape.

[0368] Embodiment 16 2. The cell culture matrix of embodiment 1, wherein the plurality of openings are arranged in a regular pattern.

[0369] Embodiment 17 2. The cell culture matrix of embodiment 1, wherein the cell culture matrix comprises a single layer substrate.

[0370] Embodiment 18 The cell culture matrix comprises a multi-layer substrate comprising at least one first substrate layer and at least one second substrate layer; 2. The cell culture matrix of embodiment 1, wherein the first substrate layer comprises a first side and a second side opposite the first side, and the second substrate layer comprises a third side and a fourth side opposite the third side, the second side facing the third side.

[0371] Embodiment 19 19. The cell culture matrix of embodiment 18, wherein the multi-layer substrate is configured such that the first substrate layer has a predetermined alignment with the second substrate layer.

[0372] Embodiment 20 20. The cell culture matrix of embodiment 19, wherein the multilayer substrate is configured such that fiber intersections of the first substrate layer face the openings of the second substrate layer.

[0373] Embodiment 21 21. The cell culture matrix of embodiment 19 or 20, wherein the openings in the first substrate layer at least partially overlap with the openings in the second substrate layer.

[0374] Embodiment 22 22. The cell culture matrix of embodiment 21, wherein the openings in the first and second substrate layers are aligned.

[0375] Embodiment 23 20. The cell culture matrix of embodiment 18, wherein the multi-layer substrate is configured such that the first substrate layer has random alignment relative to the second substrate layer.

[0376] Embodiment 24 2. The cell culture matrix of embodiment 1, wherein the cell culture matrix comprises a plurality of the substrates, each of the plurality of substrates being randomly oriented relative to the other substrates of the plurality of substrates.

[0377] Embodiment 25 2. The cell culture matrix of embodiment 1, wherein the cell culture matrix comprises a plurality of the substrates in a stacked configuration.

[0378] Embodiment 26 26. The cell culture matrix of embodiment 25, wherein the first side and the second side of one of the substrates are generally parallel to the first side and the second side of the other substrates in the stacked configuration.

[0379] Embodiment 27 2. The cell culture matrix of embodiment 1, wherein the substrate is in a cylindrical roll configuration.

[0380] Embodiment 28 2. The cell culture matrix of embodiment 1, wherein the cell culture matrix comprises a plurality of said substrates comprising woven meshes of different geometries, the different geometries differing in at least one of fiber diameter, opening diameter, or opening geometry.

[0381] Embodiment 29 29. The cell culture matrix of embodiment 28, wherein the woven meshes of different geometries are arranged in a predetermined arrangement based on desired flow characteristics within the bioreactor vessel.

[0382] Embodiment 30 30. The cell culture matrix of embodiment 29, wherein the desired flow characteristics include at least one of uniform perfusion of liquid medium throughout the cell culture matrix and distribution of cell growth throughout the cell culture matrix.

[0383] Embodiment 31 the woven meshes of different geometries include a first mesh having a first geometry and a second mesh having a second geometry; 30. The cell culture matrix of embodiment 29, wherein the predetermined arrangement comprises the first mesh being upstream of the second mesh with respect to a desired bulk flow direction of cell culture medium through the cell culture matrix.

[0384] Embodiment 32 32. The cell culture matrix of embodiment 31, wherein the predetermined arrangement comprises placing the first mesh stack upstream of the second mesh stack.

[0385] Embodiment 33 32. The cell culture matrix of embodiment 31, wherein the predetermined arrangement comprises an alternating arrangement of multiple stacks of the first mesh and multiple stacks of the second mesh along the bulk flow direction.

[0386] Embodiment 34 2. The cell culture matrix of embodiment 1, wherein the cell culture matrix is ​​configured for the culture and / or harvest of at least one of cells, proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0387] Embodiment 35 2. The cell culture matrix of embodiment 1, wherein the substrate comprises a functionalized surface, the functionalized surface being physically or chemically modified to improve adhesion of the adherent cells to the polymer mesh material.

[0388] Embodiment 36 2. The cell culture matrix of embodiment 1, wherein the cell culture matrix comprises a surface configured for adsorption or absorption of components in the culture medium onto the surface of the mesh.

[0389] Embodiment 37 2. The cell culture matrix of embodiment 1, wherein the cell culture matrix comprises a coating on a surface of the polymer mesh material, the coating being configured to promote attachment of the adherent cells.

[0390] Embodiment 38 38. The cell culture matrix of embodiment 37, wherein the cells adhere to the coating.

[0391] Embodiment 39 38. The cell culture matrix of embodiment 37, wherein the coating is a biological molecule or a synthetic bioactive molecule configured to promote cell attachment to the cell culture matrix.

[0392] Embodiment 40 38. The cell culture matrix of embodiment 37, wherein the coating is at least one of a hydrogel, collagen, Matrigel, a bioactive molecule or peptide, and a biological protein.

[0393] Embodiment 41 38. The cell culture matrix of embodiment 37, wherein the functionalized surface is plasma treated.

[0394] Embodiment 42 2. The cell culture matrix of embodiment 1, wherein the cells comprise at least one of adherent cells, suspension cells, and weakly adherent cells that adhere to the woven mesh. [Explanation of symbols]

[0395] 100 cell culture substrate 102 first plurality of fibers 104 second plurality of fibers 106 Aperture 108 first side 110 second side 200 Multilayer substrate, matrix 202 first mesh substrate layer 204 Second mesh substrate layer 206 Aperture 300, 320, 360, 400, 420 Cell Culture Systems 302, 322, 362, 402 Bioreactor vessels 304, 324 Cell culture chamber 306, 364 Cell culture matrix 308, 328 Base material layer, base material 310, 330, 408 entrance 312, 332, 410 exits 350 cylindrical roll 352 Mesh base material 366 Support, central support member 404, 424 Medium conditioning container 406 Cell Culture Media 412 Sensors 414 Flow control unit, signal processing unit 416 Pumps, peristaltic pumps 418 Medium conditioning control unit 422 Packed-bed bioreactor, bioreactor 426 Controller 428 Perfusion circuit 500 roller bottles 501 Bottle Mouth 502 Cell Culture Mesh 503 Cylindrical Roll 600, 602 sample cell 620 Container 622 Filled bed region 624 Entrance 626 Exit 1801, 1801' First disc 1802, 1802' Second disc 1803, 1803' Third disc

Claims

1. 1. A perfusion bioreactor system for culturing cells, comprising: a cell culture container including at least one internal reservoir defining a cell culture space, an inlet fluidly connected to the reservoir, and an outlet fluidly connected to the reservoir; a cell culture matrix disposed within the culture space, the cell culture matrix including a substrate including a plurality of layers of a woven fabric substrate in a laminated configuration; Including, the substrate is configured to adhere cells, and includes a first surface, a second surface opposite the first surface, a width defined by the widths of the first surface and the second surface, a thickness separating the first surface and the second surface, a plurality of pores extending through the thickness of the substrate and arranged in an array, and monofilament fibers configured to maintain the pores and / or shape under fluid flow; the cell culture vessel is configured to allow fluid to flow from the inlet, through the cell culture matrix, and to the outlet in a direction generally parallel to a direction from the inlet to the outlet; the first surface and the second surface of each of the plurality of layers are oriented substantially perpendicular to a direction of fluid flow within the internal reservoir; The cell culture space has a width, and the width of the substrate extends across the width of the cell culture space; A bioreactor system wherein at least a portion of said plurality of layers are in physical contact with each other.

2. 10. The bioreactor system of claim 1, wherein each layer of the plurality of layers is in physical contact with an adjacent layer of the plurality of layers.

3. 10. The bioreactor system of claim 1, wherein the cell culture matrix has a uniform porosity per unit volume throughout.

4. 10. The bioreactor system of claim 1, wherein the multi-layer substrate comprises a multi-layer woven fabric substrate made from woven fibers defining openings therebetween, the multi-layer woven fabric substrate comprising woven fabric substrates of different geometries, the different geometries differing in at least one of fiber diameter, opening diameter, or opening geometry.

5. 10. The bioreactor system of claim 1, wherein the multi-layer substrate comprises a plurality of disks.

Images