Weaving cell culture substrate

Abstract

A cell culture substrate is provided, the cell culture substrate having 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 extending through the thickness of the substrate. The plurality of openings permit at least one of cell culture medium, cells, or cell products to flow through the thickness of the substrate and provide a uniform, effective, and scalable substrate for cell seeding, proliferation, and culture. The substrate can be formed of a woven polymeric mesh material that provides a high surface area to volume ratio for the cells and provides excellent fluid flow through the matrix. Also provided are bioreactor systems and related methods that include the cell culture substrate.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 910,696, filed Oct. 4, 2019, and U.S. Provisional Application No. 62 / 801,325, filed Feb. 5, 2019, under 35 U.S.C. § 120. This application is based on the content of both and incorporates the full text thereof by reference herein. Technical Field

[0003] The present disclosure generally relates to substrates for culturing cells, as well as systems and methods for culturing cells. Specifically, the present disclosure relates to cell culture substrates, bioreactor systems comprising such substrates, and methods of culturing cells using such substrates. Background Art

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

[0005] Most of the cells used in bioprocessing are anchorage - dependent, meaning that the cells require a surface to adhere to in order to grow and function. Traditionally, the culture of adherent cells has been carried out on two - dimensional (2D) surfaces that adhere cells, which are contained in one of a variety of container forms, such as T - flasks, Petri dishes, cell factories, cell stack containers, roller bottles, and containers. These methods may have significant drawbacks, including difficulty in achieving a sufficiently high cell density to make them suitable for large - scale production of cells or therapies.

[0006] Alternative methods have been proposed to increase the volumetric density of the cultured cells. These include microcarrier culture carried out in stirred tanks. In this method, cells adhered to the surface of the microcarriers are subjected to constant shear stress, resulting in a significant impact on proliferation and culture performance. Another example of a high - density cell culture system is a hollow - fiber bioreactor, in which cells can form large three - dimensional aggregates as they proliferate in the fiber interstitial space. However, cell growth and performance are significantly inhibited due to the lack of nutrients. To alleviate this problem, these bioreactors are made smaller, which is not suitable for large - scale manufacturing.

[0007] Another example of a high-density culture system for anchorage-dependent cells is a packed-bed bioreactor system. In this type of bioreactor, a cell substrate is used to provide a surface for adherent cells to attach to. The culture medium is perfused along the surface or through a semi-porous substrate to provide the nutrients and oxygen required for cell growth. For example, a packed-bed bioreactor system comprising a substrate or matrix system for capturing cells in a packed bed has been previously disclosed in U.S. Patent Nos. 4,833,083; 5,501,971 and 5,510,262. Packed-bed matrices are often made of porous particles used as substrates or non-woven microfibers of polymers. Such bioreactors serve to recycle and circulate the bioreactor. One of the major problems with such bioreactors is the non-uniform distribution of cells in the packed bed. For example, the packed bed serves as a depth filter and cells are mainly captured at the inlet region, resulting in a cell distribution gradient during the seeding step. In addition, due to the random packing of the fibers, the flow resistance and cell capture efficiency across the cross-section of the packed bed are non-uniform. For example, the culture medium flows rapidly through regions with a low cell packing density, while flowing slowly through regions with a high resistance due to a higher number of captured cells. This results in a channeling effect, where nutrients and oxygen are more effectively delivered to regions with a lower cell volume density, while regions with a higher cell density remain under sub-optimal culture conditions.

[0008] Another significant drawback of the packed-bed systems disclosed in the prior art is the inability to efficiently harvest intact live cells at the end of the culture process. Cell harvesting is important if the final product is cells, or if the bioreactor is used as part of a "seed train", where a cell population is grown in one vessel and then transferred to another vessel for further cell population growth. U.S. Patent No. 9,273,278 discloses a bioreactor design for improving the efficiency of cell recovery from a packed bed during the cell harvesting step. It is based on loosening the packed-bed matrix and agitating or stirring the packed-bed particles to cause the porous matrix to collide and thus detach the cells. However, this method is laborious and may cause significant cell damage, thus reducing the overall cell viability.

[0009] An example of a packed-bed bioreactor currently on the market is produced by Pall Corporation The iCellis uses small strips of cell substrate material composed of randomly oriented fibers in a non-woven arrangement. 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 also has drawbacks. Specifically, the non-uniform packing of the substrate strips creates visible channels within the packed bed, resulting in preferential flow of the culture medium and nutrients through the packed bed and non-uniform distribution. The research shows "systematic non-uniform distribution of cells, and the number of cells increases from the top to the bottom of the fixed bed", as well as "nutrient gradients... lead to limited cell growth and reproduction", all of which result in "non-uniform cell distribution, which may weaken transfection efficiency." [Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells. Biotechnol. J. 2016, 11, 290–297]. The study points out that agitation of the packed bed may improve dispersion, but there are also other drawbacks (i.e., "agitation necessary for better dispersion during seeding and transfection leads to increased shear stress, which in turn leads to reduced cell viability." Ibid.). Another study on points out that the non-uniform distribution of cells makes it difficult to monitor cell populations using biomass sensors ("... if the cell distribution is non-uniform, the biomass signal from cells on the top carrier may not represent the overall bioreactor." Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale. Human Gene Therapy, Vol. 26, No. 8, 2015).

[0010] In addition, due to the random arrangement of fibers in the substrate strips and the variability of strip packing between one packed bed and another packed bed, customers may have difficulty predicting cell culture performance because the substrates between cultures are different. Additionally, the packed substrate makes it very difficult or impossible to effectively harvest cells because it is believed that cells are retained by the packed bed.

[0011] Roll bottles have several advantages, such as ease of handling and the ability to monitor cells on an adherent surface. However, from a production perspective, the main disadvantages are the low surface area to volume ratio and the fact that the roll bottle configuration occupies a large area of manufacturing floor space. Various methods have been used to increase the surface area available to adherent cells in the roll bottle format. Some solutions have been implemented in commercially available products, but there is still room for improvement to further increase roll bottle productivity. Traditionally, roll bottles are produced as a single structure by blow molding. This manufacturing simplicity makes roll bottles economically viable in the bioprocessing industry. Some roll bottle improvements to increase the available surface area for cell culture can be achieved without changing the manufacturing process, but the improved roll bottle surface area only achieves a marginal increase. Other changes to the roll bottle design significantly increase the complexity of the manufacturing process, making it economically unviable in the bioprocessing industry. Therefore, there is a need to provide roll bottles with increased surface area and bioprocess productivity while using the same blow molding process for manufacturing.

[0012] While it is possible to produce viral vectors for early clinical trials using existing platforms, there is a need for platforms that can produce more high-quality products to reach late-stage commercial production scale.

[0013] There is a need for cell culture substrates, systems, and methods that can culture cells in a high-density form, have a uniform cell distribution, and are easy to access and harvest with increased yields. SUMMARY OF THE INVENTION

[0014] According to one embodiment of the present disclosure, a cell culture substrate is provided. The cell culture substrate 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 are configured to allow at least one of a cell culture medium, cells, or cell products to flow through the thickness of the substrate. The substrate can be at least one of a molded polymeric lattice sheet, a 3D printed lattice sheet, and a woven mesh. The substrate has a rectangular ordered structure and provides a surface for cell adhesion, growth, and eventual cell release.

[0015] According to one embodiment of the present disclosure, a bioreactor system for cell culture is provided. The system includes a cell culture vessel having at least one reservoir; and a cell culture substrate disposed in the at least one reservoir, the cell culture substrate including a woven substrate having a plurality of interwoven fibers, and the surface of the fibers being configured for cell adhesion.

[0016] According to one or more embodiments, a cell culture system is provided. The system includes a bioreactor vessel; and a cell culture substrate disposed in the bioreactor vessel and configured to culture cells. The cell culture substrate includes a substrate that includes 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 extending through the thickness of the substrate, the plurality of openings being configured to allow at least one of a cell culture medium, cells, or cell products to flow through the thickness of the substrate.

[0017] 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 substrate disposed in the at least one reservoir. The cell culture substrate has a plurality of woven substrates, each woven substrate including a plurality of interwoven fibers, and the surface of the fibers being configured for cell adhesion. The bioreactor system is configured to allow material to flow through the at least one reservoir in a flow direction from the first end to the second end, and the substrates in the plurality of woven substrates are stacked such that each woven substrate is substantially parallel to each other woven substrate and substantially perpendicular to the flow direction.

[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 substrate disposed in the at least one reservoir, the cell culture substrate including a plurality of woven substrates, each woven substrate having a plurality of interwoven fibers, and the surface of the fibers being configured for cell adhesion. The bioreactor system is configured to allow material to flow through the at least one reservoir in a flow direction from the first end to the second end, and the substrates in the plurality of woven substrates are stacked such that each woven substrate is substantially parallel to each other woven substrate and substantially parallel 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 substrate disposed in the at least one reservoir. The cell culture substrate includes a woven substrate that includes a plurality of interwoven fibers and a surface configured for cell adhesion, the woven substrate being disposed in the at least one reservoir in a wound configuration to provide a cylindrical cell culture substrate, and the surface of the woven substrate being parallel to the longitudinal axis of the cylindrical cell culture substrate.

[0020] According to another embodiment, a method of culturing cells in a bioreactor is provided. The method includes: providing a bioreactor vessel having a cell culture chamber within the bioreactor vessel and a cell culture substrate disposed in the cell culture chamber. The cell culture substrate is provided for culturing cells thereon. The cell culture substrate 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 extending through the thickness of the substrate. The method further includes: inoculating cells on the cell culture substrate; culturing cells on the cell culture substrate; and harvesting the product of the cell culture. The plurality of openings in the substrate allow at least one of cell culture medium, cells, or cell products to flow through the thickness of the substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1A According to one or more embodiments of the present disclosure, a perspective view of a three-dimensional model of a cell culture substrate is shown.

[0022] Figure 1B is Figure 1A a two-dimensional plan view of the substrate.

[0023] Figure 1C is Figure 1B a cross-section of the substrate in along line A-A.

[0024] Figure 2A According to some embodiments, an example of a cell culture substrate is shown.

[0025] Figure 2B According to some embodiments, an example of a cell culture substrate is shown.

[0026] Figure 2C According to some embodiments, an example of a cell culture substrate is shown.

[0027] Figure 3A According to one or more embodiments, a perspective view of a multi-layer cell culture substrate is shown.

[0028] Figure 3B According to one or more embodiments, a plan view of a multi-layer cell culture substrate is shown.

[0029] Figure 4 According to one or more embodiments, shown is Figure 3B a cross-sectional view of the multi-layer cell culture substrate along line B-B.

[0030] Figure 5 According to one or more embodiments, shown is Figure 4 a cross-sectional view of the multi-layer cell culture substrate along line C-C.

[0031] Figure 6 According to one or more embodiments, a schematic diagram of a cell culture system is shown.

[0032] Figure 7 According to one or more embodiments, a schematic diagram of a cell culture system is shown.

[0033] Figure 8 According to one or more embodiments, a cell culture substrate in a rolled cylindrical configuration is shown.

[0034] Figure 9 According to one or more embodiments, a cell culture system comprising a rolled cylindrical cell culture substrate is shown.

[0035] Figure 10A According to one or more embodiments, it is a schematic representation of a cell culture system.

[0036] Figure 10B According to one or more embodiments, it is a detailed schematic diagram of a cell culture system.

[0037] Figure 11A According to one or more embodiments, a process flow diagram for culturing cells on a cell culture system is shown.

[0038] Figure 11B According to one or more embodiments, an operation for controlling the perfusion flow rate of a cell culture system is shown.

[0039] Figure 12 According to one or more embodiments, is a micrograph of HEK293T stained cells on a cell culture substrate.

[0040] Figure 13A is a graph showing cell growth and expansion data of HEK293T cells on the substrate of Figure 12.

[0041] Figure 13B is a bar graph showing the viability of cells from Figures 12 and 13A.

[0042] Figure 14A is a photograph of a layer of a cell culture substrate with HEK293T stained cells, the HEK293T stained cells being from a bioreactor inoculated under static conditions.

[0043] Figure 14B is a photograph of a layer of a cell culture substrate with HEK293T stained cells, the HEK293T stained cells being from a bioreactor inoculated by the cell tumbling method.

[0044] Figure 15Cross-sectional view of a cell culture system with an extended cell culture substrate, according to one or more embodiments.

[0045] Figure 16 Cross-sectional view of a roller bottle type cell culture container with a multi-layer cell culture substrate, according to one or more embodiments.

[0046] Figure 17A Photograph of a cell culture substrate with stained cells after seeding in a roller bottle type cell culture system using a slow rotation speed during cell seeding, according to one or more embodiments.

[0047] Figure 17B Photograph of a cell culture substrate with stained cells after seeding in a roller bottle type cell culture system using a faster rotation speed during cell seeding, according to one or more embodiments.

[0048] Figure 18A Photograph of a disc from a cell culture matrix with stained cells after seeding and growth but before cell harvest, according to one or more embodiments.

[0049] Figure 18B Photograph of a disc from Figure 18A after cell harvest, according to one or more embodiments.

[0050] Figure 19A Shows experimental results of the total cells harvested from two examples according to embodiments of the present disclosure compared to a HYPER flask.

[0051] Figure 19B Shows experimental results of the total genomic copies / container in two examples according to embodiments of the present disclosure compared to a HYPER flask.

[0052] Figure 19C Shows experimental results of the genomic copies / surface area in two examples according to embodiments of the present disclosure compared to a HYPER flask.

[0053] Figure 20A Plan view of a molded multi-layer woven mesh cell culture substrate in a tightly packed arrangement, according to one or more embodiments of the present disclosure.

[0054] Figure 20B According to one or more embodiments of the present disclosure, shows Figure 20A side cross-sectional view of a multi-layer woven mesh cell culture substrate.

[0055] Figure 21A Plan view of a molded multi-layer woven mesh cell culture substrate in a loosely packed arrangement, according to one or more embodiments of the present disclosure.

[0056] Figure 21B According to one or more embodiments of the present disclosure, a side cross-sectional view of a multi-layer woven mesh cell culture substrate is shown. Figure 21A of

[0057] Figure 22A Shown is Figure 20A and 20B the molded empty space in the dotted-line volume shown in

[0058] Figure 22B Shown is Figure 21A and 21B the molded empty space in the dotted-line volume shown in

[0059] Figure 23 shows photographs of various mesh samples A - F from Table 5 according to one or more embodiments of the present disclosure.

[0060] Figure 24 is a bar graph of the permeability of the woven mesh samples A - F from Figure 23.

[0061] Figure 25 Shows the results of the pressure drop test using samples A - C from Figure 23.

[0062] Figure 26 According to one or more embodiments of the present disclosure, it is a schematic diagram of the seed domestication process.

[0063] Figure 27A According to one or more embodiments of the present disclosure, a flow uniformity model of a bioreactor with a woven mesh substrate is shown.

[0064] Figure 27B is Figure 27A an enlarged view of the flow uniformity model of

[0065] Figure 28 is a bar graph of the permeability measurements of woven and non-woven cell culture substrates.

[0066] Figure 29A Shows the simulated flow velocity around a non-woven mesh substrate sheet arranged at 90° to the flow direction.

[0067] Figure 29B Shows the simulated flow velocity around a non-woven mesh substrate sheet arranged at 45° to the flow direction.

[0068] Figure 30A Shows the simulated flow velocity around a non-woven mesh substrate sheet with a 1 mm gap between all adjacent sheets.

[0069] Figure 30BShows the simulated flow velocity around an open-weave mesh with 1 mm gaps between all adjacent sheets.

[0070] Figure 31 Is a schematic diagram of an experimental setup for measuring the residence time distribution of different cell culture substrate samples.

[0071] Figure 32 Is a graph showing the relationship between the change in dye concentration and time for woven and non-woven cell culture substrates during the residence time distribution test. Detailed Description of the Invention

[0072] The various embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings (if any). Referring to the various embodiments does not limit the scope of the present invention, which is limited only by the scope of the appended claims. In addition, any examples set forth in this specification are not restrictive and only list some of the many possible embodiments of the claimed invention.

[0073] Embodiments of the present disclosure relate to cell culture substrates, and cell culture or bioreactor systems comprising such substrates, and to methods of culturing cells using such substrates and bioreactor systems.

[0074] In conventional large-scale cell culture bioreactors, different types of packed bed bioreactors have been used. Generally, these packed beds contain a porous matrix to retain adherent or suspended cells and support growth and proliferation. The packed bed matrix provides a high surface area / volume ratio, so the cell density can be higher than in other systems. However, the packed bed often acts as a depth filter, where cells are physically trapped or entangled in the fibers of the matrix. Thus, due to the linear flow of the cell inoculum through the packed bed, the cells are unevenly distributed within the packed bed, resulting in a variation in cell density along the depth or width of the packed bed. For example, the cell density may be higher at the inlet region of the bioreactor and significantly lower closer to the outlet of the bioreactor. This non-uniform distribution of cells within the packed bed significantly hinders the scalability and predictability of such bioreactors in bioprocess manufacturing and may even result in a reduction in the efficiency of cell growth or virus vector production per unit surface area or volume of the packed bed.

[0075] Another problem encountered in the packed-bed bioreactors disclosed by the prior art is the channeling effect. Due to the random nature of the packed non-woven fibers, the local fiber density is non-uniform at any given cross-section of the packed bed. The culture medium flows rapidly in regions of low fiber density (high bed permeability) and significantly slower in regions of high fiber density (lower bed permeability). The non-uniform perfusion of the culture medium across the packed bed creates a channeling effect, which manifests as significant nutrient and metabolite gradients, negatively impacting cell culture and the overall performance of the bioreactor. Cells located in regions of low culture medium perfusion starve and often die due to lack of nutrients or metabolite poisoning. When using a bioreactor packed with a non-woven fiber scaffold, cell harvesting is another problem encountered. Since the packed bed acts as a depth filter, the cells released at the end of the cell culture process are retained within the packed bed and the cell recovery rate is extremely low. This greatly limits the utilization of such bioreactors in bioprocesses where live cells are the product. Thus, the non-uniformity results in varying degrees of exposure of different regions to flow and shear, effectively reducing the available cell culture area, causing non-uniform culture, and interfering with transfection efficiency and cell release.

[0076] To address these and other problems of existing cell culture solutions, embodiments of the present disclosure provide cell growth substrates, the matrices of these substrates, and / or packed-bed systems using these substrates that are capable of achieving efficient and high-yield cell culture of anchorage-dependent cells, as well as the production of cell products (e.g., proteins, antibodies, virus particles). Embodiments include a porous cell culture matrix made of an ordered and regular array of porous substrate materials, the porous cell culture matrix capable of achieving uniform cell seeding and culture medium / nutrient perfusion, as well as efficient cell harvesting. Embodiments are also capable of providing scalable cell culture solutions, where the substrates and bioreactors can inoculate and grow cells and / or harvest cell products from process development scale to full production scale without sacrificing the uniform performance of the embodiments. For example, in some embodiments, the bioreactor can be easily scaled up from process development scale to production scale and have a comparable virus genome / substrate unit surface area (VG / cm 2 ) throughout the production scale. The harvestability and scalability of the embodiments herein enable their use in efficient seeding and domestication of cell populations grown at multiple scales on the same cell substrate. Additionally, the embodiments herein provide a cell culture matrix having a high surface area, which in combination with the other features described enables a high-yield cell culture solution. In some embodiments, for example, the cell culture substrates and / or bioreactors described herein can produce 10 16 to 10 18 virus genomes (VG) per batch.

[0077] In one embodiment, the matrix has a structurally defined surface area for adherent cell attachment and proliferation, and when assembled in a packed bed or other bioreactor, the matrix has good mechanical strength and forms a highly uniform multi-interconnected fluid network. In a specific embodiment, a mechanically stable non-degradable woven mesh can be used as a substrate to support adherent cell production. The cell culture matrix disclosed herein supports the adhesion and propagation of anchorage-dependent cells in a high volumetric density form. Such a matrix enables uniform cell seeding, as well as efficient harvesting of cells or other products of the bioreactor. Additionally, embodiments of the present disclosure support this cell culture to provide a uniform cell distribution during the seeding step and achieve confluent monolayer or multilayer adherent cells on the disclosed matrix, and can avoid the formation of large and / or uncontrollable 3D cell aggregates with limited nutrient perfusion and increased metabolite concentrations. Thus, the matrix eliminates diffusion limitations during bioreactor operation. Moreover, the matrix enables easy and efficient harvesting of cells from the bioreactor. The structurally defined matrix of one or more embodiments enables complete recovery of cells from the packed bed of the bioreactor and achieves consistent cell harvesting.

[0078] According to some embodiments, there is also provided a method of using a bioreactor having a matrix for cell culture for the bioprocess production of therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0079] Unlike currently used cell culture substrates for bioreactors (i.e., non-woven substrates of randomly arranged fibers), embodiments of the present disclosure include cell culture substrates having a defined and ordered structure. This defined and ordered structure allows for consistent and predictable cell culture results. Additionally, the substrate has an open porous structure that prevents cell entrapment and enables uniform flow through the packed bed. This configuration can improve cell seeding, nutrient delivery, cell growth, and cell harvesting. According to one or more specific embodiments, the matrix is formed from a substrate material having a thin sheet-like configuration with a first side and a second side separated by a relatively small thickness such that the thickness of the sheet is small relative to the width and / or length of the first and second sides of the substrate. Further, a plurality of holes or openings are formed through the thickness of the substrate. The size and geometry of the substrate material between the openings allow cells to adhere to the surface of the substrate material as if it were an approximately two-dimensional (2D) surface while also allowing sufficient fluid to flow around and through the openings in the substrate material. In some embodiments, the substrate is a polymer-based material and can be formed as a molded polymer sheet; a polymer sheet having openings that penetrate the thickness; a plurality of filaments fused into a mesh layer; a 3D printed substrate; or a plurality of filaments woven into a mesh layer. The physical structure of the matrix has a high surface / volume ratio for culturing anchorage-dependent cells. According to various embodiments, the matrix can be arranged or packed in a bioreactor in certain ways described herein for uniform cell seeding and growth, uniform medium perfusion, and efficient cell harvesting.

[0080] Embodiments of the present disclosure enable a practical-sized viral vector platform that can produce viral genomes on the following scales: greater than about 10 14 viral genomes per batch, greater than about 10 15 viral genomes per batch, greater than about 10 16 viral genomes per batch, greater than about 10 17 viral genomes per batch, or up to or greater than about 10 16 viral genomes per batch. In some embodiments, the productivity is from about 10 15 to about 10 18 or more viral genomes per batch. For example, in some embodiments, the viral genome yield can be from about 10 15 to about 10 16 viral genomes per batch, or from about 10 16 to about 10 19 viral genomes per batch, or from about 10 16 -10 18 viral genomes per batch, or from about 10 17 to about 10 19a viral genome, or about 10 per batch 18 to about 10 19 viral genomes, or about 10 18 or more viral genomes.

[0081] In addition, the embodiments disclosed herein are not only capable of enabling cells to adhere to a cell culture substrate and grow, but also of harvesting the cultured cells alive. The inability to harvest live cells is a significant drawback of existing platforms and it has led to difficulties in building and maintaining a sufficient number of cells for production capacity. According to one aspect of the embodiments of the present disclosure, live cells can be harvested from the cell culture substrate, including 80% to 100% live cells, or about 85% to about 99% live cells, or about 90% to about 99% live cells. For example, at least 80% of the harvested cells are alive, at least 85% are alive, at least 90% are alive, at least 91% are alive, at least 92% are alive, at least 93% are alive, at least 94% are alive, at least 95% are alive, at least 96% are alive, at least 97% are alive, at least 98% are alive, or at least 99% are alive. Cells can be released from the cell culture substrate using, for example, trypsin, TrypLE, or Accutase.

[0082] Figure 1A and 1B Examples according to one or more embodiments of the present disclosure respectively show a three-dimensional (3D) perspective view and a two-dimensional (2D) plan view of a cell culture substrate 100. The cell culture substrate 100 is a woven mesh layer made of a first plurality of fibers 102 traveling in a first direction and a second plurality of fibers 104 traveling in a second direction. The woven fibers of the substrate 100 form a plurality of openings 106, and the plurality of openings 106 can be defined by one or more widths or diameters (e.g., D1, D2). The size and shape of the openings can vary based on the type of weaving (e.g., the number, shape, and size of the filaments; the angle between intersecting filaments, etc.). The woven mesh can be characterized as a two-dimensional sheet or layer at the macroscopic scale. However, upon closer inspection of the woven mesh, it is found that due to the rising and falling of the intersecting fibers of the mesh, it has a three-dimensional structure. Thus, as Figure 1C shown, the thickness T of the woven mesh 100 can be thicker than the thickness of a single fiber (e.g., t1). As used herein, the thickness T is the maximum thickness between the first side 108 and the second side 110 of the woven mesh. Without being bound by theory, it is believed that the three-dimensional structure of the structure 100 is advantageous because it provides a large surface area for culturing adherent cells, and the structural rigidity of the mesh can provide a consistent and predictable cell culture matrix structure that enables uniform fluid flow.

[0083] In Figure 1BIn [the context], the opening 106 has a diameter D1 and a diameter D2. The diameter D1 is defined as the distance between opposite fibers 102, and the diameter D2 is defined as the distance between opposite fibers 104. Depending on the weaving geometry, D1 and D2 may be equal or unequal. In the case where D1 and D2 are unequal, the larger one may be referred to as the major diameter, and the smaller one may be referred to as the minor diameter. In some embodiments, the diameter of the opening may refer to the widest part of the opening. Unless otherwise specified, the opening diameter used herein shall refer to the distance between parallel fibers on opposite sides of the opening.

[0084] A given fiber among the plurality of fibers 102 has a thickness t1, and a given fiber among the plurality of fibers 104 has a thickness t2. In the case of fibers with a circular cross-section, as Figure 1A or as shown in other three-dimensional cross-sectional views, the thicknesses t1 and t2 are the maximum diameters or thicknesses of the fiber cross-sections. According to some embodiments, all of the plurality of fibers 102 have the same thickness t1, and all of the plurality of fibers 104 have the same thickness t2. Additionally, t1 and t2 may be equal. However, in one or more embodiments, t1 and t2 are not equal. For example, when the plurality of fibers 102 are different from the plurality of fibers 104. Furthermore, each of the plurality of fibers 102 and the plurality of fibers 104 may comprise two or more different thicknesses of fibers (e.g., t 1a 、t 1b etc. and t 2a 、t 2b etc.). According to the embodiments, the thicknesses t1 and t2 are large relative to the size of the cells cultured thereon. Thus, from the perspective of the cells, the fibers provide a substantially flat surface, which enables better cell adhesion and growth compared to some other solutions where the fiber size is small (e.g., on the scale of the cell diameter). Due to the three-dimensional nature of the woven mesh, as Figure 1A - 1C shown, the 2D surface area of the fibers available for cell adhesion and proliferation exceeds the surface area that would adhere on a comparable planar 2D surface.

[0085] In one or more embodiments, the diameter of the fibers can be in the range of: about 50 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μm to about 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400 μm; about 200 μm to about 300 μm; or about 150 μm to about 300 μm. At the microscale level, since the fibers are larger than the scale of cells (e.g., fiber diameter is greater than cell), the surface of the monofilament fibers exists as a substantially 2D surface for adherent cells to adhere and proliferate. The fibers can be woven into a mesh, and the openings are in the range of about 100 μm x 100 μm to about 1000 μm x 1000 μm. In some embodiments, the diameter o of the openings 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. These ranges of filament diameters and opening diameters are examples of some embodiments, but they are not intended to limit the possible characteristic dimensions of the mesh for all embodiments. The combination of fiber diameter and opening diameter is selected to provide an effective and uniform fluid flow through the substrate, e.g., when the cell culture substrate includes multiple adjacent mesh layers (e.g., a stack of individual layers or a rolled mesh layer).

[0086] Factors such as fiber diameter, aperture diameter, and weave type / pattern will determine the surface area available for cell adhesion and growth. Additionally, when the cell culture substrate comprises a stack, roll, or other arrangement of overlapping substrates, the packing density of the cell culture substrate will affect the surface area of the packed bed substrate. The packing density can vary with the packing thickness of the substrate material (e.g., the space required for a layer of the substrate). For example, if a stack of cell culture substrates has a certain height, each layer of the stack can be said to have a packing thickness that is determined by dividing the total height of the stack by the number of layers in the stack. The packing thickness will vary based on the fiber diameter and weave, but can also vary based on the arrangement of adjacent layers in the stack. For example, due to the three-dimensional nature of the woven layers, adjacent layers can accommodate a certain amount of interlocking or overlap based on their arrangement relative to each other. In a first arrangement, adjacent layers can be closely adjacent to each other, but in a second arrangement, adjacent layers can have zero overlap, e.g., when the lowest point of the upper layer is in direct contact with the highest point of the lower layer. For certain applications, it may be desirable to provide a cell culture substrate with a lower density layer packing (e.g., when higher permeability is a priority), or it may be desirable to provide a cell culture substrate with a higher density packing (e.g., when maximizing the substrate surface area is a priority). According to one or more embodiments, the packing thickness can be from about 50 μm to about 1000 μm; from about 100 μm to about 750 μm; from about 125 μm to about 600 μm; from about 150 μm to about 500 μm; from about 200 μm to about 400 μm; from about 200 μm to about 300 μm.

[0087] The above-described structural factors can determine the surface area of the cell culture substrate, whether it is a cell culture substrate with a single layer of cell culture material or a cell culture substrate with multiple substrate layers. For example, in one specific embodiment, a single layer of a woven mesh substrate having a circular shape and a 6 cm diameter can have an effective surface area of about 68 cm 2 . As used herein, "effective surface area" is the total surface area of the fibers in a portion of the substrate material that is available for cell adhesion and growth. Unless otherwise specified, "surface area" refers to this effective surface area. According to one or more embodiments, a single layer of a woven mesh substrate having a 6 cm diameter can have an effective surface area of from about 50 cm 2 to about 90 cm 2 ; from about 53 cm 2 to about 81 cm 2 ; about 68 cm 2 ; about 75 cm 2 ; or about 81 cm 2 . These ranges of effective surface areas are provided for illustration only, and some embodiments can have different effective surface areas. The cell culture substrate can also be characterized in terms of porosity, as discussed in the examples herein.

[0088] The substrate mesh can be made of monofilament or multifilament fibers of polymer materials that are compatible in cell culture applications, such as polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. The mesh substrate can have different patterns or weaving forms, for example, including knitting, warp knitting, or weaving (such as plain weave, twill weave, Dutch weave, five-needle weave).

[0089] It may be necessary to modify the surface chemistry of the mesh filaments to provide the desired cell adhesion properties. These modifications can be achieved by chemical treatment of the polymer material of the mesh or by grafting cell adhesion molecules to the filament surface. Alternatively, the mesh can be coated with a thin layer of a biocompatible hydrogel (e.g., including collagen or ) that exhibits cell adhesion properties. Alternatively, the surface of the mesh filament fibers can be made to have cell adhesion properties by a treatment process using various types of plasmas, process gases, and / or chemicals known in the industry. However, in one or more embodiments, the mesh is capable of providing an effective cell growth surface without surface treatment.

[0090] Figure 2A - 2C Some contemplated embodiments according to the present disclosure illustrate different examples of woven meshes. The fiber diameters and opening sizes of these meshes, as well as the approximate magnitude of the increase in the cell culture surface area provided by a single layer of the corresponding mesh relative to a comparable 2D surface, are summarized in Table 1 below. In Table 1, Mesh A refers to Figure 2A the mesh of Figure 2B and Mesh B refers to Figure 2C the mesh of

[0091]

[0092]

[0093] Table 1 : Figure 2A - 2C Comparison of the meshes of

[0094] As shown in the table above, compared to a planar 2D surface of comparable dimensions, the three-dimensional nature of the mesh provides an increased surface area for cell adhesion and proliferation. This increased surface area contributes to the scalable performance enabled by the embodiments of the present disclosure. For process development and process validation studies, small-scale bioreactors are often desired to conserve reagent costs and increase experimental throughput. Embodiments of the present disclosure can be applied to such small-scale studies, but can also be scaled up to industrial or production scale. For example, if a 100-layer mesh C in the form of a 2.2 cm diameter circle is packed into a cylindrical packed bed with an inner diameter of 2.2 cm, the total surface area available for cell adhesion and proliferation is equal to approximately 935 cm 2 . To scale this bioreactor up by a factor of ten, a similar setup with a cylindrical packed bed having an inner diameter of 7 cm and 100 layers of the same mesh can be used. In this case, the total surface area will be equal to 9,350 cm 2 . In some embodiments, the available surface area is equal to or greater than approximately 99,000 cm 2 / L. Due to the plug-flow perfusion in the packed bed, the same flow rate can be used in both smaller and larger scale bioreactor formats, which is expressed in ml / min / cm 2 (cm 2 being the cross-sectional surface area of the packed bed). The larger surface area allows for higher seeding densities and higher cell growth densities. According to one or more embodiments, the cell culture substrates described herein exhibit cell seeding densities up to 22,000 cells / cm 2 or greater. For reference, Corning's has a seeding density of approximately 20,000 cells / cm 2 on a two-dimensional surface.

[0095] Another advantage of the higher surface area and high cell seeding or growth densities is that the cost of the embodiments disclosed herein can be the same as or less than that of competitive solutions. Specifically, the cost per cell product (e.g., per cell or per viral genome) can be equal to or less than the cost of cell products of other packed bed bioreactors.

[0096] In additional embodiments of the present disclosure described below, the woven mesh substrate can be packed inside the bioreactor in the form of a cylindrical roll (see Figure 8 and 9)。In such an embodiment, the scalability of the packed bed bioreactor can be achieved by increasing the total length and height of the mesh strips. The amount of mesh used for such a cylindrical roll configuration can vary based on the desired packing density of the packed bed. For example, the cylindrical roll can be tightly wound for dense packing or loosely wound for loose packing. The packing density will often be determined by the required surface area of the cell culture substrate needed for a given application or scale. In one embodiment, the required length of the mesh can be calculated according to the diameter of the packed bed bioreactor using the following formula:

[0097]

[0098] where L is the total length of the mesh required for the packed bioreactor (i.e., Figure 8 the H in Figure 9 ), R is the inner radius of the packed bed culture chamber, r is the radius of the internal support member around which the mesh is wound ( Figure 8 the support member 366 in

[0099] ), and t is the thickness of one layer of the 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., Figure 8 the W in

[0099] ) and / or increasing the height H of the packed bed cylindrical roll, and thus providing more substrate surface area for seeding and growing adherent cells.

[0100] Figure 3A An embodiment of the substrate with a multi-layer substrate 200 is shown, 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. Although the first substrate layer 202 and the second substrate layer 204 overlap, the geometric structure of the mesh (e.g., the ratio of the opening diameter to the fiber diameter) is such that the openings of the first substrate layer 202 and the second substrate layer 204 overlap and provide a path for fluid to flow through the entire thickness of the multi-layer substrate 200, as shown in Figure 3Bas shown by the fibril-free opening 206 in

[0101] Figure 4 shows a cross-sectional view of the multi-layer substrate 200 at line B-B in Figure 3B . The arrow 208 shows a possible fluid flow path through the opening in the second substrate layer 204 and then around the fibrils in the first substrate layer 202. The geometry of the mesh substrate layer is designed to allow efficient and uniform flow through one or more substrate layers. In addition, the structure of the matrix 200 may allow fluid to flow through the matrix in multiple orientations. For example, as Figure 4 shown, the overall fluid flow direction (as shown by arrow 208) is perpendicular to the main side surfaces of the first substrate layer 202 and the second substrate layer 204. However, the matrix can also be oriented relative to the flow such that the side surfaces of the substrate layers are parallel to the overall flow direction. Figure 5 shows a cross-sectional view of the multi-layer substrate 200 along line C-C in Figure 4 . The structure of the matrix 200 allows fluid flow (arrow 210) through the fluid path in the multi-layer substrate 200. In addition to fluid flow perpendicular or parallel to the first and second side surfaces of the mesh layer, the matrix can be arranged such that multiple substrate sheets are at intermediate angles, or even randomly arranged relative to the fluid flow. This flexibility in orientation is achieved through the substantially isotropic flow behavior of the woven substrate. In contrast, the substrates for adherent cells in existing bioreactors do not exhibit this behavior. Instead, their packed beds tend to produce preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the matrix of the present disclosure allows it to be used in a variety of applications and bioreactor or vessel designs, while achieving better and more uniform permeability throughout the bioreactor vessel.

[0102] As discussed herein, according to one or more embodiments, a cell culture substrate can be used within a bioreactor vessel. For example, the substrate can be used in a packed bed bioreactor configuration, or other configurations within a three-dimensional culture chamber. However, the embodiments are not limited to three-dimensional culture spaces, and it is contemplated that the substrate can be used in configurations that can be considered two-dimensional culture surface configurations, where one or more layers of the substrate are laid flat, for example, in a flat-bottom culture dish, to provide a culture substrate for cells. Due to contamination issues, the container can be a disposable container and can be discarded after use.

[0103] According to one or more embodiments, a cell culture system is provided, wherein a cell culture matrix is used within the culture chamber of a bioreactor vessel. Figure 6An example of a cell culture system 300 is shown. The cell culture system 300 includes a bioreactor vessel 302 having a cell culture chamber 304 within the interior of the bioreactor vessel 302. Within the cell culture chamber 304 is a cell culture substrate 306 made of a stack of substrate layers 308. The substrate layers 308 are stacked such that the first or second side of a substrate layer faces the first or second side of an adjacent substrate layer. The bioreactor vessel 300 has an inlet 310 at one end for inputting media, cells, and / or nutrients into the culture chamber 304 and an outlet 312 at the opposite end for removing media, cells, or cell products from the culture chamber 304. By stacking the substrate layers in this manner, the system can be easily scaled up, and there is no adverse effect on cell adhesion and proliferation due to the defined structure and the efficient flow of fluid through the stacked substrates. Although the vessel 300 can generally be described as having an inlet 310 and an outlet 312, some embodiments may use one or both of the inlet 310 and the outlet 312 to allow media, cells, or other contents to flow into and out of the culture chamber 304. For example, the inlet 310 can be used to allow media or cells to flow into the culture chamber 304 during cell seeding, perfusion, or culturing phases, but can also be used to remove one or more of media, cells, or cell products through the inlet 310 during the harvest phase. Thus, the terms "inlet" and "outlet" are not intended to limit the function of these openings.

[0104] In one or more embodiments, the flow resistance and bulk density of the packed bed can be controlled by staggering substrate layers having different geometries. Specifically, the mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the flow resistance in the packed bed form. By staggering meshes having different sizes and geometries, the flow resistance can be controlled or altered in one or more specific portions of the bioreactor. This will enable better uniformity of liquid perfusion in 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 start with 10 layers of Mesh B, followed by 50 layers of Mesh C, followed by 10 layers of Mesh B. This repeating pattern can continue until the entire bioreactor is filled with meshes. These are merely examples and are for illustrative purposes only and are not intended to limit the possible combinations. In fact, various combinations of meshes having different sizes are possible to obtain different distributions of bulk density of the cell growth surface and flow resistance. For example, a packed bed column having regions with varying cell bulk density (e.g., a series of regions producing a low / high / low / high etc. density pattern) can be assembled by staggering meshes of different sizes.

[0105] In Figure 6In [the figure], the overall flow direction is in the direction from the inlet 310 to the outlet 312, and in this example, the first major side and the second major side of the substrate layer 308 are perpendicular to the overall flow direction. In contrast, Figure 7 The illustrated example is an embodiment in which the system 320 includes a bioreactor vessel 322 and a stack of substrates 328 within a culture space 324, the substrates 328 having a first side and a second side parallel to the overall flow direction, the overall flow direction corresponding to the direction shown by the flow path entering the inlet 330 and exiting the outlet 332. Thus, the substrates of the embodiments of the present disclosure can be used in either configuration. In each of the systems 300 and 320, the size and shape of the substrates 308, 328 are adjusted to fill the internal space defined by the culture chambers 304, 324 such that the culture space in each vessel is filled for a cell growth surface, thereby maximizing efficiency in terms of cells / unit volume. Although Figure 7 Multiple inlets 330 and multiple outlets 332 are shown, but it should be considered that the system 320 can be fed through a single inlet and have a single outlet. However, according to various embodiments herein, a distribution plate can be used to assist in distributing the culture medium, cells, or nutrients across the cross-section of the packed bed and thus improve the uniformity of fluid flow through the packed bed. Thus, the multiple inlets 330 represent how a distribution plate can be equipped with multiple holes across the cross-section of the packed bed to establish a more uniform flow.

[0106] Figure 8 An embodiment of the substrate is shown, in which the substrate is formed as a cylindrical roll 350. For example, a sheet of substrate 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 perpendicular to the central longitudinal axis y. In one or more preferred embodiments, the cylindrical roll 350 is designed to be within the bioreactor vessel such that the central longitudinal axis y is parallel to the overall flow direction F of fluid through the bioreactor or the culture chamber containing the cylindrical roll. Figure 9 A cell culture system 360 having a bioreactor vessel 362 is shown, the bioreactor vessel 362 containing a cell culture substrate 364 in the form of the cylindrical roll configuration. Similar to Figure 8 the cylindrical roll 350 in [the figure], the cell culture substrate 364 has a central longitudinal axis, and in Figure 9In this case, the central longitudinal axis extends into the page. The system 360 also includes a central support member 366 around which the cell culture substrate 364 is positioned. According to some embodiments, the central support member 366 may simply provide physical support and / or arrangement of the cell culture substrate 364, but may also provide other functions. For example, the central support member 366 may be equipped with one or more openings to supply culture medium to the cell culture substrate 364 along the length H of the substrate. In other embodiments, the central support member 366 may include one or more attachment sites for holding one or more portions of the cell culture substrate 364 inside the cylindrical roll. These attachment sites may be hooks, clasps, posts, clips, or other means of attaching the mesh to the central support member 366.

[0107] Figure 10A A cell culture system 400 is shown in accordance with one or more embodiments. The system 400 includes a bioreactor 402 that houses the cell culture substrate of one or more embodiments disclosed herein. The bioreactor 402 may be fluidly connected to a culture medium conditioning vessel 404, and the system is capable of supplying the cell culture medium 406 within the conditioning vessel 404 to the bioreactor 402. The culture medium conditioning vessel 404 may include sensors and control portions seen in typical bioreactors used in the bioprocessing industry for suspension batch, fed batch, or perfusion cultures. These include, but are not limited to, DO oxygen sensors, pH sensors, oxygenator / gas bubbling units, temperature probes, and nutrient addition and base addition ports. The gas mixture supplied to the bubbling unit may be controlled by gas flow controllers for N2, O2, and CO2 gases. The culture medium conditioning vessel 404 also contains an impeller for medium mixing. All of the culture medium parameters measured by the sensors listed above may be controlled by a culture medium conditioning control unit 418 that communicates with the culture medium conditioning vessel 404 and is capable of measuring and / or adjusting the conditions of the cell culture medium 406 to a desired level. As Figure 10A shown, the culture medium conditioning vessel 404 is provided as a vessel separate from the bioreactor vessel 402. This can be advantageous in being able to condition the culture medium in an area independent of the cultured cells and then supply the conditioned medium to the cell culture space. However, in some embodiments, the culture medium conditioning may be performed within the bioreactor vessel 402.

[0108] The culture medium from the culture medium regulating container 404 is conveyed to the bioreactor 402 through the inlet 408. It may also include an injection port for cell seeding, thereby seeding cells and starting cell culture. The bioreactor container 402 may also include one or more outlets 410 through which the cell culture medium 406 exits the container 402. In addition, cells or cell products may be output through the outlet 410. To analyze the content of the effluent from the bioreactor 402, one or more sensors 412 may be provided in the line. In some embodiments, the system 400 includes a flow control unit 414 for controlling the flow rate into the bioreactor 402. For example, the flow control unit 414 may receive signals from the one or more sensors 412 (e.g., O2 sensors), and based on the signals, adjust the flow rate into the bioreactor 402 by sending signals to a pump 416 (e.g., a peristaltic pump) upstream of the inlet 408 of the bioreactor 402. Thus, based on one factor or a combination of factors measured by the sensor 412, the pump 416 can control the flow rate into the bioreactor 402 to obtain desired cell culture conditions.

[0109] The culture medium perfusion rate is controlled by the signal processing unit 414, which collects and compares the sensor signals from the culture medium regulating container 404 and the sensors located at the outlet 410 of the packed bed bioreactor. Due to the plug flow nature of the culture medium perfusion through the packed bed bioreactor 402, nutrient, pH, and oxygen gradients develop along the packed bed. According to the flowchart of FIG. 11, the perfusion flow rate of the bioreactor can be automatically controlled by the flow control unit 414 operatively connected to the peristaltic pump 416.

[0110] One or more embodiments of the present disclosure provide a cell seeding step different from conventional methods. In conventional methods, a packed bed with a conventional matrix is filled with culture medium and a concentrated inoculum is injected into the culture medium circulation loop. The cell suspension is pumped through the bioreactor at an increased flow rate, thereby reducing the non-uniformity of cell seeding by capture on the conventional packed bed matrix. In such conventional methods, the cells are pumped into the circulation loop at an elevated flow rate for perhaps several hours until most of the cells are captured in the packed bed bioreactor. However, due to the non-uniform deep bed filtration properties of conventional packed bed bioreactors, the cells are non-uniformly distributed within the packed bed, with a higher cell density at the inlet region of the bioreactor and a lower cell density at the outlet region of the bioreactor.

[0111] In contrast, according to an embodiment of the present disclosure, a cell inoculum having a volume equal to the interstitial volume of the culture chamber of the bioreactor is directly injected into the packed bed through a cell inoculum injection port at the inlet 408 of the bioreactor 402 ( Figure 10A ). Due to the uniform and continuous fluid channels present in the cell culture matrix described herein, the cell suspension then becomes evenly distributed within the packed bed. To prevent cell sedimentation due to gravity during the initial inoculation phase, media perfusion can be started immediately after inoculum injection. The perfusion flow rate is maintained below a preset threshold to counteract gravity and avoid washing cells out of the packed bed bioreactor. Thus, during the initial cell adhesion phase, the cells are gently tumbled within the packed bed and an even cell distribution and adhesion on the available substrate surfaces is achieved.

[0112] Figure 10B According to one or more embodiments, a more detailed schematic illustration of a cell culture system 420 is shown. The basic configuration of system 420 is similar to Figure 10A system 400, where the packed bed bioreactor 422 has a container that contains a packed bed of cell culture material (e.g., a PET woven mesh), and a separate media conditioning container 424. However, compared to system 400, system 420 shows details of the system, including sensors, user interface and controls, and various inlets and outlets for media and cells. According to some embodiments, the media conditioning container 424 is controlled by a controller 426 to provide appropriate temperature, pH, O2, and nutrients. While in some embodiments the bioreactor 422 may also be controlled by the controller 426, in other embodiments the bioreactor 422 is provided in a separate perfusion loop 428, where the media flow rate through the perfusion loop 428 is controlled using a pump based on the detection of O2 at or near the outlet of the bioreactor 422.

[0113] Figure 10A and 10B systems can be operated according to the process steps of one or more embodiments. As Figure 11A shown, these process steps can include process preparation (S1), inoculation and cell adhesion (S2a, S2b), cell expansion (S3), transfection (S4a, S4b), production of viral vectors (S5a, S5b), and harvest (S6a, S6b).

[0114] FIG. 11 shows a control for a perfusion bioreactor system (e.g., Figure 10AAn example of a method 450 for regulating the flow rate of system 400 or 10B. According to method 450, certain parameters of system 400 are pre-determined by optimizing the operation of the bioreactor at step S21. Based on these optimized operations, the values of pH1, pO1, [glucose]1, pH2, pO2, [glucose]2, and the maximum flow rate can be determined. At step S22, the values of pH1, pO1, and [glucose]1 are measured in the cell culture chamber of bioreactor 402, and at step S23, pH2, pO2, and [glucose]2 are measured by sensor 412 at the outlet of bioreactor 402. Based on these values at S22 and S23, the perfusion pump control unit makes a decision at S24 to maintain or adjust the perfusion flow rate. For example, if pH2 ≥ pH 2最小 , pO2 ≥ pO 2最小 , and [glucose]2 ≥ [glucose] 2最小 are satisfied for at least one of them, the perfusion flow rate of the cell culture medium reaching the cell culture chamber can continue at the current rate (S25). If the current flow rate is less than or equal to the predetermined maximum flow rate of the cell culture system, the perfusion flow rate is increased (S27). Further, if the current flow rate is not less than or equal to the predetermined maximum flow rate of the cell culture system, the controller of the cell culture system can re-evaluate at least one of the following: (1) pH 2最小 , pO 2最小 , and [glucose] 2最小 ; (2) pH1, pO1, and [glucose]1; and (3) the height of the bioreactor vessel (S26).

[0115] Depending on the desired system, the cell culture medium can be arranged in the culture chamber in a variety of configurations. For example, in one or more embodiments, the system includes one or more substrate layers, and its width extends across the entire width of the defined cell culture space in the culture chamber. Multiple substrate layers can be stacked in this way to a predetermined height. As described above, the substrate layers can be arranged such that the first and second sides of one or more layers are perpendicular to the overall flow direction of the culture medium through the defined culture space in the culture chamber, or the first and second sides of one or more layers can be parallel to the overall flow direction. In one or more embodiments, the cell culture medium includes one or more substrate layers oriented in a first orientation relative to the overall flow, and one or more other layers in a second orientation different from the first orientation. For example, each layer can have a first side and a second side that are parallel or perpendicular to the overall flow direction, or at an angle between parallel and perpendicular.

[0116] In one or more embodiments, the cell culture system includes a plurality of discrete cell culture substrate sheets in a packed bed configuration, wherein the length and / or width of the substrate sheet is relatively small with respect to the culture chamber. As used herein, a substrate sheet is considered to have a relatively small length and / or width with respect to the culture chamber when the length and / or width of the substrate sheet is equal to or less than 50% of the length and / or width of the culture space. Thus, the cell culture system can include a plurality of substrate sheets packed into the culture space in a desired arrangement. The arrangement of the substrate sheets can be random or semi-random, or can have a predetermined order or arrangement pattern, e.g., the substrate sheets are oriented in a substantially similar orientation (e.g., horizontal, vertical, or at an angle between 0° and 90° with respect to the overall flow direction).

[0117] As used herein, "defined culture space" refers to the space within the culture chamber that is occupied by the cell culture substrate and in which cell seeding and / or culturing will occur. The defined culture space can fill approximately the entire culture chamber, or can occupy a portion of the space within the culture chamber. As used herein, "overall flow direction" is defined as the direction of the overall mass flow of fluid or medium through or over the cell culture substrate during cell culture and / or during the inflow or outflow of medium into or out of the culture chamber.

[0118] In one or more embodiments, the cell culture substrate is secured within the culture chamber by a securing mechanism. The securing mechanism can secure a portion of the cell culture substrate to the culture chamber wall surrounding the substrate, or to the chamber wall at one end of the culture chamber. In some embodiments, the securing mechanism adheres a portion of the cell culture substrate to a member traveling through the culture chamber, e.g., a member traveling parallel to the longitudinal axis of the culture chamber, or to a member traveling perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture substrate can be contained within the culture chamber and not fixedly attached to the walls of the chamber or bioreactor vessel. For example, the substrate can be contained within the chamber by the boundaries of the culture chamber or other structural members to hold the substrate within a predetermined region of the bioreactor vessel and not fixedly secure the substrate to these boundaries or structural members.

[0119] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. According to one or more embodiments of the present disclosure, the culture chamber can contain a cell culture substrate and a substrate. In a roller bottle configuration, the bioreactor vessel can be operatively attached to means for moving the bioreactor vessel about a central longitudinal axis of the vessel. For example, the bioreactor vessel can rotate about the central longitudinal axis. The rotation can be continuous (e.g., continuously in one direction), or discontinuous (e.g., intermittent rotation in a single direction or alternating directions, or oscillating in a back-and-forth rotational direction). In operation, the rotation of the bioreactor vessel causes movement of the cells and / or fluid within the chamber. This movement can be considered movement relative to the chamber wall. For example, when the bioreactor vessel rotates about its central longitudinal axis, gravity can cause the fluid, culture medium, and / or non-adherent cells to remain towards the lower part of the chamber. However, in one or more embodiments, the cell culture substrate is substantially fixed relative to the vessel and thus rotates with the vessel. In one or more other embodiments, the cell culture substrate can be non-attached and can move freely relative to the vessel to a desired extent when the vessel rotates. The cells can adhere to the cell culture substrate, and at the same time, the movement of the vessel causes the cells to be exposed to the cell culture medium or liquid, as well as oxygen or other gases in the culture chamber.

[0120] By using the cell culture substrates of the embodiments of the present disclosure, for example, substrates including woven or mesh substrates, the roller bottle container has an increased surface area available for adherent cell adhesion, proliferation, and functionalization. Specifically, when using a woven mesh substrate of a monofilament polymer material within a roller bottle, the surface area can be increased by about 2.4 to about 4.8 times, or up to about 10 times that of a standard roller bottle. As described herein, each monofilament strand of the mesh substrate can present itself as a 2D surface for adherent cell adhesion. In addition, multiple mesh layers can be arranged in the roller bottle, such that the total available surface area is increased by about 2 to 20 times compared to a standard roller bottle. Thus, by adding the improved cell culture substrates disclosed herein, existing roller bottle facilities and processes (including cell seeding, medium exchange, and cell harvesting) can be altered with minimal impact on existing operating infrastructure and processing steps.

[0121] The bioreactor vessel optionally includes one or more outlets that can be attached to inlet and / or outlet means. Through the one or more outlets, liquid, culture medium, or cells can be supplied to the chamber, or liquid, culture medium, or cells can be removed from the chamber. A single port in the vessel can serve as both an inlet and an outlet simultaneously, or multiple ports can be provided for dedicated inlets and outlets.

[0122] The packed bed cell culture substrate of one or more embodiments can consist of a cell culture woven mesh substrate, and no other form of cell culture substrate is provided therein or no other form of cell culture substrate is interleaved with the cell culture substrate. That is, the cell culture woven mesh substrate of the embodiments of the present disclosure is an effective cell culture substrate and does not require the irregular, non-woven substrate types used in existing solutions. This enables a cell culture system with simplified design and construction while providing the other advantages described herein regarding flow uniformity, harvestability, etc. to the high-density cell culture substrate.

[0123] As described herein, the cell culture substrate and the bioreactor system provide multiple advantages. For example, the embodiments of the present disclosure can support the production of any one of many viral vectors (such as AAV (all serotypes) and lentivirus) and can be applied for in vivo and in vitro gene therapy applications. Uniform cell seeding and distribution maximize the viral vector yield per container, and the design enables the harvesting of live cells, which can be useful for seed taming consisting of multiple amplification stages using the same platform. In addition, the embodiments herein can be scaled from the process development scale to the production scale, which ultimately saves development time and cost. The methods and systems disclosed herein also allow for the automation and control of the cell culture process to maximize vector yield and improve reproducibility. Finally, compared to other cell culture solutions, the number of containers required to reach the production scale level of viral vectors (e.g., 10 16 to 10 18 AAV VGs) per batch can be greatly reduced.

[0124] The embodiments are not limited to the rotation of the container around the central longitudinal axis. For example, the container can rotate around an axis that is not centered relative to the container. In addition, the rotation axis can be a horizontal or vertical axis.

[0125] Examples

[0126] To demonstrate the efficacy of the cell culture substrate, cell culture system, and related methods of the present disclosure, studies on cell seeding and culture were conducted according to the following examples.

[0127] Example 1

[0128] In Example 1, a cell culture substrate with a polyethylene terephthalate (PET) woven mesh substrate (see Figure 12) was tested under static cell culture conditions. The PET mesh was washed in ethanol and plasma-treated in oxygen RF plasma. Gelatin was adsorbed on the surface of the mesh filaments to promote cell adhesion. A disc-shaped piece of the mesh was placed into an ultra-low adhesion (ULA) six-well plate. At different seeding densities (50K / cm 2, 75 K / cm 2 , 100 K / cm 2 ) HEK293T cells were seeded onto the mesh disk and cell culture was carried out for three days. The cells on the surface of the fibrils were stained with a green fluorescent cell tracking dye. Figure 12 shows such a visualization result of the cells on the surface of the fibrils. The size of the mesh fibrils relative to the cell size enables single fibril fibers to effectively serve as a two-dimensional surface for cell adhesion and proliferation. By harvesting the cells from the mesh and counting them on a cell counter from Beckman Coulter, cell proliferation was measured. The results showed excellent cell adhesion and proliferation on the cell culture substrate under static cell culture conditions. For example, Figure 13A shows the total cells / well of each seeded mesh after 24, 48, and 72 hours. In addition to the cell number, cell viability is shown in Figure 13B , which demonstrates extremely high viability at each seeding density.

[0129] Example 2

[0130] In Example 2, cells were cultured in a packed bed bioreactor system (e.g., the system shown in Figure 6 ). The packed bed has a cylindrical shape and is made of a stack of cell culture substrates, each of which has a circular or disk shape. Specifically, in Example 2, the height of the packed bed is about 25 mm and it includes one hundred PET woven mesh substrates disks, each with a diameter of about 20 mm. The mesh used corresponds to Mesh C in Table 1. The estimated total two-dimensional surface area available for cell adhesion is about 760 cm 2 . To inoculate the bioreactor, 8 ml of a HEK293T cell suspension (two million cells / ml) was directly injected into the packed bed. Medium perfusion was started immediately after the introduction of the cell suspension, and the perfusion flow rate was set to 3 ml / min (milliliters per minute). Perfusion was continued at this flow rate for 24 hours, and then the flow rate was reduced to 1 ml / min. After that, the perfusion flow rate was adjusted to maintain pO2 ≥ 50% saturation pressure and pH ≥ 7 at the bioreactor outlet. After two to three days, the cells were stained with crystal violet and the bioreactor was disassembled to demonstrate the uniformity of cell adhesion within the matrix. Figure 14A and 14B show every other packed bed matrix disk, and the attached HEK293T cells are stained with the crystal violet stain. Figure 14A shows the results of inoculation in the bioreactor under static conditions. Based on the change in staining, uneven cell adhesion was visible after 3 days of culture. Specifically, at the bottom of the packed bed (corresponding to Figure 14AThe disk at the bottom of the image of Figure 14A has a higher cell concentration, while at the top part of the packed bed (corresponding to the disk at the top of the image of Figure 14B ), there are fewer cells. Figure 14B shows the results from a bioreactor inoculated using the inoculation method of the preferred embodiment, where during the initial adhesion phase, the cells tumble continuously within the packed bed. As a result, after two days of cell culture, a uniform cell distribution is observed in all parts of the packed bed, which is demonstrated by consistent cell staining in the disks from the top to the top (and from the top to the bottom of the image of

[0131] Example 3

[0132] In Example 3, cells were cultured in a packed bed bioreactor system, and HEK293T cells were transfected in the bioreactor for adeno-associated virus (AAV) production. The same bioreactor setup as in Example 2 was used in Example 3 (see, for example Figure 6 ). The packed bed contained disks of 100 PET meshes (Mesh C in Table 1). The diameter of each disk was approximately 20 mm, and the bed height was approximately 25 mm, with a total two-dimensional surface area available for cell adhesion and proliferation of approximately 760 cm 2 . To inoculate the bioreactor, 8 ml of a HEK293T cell suspension (two million cells / ml) was directly injected into the packed bed. A medium storage container containing approximately 50 ml of medium was fluidly connected to the bioreactor container. The cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) medium (with +10% FBS and +6 mM L-glutamine). When the pH of the medium in the storage container dropped below 7, the medium was replaced with fresh medium feed. Accordingly, the perfusion flow rate was adjusted to maintain pO2 ≥ 50% saturation pressure and pH ≥ 7 at the bioreactor outlet. After 72 hours, 50 ml of DMEM (15 - 018) (with +10% FBS, +6 mM L - glutamine) was changed to a medium, and a transfection reagent was added to obtain a final concentration with a ratio of 2 μg / ml of AAV2 to PEIpro of 1:2. During the next 72 hours, if the pH in the reservoir dropped below 7, the medium was changed to fresh feed. Accordingly, the perfusion flow rate was adjusted to maintain pO2 ≥ 50% saturation pressure and pH ≥ 7 at the bioreactor outlet. Cells were harvested using 5X TrypLE. Transfection efficiency was analyzed by fluorescence flow cytometry, and viral particle and viral genome titers were analyzed by ELISA and PCR assays. The cell culture results are shown in Table 2, where "VP" represents viral protein and "GC" represents genomic copy.

[0133]

[0134] Table 2 : Results of transfection and AAV production of HEK 293T cells in a packed - bed bioreactor.

[0135] Example 4

[0136] In Example 4, the implementation of a roller - bottle cell - culture system was tested. Using roller - bottle #430195, with a surface area of 490 cm 2 . To prevent cell adhesion to the tissue - culture - treated surface, the roller - bottle was treated with 0.5% BSA solution for at least 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 that uses trypsin / EDTA solution to release cells from the surface, followed by inactivating the trypsin / EDTA solution by adding complete medium containing fetal bovine serum (FBS). Then the cells were counted using a cell counter and seeded into roller - bottles with and without a cell - culture mesh at a concentration of approximately 5×10 4 cells / cm 2 and a total volume of 200 mL. As Figure 15 and 16 shown, for roller - bottle 500 with cell - culture mesh 502, the mesh was rolled into a tight cylindrical roll 503 so that it could be inserted through the mouth 501 of the bottle. Once inserted into the interior of the bottle, the cell - culture mesh partially unfurled and expanded towards the roller - bottle wall (as shown by arrow 504). The length of the cell - culture mesh was long enough so that after expansion inside the roller - bottle, a double - layer cell - culture mesh was provided around the inner circumference of the roller - bottle, as Figure 15 and 16As shown. In an incubator at 37 °C with 5% CO2 and 95% relative humidity, cells were allowed to adhere to the surface at various rotation speeds (0.5 to 4 rpm) for 16+ hours. After cell adhesion, the speed was reduced to approximately 1 rpm for standard cell growth in roller bottles. Medium assays were performed periodically to determine when medium replacement was needed. Crystal violet in a solution containing methanol and paraformaldehyde was used in the roller bottle to visualize the cells adhered to the mesh surface. After cell staining, the mesh was removed from the roller bottle and imaged.

[0137] Figure 17A and 17B shows the mesh removed from the roller bottle in Example 4 and demonstrates the presence of HEK293 stained cells adhered to the double-layer mesh inside the roller bottle. According to one embodiment of the present disclosure, the total available surface area for adherent cells to adhere in the roller bottle is 2450 cm 2 , which is in contrast to 490 cm 2 of a conventional roller bottle without a mesh substrate. In Figure 17A , crystal violet-stained cells adhered to a PET mesh (corresponding to Mesh C in Table 1) that self-assembled in a double-layer structure inside the roller bottle. Cells were inoculated at a roller bottle speed of 0.5 rpm. Note that mainly the outer layer of the mesh facing the bottle wall was inoculated with cells. Figure 17B shows the crystal violet staining of cells adhered to a PET mesh (corresponding to Mesh C in Table 1) that self-assembled in a double-layer structure inside the roller bottle. Cells were inoculated at a roller bottle speed of 4 rpm. Note that both mesh layers were evenly inoculated with cells. As can be seen from Figure 17A and 17B , the uniformity of cell inoculation depends on the rotation speed of the roller bottle during the inoculation step. Contrary to the conventional roller bottle inoculation protocol, a fast rotation speed is required to evenly inoculate cells on the available adhesion surface of the mesh.

[0138] The embodiments disclosed herein have several advantages over existing platforms for cell culture and viral vector production. It should be noted that the embodiments of the present disclosure can be used to produce various types of cells and cell by-products, including, for example, adherent or semi-adherent cells, human embryonic kidney (HEK) cells (such as HEK23), including transfected cells, viral vectors such as lentivirus (stem cells, CAR-T) and adeno-associated virus (AAV). These are examples of some common applications of the bioreactor or cell culture substrate disclosed herein, but they are not intended to limit the use or application of the disclosed embodiments, as those of ordinary skill in the art will understand that the embodiments can be applied to other uses.

[0139] Example 5

[0140] As described above, one advantage of the embodiments of the present disclosure is the flow uniformity of the cell culture substrate. Without being bound by theory, it is believed that the regular and uniform structure of the cell culture substrate provides a consistent and uniform body through which the culture medium can pass. In contrast, existing platforms mainly rely on irregular or random substrates, such as felt-like or non-woven fibrous materials. The uniform nature of the substrate of the present disclosure can be illustrated by examining the uniform and consistent cell seeding achieved on the substrate. For example, Figure 18A Three discs (1801, 1802, 1803) of the substrate material from Example 5 are shown according to some embodiments of the present disclosure. Figure 18A The discs in are PET woven mesh materials as described herein, and each disc has a diameter of about 60 mm. The surface area of a bioreactor filled with 10 to 300 layers of similar discs will be about 678 to 20,300 cm 2 . In this example, a stack of 100 discs was used for cell culture. The first disc 1801 is the top disc in the stack of these discs in the bioreactor, the second disc 1802 is the middle disc of the stack, and the third disc 1803 is the bottom disc of the stack. Although located at different positions within the stack, Figure 18A the staining in shows significantly consistent cell adhesion.

[0141] In the experiment to generate Figure 18A and 18B images, the bioreactor was pre-filled with cell culture medium, and the system was pre-conditioned overnight to reach a steady state of pH 7.2, D.O. 100%, and 37 °C. The entire bioreactor system was filled with 400 ml of ATCC DMEM medium + 10% FBS + 6 mM L-glutamine. 30 ml of suspended HEK293T cells (five million cells / ml) were directly injected into the packed bed through a three-way port to form an inoculum. The bioreactor was perfused with the pre-conditioned medium at a rate of 30 mL / min for the first 48 hours to achieve a uniform cell distribution, adhesion, and initial growth in 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 the DO at the bioreactor outlet 外≥45% of the culture medium is saturated. At 72 hours post-inoculation, replace the culture medium with 500 ml of Corning DMEM (15-018) + 10% FBS + 6 mM L-glutamine and allow perfusion for 2 hours. Add the transfection mixture (a complex of plasmid DNA and PEI at a ratio of 1:2; 0.8 μg of total DNA per million cells) to achieve a final concentration of 2 μg of total DNA / ml of culture medium at 24 hours post-transfection. Replace the culture medium with 500 ml of fresh complete Corning DMEM (15-018) medium to replenish the consumed nutrients. Automatically adjust the perfusion flow rate to maintain DO at the bioreactor outlet. 外 ≥45% saturated. Monitor the glucose level during the subsequent 48-hour culture period and supplement as needed by medium addition or exchange to maintain a level above 0.3 g / L. At 72 hours post-transfection, wash the cells with DPBS and harvest the cells using 1X Accutase solution. Analyze the transfection efficiency by fluorescence flow cytometry and analyze the virus particle and virus genome titers by ELISA and qPCR assays.

[0142] Use crystal violet staining to highlight Figure 18A the uniform growth of cells on the entire surface of the disks in Figure 18A The image in Figure 18B was taken 72 hours after culturing and before harvesting the cells from the substrate. Figure 18B Shows the same three disks (1801′, 1802′, and 1803′) after harvesting the cells. As 2 shown by the relative absence of crystal staining in 2 , the cells have been uniformly harvested on the surface of each disk and on the three disks of the cell culture matrix stack. Based on the analysis, more than 95% of the cells were recovered from the bioreactor. Table 3 below shows the cell culture results for AAV production in a stack / container of substrates with a diameter of 60 mm and a total surface area of 6780 cm

[0143]

[0144] Table 3 : Transfected cell yield, transfection efficiency, and virus genome yield per cm 2 from the 60-mm bioreactor.

[0145] Table 4 below shows the above results in the context of multiple experiments involving bioreactor vessels of different diameters (29 mm and 60 mm). The data show good scalability between smaller (e.g., 29 mm diameter, 1600 cm 2 surface area) and larger (e.g., 60 mm diameter, 6780 cm 2 surface area) vessels and / or packed bed matrices.

[0146]

[0147]

[0148] Table 4: Consistent results across the entire size of the bioreactor.

[0149] As described above, embodiments of the present disclosure can provide a packed bed cell culture matrix and / or bioreactor capable of culturing high-density cells in a relatively small and practical footprint. For example, the 60 mm cell culture matrix in the examples of Tables 3 and 4 above has a surface area of approximately 6870 cm 2 . For reference, Corning has a surface area of approximately 1720 cm 2 . The 60 mm diameter cell culture matrices of Tables 3 and 4 can be accommodated in a bioreactor smaller than a HYPER flask, but still result in higher cell counts and higher total genomic copy numbers (GC or viral genome (VG)) / vessel at harvest. Figure 19 shows these numbers for two bioreactor vessels with a 60 mm diameter substrate from Tables 3 and 4 compared to a HYPER flask, and shows GC / cm 2 , which, although lower than the HYPER flask, is compensated for by the higher surface area.

[0150] Example 6

[0151] To further examine 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 PET woven mesh substrates were modeled in a tight-packed configuration and a loose-packed configuration, which represent the upper and lower limits of the packing density of the substrate stack of the specific mesh sheets being modeled. Specifically, Figure 20A shows a plan view of the tight-packed configuration, Figure 20B shows a cross-sectional side view of the same stack. Figure 21A and 21BThe plan view and cross-sectional view of the loose filling structure are shown respectively. For each modeling structure, sample units 600 and 602 are defined, which enclose the same volume of mesh material to analyze the porosity in the volume of each sample unit 600 and 602. The modeled volume of the open space within each unit is shown in Figure 22A (for the tightly packed stack) and Figure 22B (for the loosely packed stack). The porosity, in terms of the percentage of the open space, is approximately 40.8% for the loosely packed unit and 61.4% for the tightly packed unit. Since Figure 20A - 21B the modeled stacks in

[0152] represent the tightest and loosest filling structures of a given mesh material, the porosities of 40.8% and 61.4% are the upper and lower limits of the porosity of this specific mesh material. Depending on the arrangement and the actual filling density when using this mesh material, the porosity can fall between these limits. However, the embodiments of the present disclosure are not limited to this porosity range, because changes in the mesh size and the arrangement of the substrate within the cell culture container can result in different porosity ranges. 3 , the volume of the PET material of the stack is calculated using the following formula:

[0153] V PET = (total weight of the stack) / (density of PET) Equation 2

[0154] Therefore, the PET volume V PET of 5.65 g PET (100 disks with a diameter of 22.4 mm) is calculated to be 4.1 ml. Then the total volume V 总 of the stack is calculated using the following formula PET - including the PET volume V

[0155] V 总 = π × (0.5 × disk diameter) × (height of the stacked bed) Equation 3

[0156] The stacked height of the stack of 100 disks is 25 ± 1 mm. Therefore, for a disk diameter of 22.4 mm, V 总 is found to be 9.85 ml. Therefore, the porosity of the stacked bed can be calculated using the following formula:

[0157] Porosity = (V 总 – V PET ) / V总 Equation 4

[0158] Using Equation 4 and the above values, the porosity was calculated to be 58.4%, which is within the range predicted by the model.

[0159] Example 7

[0160] In Example 7, the permeability of various PET woven mesh substrate materials was compared. Table 5 shows the PET mesh samples used for this comparison.

[0161]

[0162] Table 5 : Mesh substrates for permeability comparison.

[0163] Figure 23 shows photographs of the mesh samples of Samples A - F. Figure 24 Shows the permeability results of each mesh sample A - F. Figure 25 Shows the pressure drop test results of Samples A - C, where Sample A was tested for pressure drop with stacks having different arrangements and packing densities. The dotted lines represent the tightest and loosest packing densities of mesh sample A, where Sample A1 is a less densely packed stack than Sample A2. A graph of pressure drop versus Q / A is plotted in terms of pressure change (Pa) per centimeter.

[0164] Example 8

[0165] 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 cell seeding domestication to gradually increase a cell population. One problem with existing cell culture solutions is that a given bioreactor system technology cannot be part of the seeding domestication. Instead, cell populations are often scaled up on various cell culture substrates. This can adversely affect the cell population because it is thought that the cells become adapted to certain surfaces, and transferring them to different types of surfaces can result in inefficiencies. Therefore, it is desirable to minimize the transitions between cell culture substrates or technologies. By using the same cell culture substrate throughout the seeding domestication, as can be achieved by embodiments of the present disclosure, the efficiency of scaling up the cell population is increased. Figure 26 Shows an example of one or more embodiments, where the woven cell culture substrate of the present application is used as part of the seeding domestication to allow a smaller bioreactor to inoculate a larger bioreactor. Specifically, as Figure 26 shown, the seeding domestication can start with the starting cells in a vial, which are inoculated into a first container (e.g., a T175 flask from Corning), and then into a second container (e.g., from Corning ) and then inoculated into a bioreactor system at the process development scale according to the embodiments of the present invention (the effective surface area of the substrate is about 20,000 cm 2 ), and then inoculated into a larger bioreactor test system according to the embodiments of the present invention (the effective surface area of the substrate is about 300,000 cm 2 ). At the end of this seed domestication, the cells can be inoculated into a bioreactor vessel at the production scale according to the embodiments of the present disclosure, where the surface area is, for example, about 5,000,000 cm 2 . When the cell culture is completed, the harvesting and purification steps can then be carried out. As Figure 26 shown, harvesting can be accomplished via in situ cell lysis using a detergent (such as Triton X-100) or via mechanical lysis; further downstream processing can be carried out if desired.

[0166] The benefits of using the same cell culture substrate in seed domestication (e.g., from the process development level to the test level, or even to the production level) include obtaining efficiency from cells accustomed to the same surface during the seed domestication and production phases; reducing the number of manual opening operations during the seed domestication phase; more efficient utilization of the packed bed due to uniform cell distribution and fluid flow, as described herein; and flexibility in using mechanical or chemical lysis during virus vector harvesting.

[0167] Example 9

[0168] To understand the potentially increased virus production yield of the substrates of the present disclosure, the performance of the PET woven mesh substrate is compared with the performance of the substrates used for . Table 6 summarizes the total virus particle production using these substrates in a simplified bioreactor.

[0169]

[0170] Table 6: Virus particles produced using the PET woven mesh substrate material and the substrate material from iCellis.

[0171] Based on the results in Table 6, the volume of the substrate material required to produce a certain number of virus particles can be calculated. For example, if the goal of virus vector production at the production scale is 3.00E+18 virus particles, as shown in Table 7, the volume of the PET woven mesh required is approximately one-seventh of the amount of the iCellis substrate required.

[0172]

[0173] Table 7 : The PET woven mesh substrate and Comparison of Substrates

[0174] Example 10

[0175] To demonstrate the uniform flow through the open mesh substrate of the present disclosure, the fluid flow velocity through a packed bed bioreactor was modeled. Figure 27A The modeling results of container 620 are shown, which has a packed bed region 622 of a PET woven mesh disk with a diameter of 6 cm, and a bed height of 10 cm, and is composed of 357 disks of PET woven mesh substrate. The fluid velocity magnitude is plotted according to the scale shown. Although the flow velocity is high near the inlet 624 and the outlet 626, the velocity is constant throughout the packed bed region 622, including along the height and across the width of the packed bed. The region indicated by the dotted line 628 is shown in the Figure 27B magnified view in, which shows that once the fluid enters the uniform, open structure of the cell culture matrix, the velocity is relatively constant. The packed bed in this example is equivalent to a total surface area of approximately 24,214 cm 2 Given the uniform flow shown in the model, the percentage of this surface area exposed to non-uniform flow (defined as a deviation from the average velocity > 2.5%) is 0%.

[0176] To demonstrate the extent to which this uniform flow persists when the size of the container is scaled up, additional modeling similar to Figure 27A was performed, but using gradually wider containers and wider packed beds. The percentages of non-uniform flow for these larger containers are shown in Table 8. As shown, even when the reactor is scaled up to a diameter of up to 60 cm, the amount of non-uniform flow remains approximately 0.5% or less of the substrate surface area. This shows that, unlike existing cell culture substrates, the uniform, non-woven mesh structure described herein can achieve uniform flow throughout the packed bed.

[0177]

[0178] Table 8: Flow uniformity of modeling of bioreactors with packed beds of various diameters.

[0179] Example 11

[0180] To better understand the permeability differences between the woven mesh substrate of the present disclosure and the non-woven, irregular substrates on the existing market, experiments were conducted to measure the permeability of these materials. Specifically, the PET woven mesh was compared with that used for The nonwoven substrate material was compared with commercially available similar nonwoven disordered substrates. For the woven mesh substrate layer in the packed bed perpendicular to the stacked disks, the permeability was measured through the random packed bed of the nonwoven substrate and through the flow of the fixed sheet of the nonwoven substrate material. The nonwoven mesh had a fiber diameter of about 20 μm, a thickness of about 0.18 mm, and a porosity of 91%. The woven mesh substrate had a diameter of about 160 μm and an opening diameter of 250 μm.

[0181] For the mesh permeability, water was used to simulate the cell culture medium for testing. A peristaltic pump was used to control the flow rate between 15 - 50 ml / cm 2 / min to simulate the flow conditions that the substrate usually experiences in a bioreactor. Since the sample experiences a low pressure drop under the test conditions, a manometer was used to measure the pressure difference across the sample. Due to different substrate types and packing methods, the way of holding the substrate was slightly different.

[0182] To measure the flow rate through the nonwoven mesh, the sample was cut into disks with a diameter of 12 mm, and 10 layers of the mesh were placed between two open cylindrical chambers sealed with O-rings. The manometer was directly connected to the open chambers to measure the pressure drop.

[0183] To measure the flow rate through the randomly packed nonwoven mesh, the mesh was cut into strips of 5 mm x 25 mm and packed into a cylindrical chamber with a diameter of 29 mm. A total of 3 g of the mesh strips were packed into 30 ml and packed to a bed height of about 45 mm. On each side of the packed strips, two open disks of the woven mesh were used to confine the packed bed. There was an open space of about 3 mm thick on each side of the bed, and then two pieces of porous material with a thickness of 10 mm were used to redistribute the flow at the inlet and outlet.

[0184] To measure the flow rate through the open woven mesh, the mesh was cut into disks of 29 mm to fit into the cylindrical chamber. A total of 170 disks were packed layer by layer to form a bed height of 45 mm. The fiber orientations in each layer of the mesh were not aligned with each other. The flow passed through the disk mesh (i.e., perpendicular to the disk surface). There was an open space of about 3 mm thick on each side of the bed, and then two pieces of porous material with a thickness of 10 mm were used to redistribute the flow at the inlet and outlet.

[0185] Equation (5) was used to calculate the permeability:

[0186]

[0187] 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.

[0188] The finally calculated permeability is summarized inFigure 28 In the result, the nonwoven web has a significantly lower permeability of about 7.5x10 -12 m 2 , which is about 1 / 50 of the permeability through the open woven web. When the nonwoven web is cut into small strips and randomly packed, its permeability increases significantly and becomes similar to that of the open woven web. This increased permeability is considered to be the result of the above-mentioned channel effect causing the flow to mainly bypass the web strips.

[0189] Based on the measured permeability, the flow through and around the nonwoven web and the open woven web is simulated. The ANSYS Fluent v19.2 software package is used to perform this simulation. For illustrative purposes, two cases are studied: the substrate material surface is arranged at (i) 90° and (ii) 45° relative to the flow direction, as Figure 29A and 29B shown. In both cases, when the spacing between adjacent webs at the same height is 5 mm, the flow mainly occurs around the webs, and only about 0.02 - 0.005% of the flow passes through the webs. This will create a large dead zone behind the webs and result in non-uniform flow through the packed bed. When the nonwoven web sheets are not perfectly perpendicular to the flow direction, the non-uniformity becomes more severe.

[0190] In the case of the open woven web, the open structure allows for easier flow through the web and does not create a dead zone behind the open web layer. It is believed that the regular structure of the woven web also promotes a uniform flow distribution through each height. This in turn enables a more uniform flow throughout the packed bed. This comparison is clearly shown in Figure 30A (nonwoven web sheet) and Figure 30B (open woven web substrate), which show magnified views of the flow near the edge of the substrate material. In Figure 30A and 30B 's case, the gap between adjacent web sheets is shortened to 1 mm in all six directions, and only the periodic domain of one such web sheet is simulated. Figure 30A It is shown that the nonwoven web has a very low permeability because the flow mainly bypasses the substrate, and very little flow passes through the substrate itself. Only 0.17% of the total mass flow passes through the nonwoven web. In contrast, the open woven web has a significantly higher permeability, so more flow passes through it, as Figure 30B shown. When comparing the color bars in the two cases, the shortcut flow through the gap becomes weaker. For the open woven web, up to 10.7% of the total mass flow rate passes through the substrate, which proves that the open woven web has excellent permeability even when packed with gaps.

[0191] As discussed herein, multiple woven mesh disks can be randomly packed, and there are countless variations in the alignment between the disks. However, the range of possible arrangements can be reduced to two theoretical limits based on packing density (i.e., the tightest and loosest packing). These two ideal or boundary conditions allow large packed beds to be simplified into small periodic domains. Using this model, it is found that the permeability through the substrate differs by about 10 times from the tightest to the loosest packing limit. The permeability data of the experimental measurements above are just within this range, which is a good verification point. The model also shows that under two packing conditions, the permeability in the flow direction is similar to the permeability in the transverse direction. This suggests that regardless of the substrate orientation, the woven mesh of the present invention will be unlikely to change the flow direction and make the flow more uniform as found in nonwoven substrates. The improved flow uniformity of the substrate disclosed herein is further demonstrated by the residence time distribution (RTD) measurement in the following examples.

[0192] Example 12

[0193] Residence time distribution (RTD) is a useful tool for studying flow in a vessel. Its theory, measurement and analysis can be found in the following textbook: Levenspiel, O. Chemical Reaction Engineering. 3rd edition. 1999. Wiley Publishing, New York. Figure 5 is a schematic diagram of the setup for measuring RTD. The chamber was cylindrical with a diameter of 29 mm, the total packed bed volume was 36 ml, and the container chamber was filled with 3.6 g of nonwoven mesh or 200 layers of open woven mesh. A 1:2000 dilution of McCormick green food coloring was used as a tracer for the measurement. A UV-vis (photometer) with a flow cell was used to monitor changes in tracer concentration. 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 OD changes were recorded. The results are shown in Figure 32 .

[0194] The following equations are used to calculate the mean residence time t (Equation (6)) and the variance σ (Equation (7)):

[0195]

[0196]

[0197] Where F is the normalized concentration of the step tracer response. Table 9 summarizes the calculated average residence time and variance based on the measurements. The open woven mesh shows a shorter average residence time, which may be caused by the lower porosity and reduced dead zones. In the packed bed of the open woven mesh, the porosity is about 60%, while the non-woven mesh has a higher porosity, about 93%. The significantly higher normalized variance detected in the packed bed of the non-woven mesh indicates that the flow in the non-woven mesh is less uniform or ideal.

[0198] Woven mesh Non - woven mesh Average residence time (minutes) 1.37 1.97 <![CDATA[Variance (min 2 )]]> 0.21 0.84 Normalized variance 0.11 0.22

[0199] Table 9: Average residence time and variance of the non-woven mesh and open woven mesh based on the measurements.

[0200] Based on the above permeability and residence time experiments, it is shown that the non-woven, irregular cell culture substrate types currently used in bioreactors have lower permeability than the substrates disclosed herein. These non-woven substrates also have different permeabilities or flow rates depending on the direction of flow relative to the non-woven substrate, while the substrates disclosed herein exhibit substantially isotropic flow behavior. Due to the non-uniform flow and shorter residence time of the non-woven substrates, nutrients and transfection reagents may take longer to reach the cells on the surface of the substrate or on the other side of the substrate layer compared to the uniform woven mesh substrates disclosed herein. In addition, the randomly packed non-woven substrates have higher permeability, which indicates a strong channel effect, and thus the delivery of cells or nutrients is not uniform.

[0201] Exemplary implementation

[0202] The following is a description of various aspects of the disclosed subject matter. Each aspect may include one or more of the various features, characteristics, or advantages of the disclosed subject matter. The embodiments are intended to illustrate several aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible embodiments.

[0203] Aspect 1 relates to a cell culture substrate comprising: a substrate including 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, wherein the plurality of openings are configured to allow at least one of a cell culture medium, cells, or cell products to flow through the thickness of the substrate.

[0204] Aspect 2 relates to the cell culture substrate 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.

[0205] Aspect 3 relates to the cell culture substrate of Aspect 1 or Aspect 2, wherein the substrate comprises at least one of a molded polymeric lattice sheet, a 3D printed lattice sheet, and a woven mesh.

[0206] Aspect 4 relates to the cell culture substrate of Aspect 3, wherein the substrate comprises a woven mesh containing one or more fibers.

[0207] Aspect 5 relates to the cell culture substrate of Aspect 4, wherein the one or more fibers comprise a cross-sectional shape that is at least one of flat, round, rectangular, or polygonal.

[0208] Aspect 6 relates to the cell culture substrate of Aspect 4 or Aspect 5, wherein the one or more fibers comprise at least one of monofilament fibers and multifilament fibers.

[0209] Aspect 7 relates to the cell culture substrate of any one of Aspects 4-6, wherein the one or more fibers comprise a first fiber having a first fiber diameter that is 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.

[0210] Aspect 8 relates to the cell culture substrate of Aspect 7, wherein the one or more fibers further comprise a second fiber having a second fiber diameter that is 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.

[0211] Aspect 9 relates to the cell culture substrate of Aspect 8, wherein the second fiber diameter is different from the first fiber diameter.

[0212] Aspect 10 relates to the cell culture substrate of any one of Aspects 1-9, wherein the plurality of openings comprise 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.

[0213] Aspect 11 relates to the cell culture substrate 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 wherein the fiber diameter is about 270 μm to about 276 μm and the opening diameter is about 785 μm to about 795 μm.

[0214] Aspect 12 relates to the cell culture substrate 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 wherein the fiber diameter is about 215 μm to about 225 μm, and the opening diameter is about 515 μm to about 530 μm.

[0215] Aspect 13 relates to the cell culture substrate 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 wherein the fiber diameter is about 150 μm to about 165 μm, and the opening diameter is about 235 μm to about 255 μm.

[0216] Aspect 14 relates to the cell culture substrate of any one of Aspects 10 - 13, 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.

[0217] Aspect 15 relates to the cell culture substrate of any one of Aspects 1 - 14, wherein the plurality of openings include openings having a square, rectangular, rhombic, oblong, circular, or elliptical shape.

[0218] Aspect 16 relates to the cell culture substrate of any one of Aspects 1 - 15, wherein the plurality of openings are arranged in a regular pattern.

[0219] Aspect 17 relates to the cell culture substrate of any one of Aspects 1 - 16, wherein the cell culture substrate comprises a single - layer substrate.

[0220] Aspect 18 relates to the cell culture substrate of any one of Aspects 1 - 17, wherein the cell culture substrate comprises a multi - layer substrate, the multi - layer substrate at least comprises a first substrate layer and a second substrate layer, wherein the first substrate layer comprises a first side and a second side opposite to the first side, and the second substrate layer comprises a third side and a fourth side opposite to the third side, and the second side faces the third side.

[0221] Aspect 19 relates to the cell culture substrate of Aspect 18, wherein the multi - layer substrate is configured such that the first substrate layer has a predetermined arrangement relative to the second substrate layer.

[0222] Aspect 20 relates to the cell culture substrate of Aspect 19, wherein the multi - layer substrate is configured such that the intersections of the fibers on the first substrate layer face the openings in the second substrate layer.

[0223] Aspect 21 relates to the cell culture substrate of Aspect 19 or Aspect 20, wherein the openings in the first substrate layer and the openings in the second substrate layer at least partially overlap.

[0224] Aspect 22 relates to the cell culture substrate of Aspect 21, wherein the openings in the first substrate layer and the openings in the second substrate layer are aligned.

[0225] Aspect 23 relates to the cell culture substrate of Aspect 28, wherein the multi-layer substrate is configured such that the first substrate layer has a random arrangement relative to the second substrate layer.

[0226] Aspect 24 relates to the cell culture substrate of any one of Aspects 1-23, wherein the cell culture substrate includes a plurality of substrates, and each substrate among the plurality of substrates has a random orientation relative to the other substrates among the plurality of substrates.

[0227] Aspect 25 relates to the cell culture substrate of any one of Aspects 1-23, wherein the cell culture substrate includes a plurality of substrates arranged in a stack.

[0228] Aspect 26 relates to the cell culture substrate of Aspect 25, wherein the first side and the second side of one substrate among the plurality of substrates are substantially parallel to the first side and the second side of the other substrates in the stack arrangement.

[0229] Aspect 27 relates to the cell culture substrate of any one of Aspects 1-23, wherein the substrate is in the configuration of a cylindrical roll.

[0230] Aspect 28 relates to the cell culture substrate of Aspect 27, wherein the cylindrical roll is configured such that when it is disposed in the culture chamber, it expands into the shape of the culture chamber in the bioreactor vessel by partial unwinding of the cylindrical roll.

[0231] Aspect 29 relates to the cell culture substrate of Aspect 28, wherein the cylindrical roll is configured to be inserted into the culture space when the cylindrical roll is in a contracted state, and it expands in the culture space when it is disposed in the culture space.

[0232] Aspect 30 relates to the cell culture substrate of any one of Aspects 1-29, wherein the cell culture substrate includes a plurality of substrates, and the plurality of substrates include woven meshes having different geometric structures, wherein the different geometric structures differ in at least one of fiber diameter, opening diameter, or opening geometry.

[0233] Aspect 31 relates to the cell culture substrate of Aspect 30, wherein the woven meshes having different geometric structures are arranged in a predetermined arrangement based on the desired flow characteristics within the bioreactor vessel.

[0234] Aspect 32 relates to the cell culture substrate of Aspect 31, wherein the desired flow characteristics include at least one of the following: the liquid culture medium is uniformly perfused through the cell culture substrate, and cell growth is distributed throughout the cell culture substrate.

[0235] Aspect 33 relates to the cell culture matrix of Aspect 31 or Aspect 32, wherein the woven mesh with different geometric structures includes a first mesh with a first geometric structure and a second mesh with a second geometric structure, and wherein the predetermined arrangement includes: relative to the desired overall flow direction of the cell culture medium through the cell culture matrix, the first mesh is upstream of the second mesh.

[0236] Aspect 34 relates to the cell culture matrix of Aspect 33, wherein the predetermined arrangement includes that a stack of the first meshes is arranged upstream of a stack of the second meshes.

[0237] Aspect 35 relates to the cell culture matrix of Aspect 33 or Aspect 34, wherein the predetermined arrangement includes that stacks of the first meshes and stacks of the second meshes are arranged alternately along the overall flow direction.

[0238] Aspect 36 relates to the cell culture matrix of any one of Aspects 1 - 35, wherein the cell culture matrix is configured to culture and / or harvest at least one of cells, proteins, antibodies, viruses, viral vectors, virus - like particles (VLPs), microvessicles, exosomes, and polysaccharides.

[0239] Aspect 37 relates to the cell culture matrix of any one of Aspects 1 - 36, wherein the substrate includes a functionalized surface, and the functionalized surface is physically or chemically modified to improve the adhesion of adherent cells to the polymer mesh material.

[0240] Aspect 38 relates to the cell culture matrix of any one of Aspects 1 - 37, wherein the cell culture matrix includes a surface configured to adsorb or absorb components in the culture medium onto the mesh surface.

[0241] Aspect 39 relates to the cell culture matrix of any one of Aspects 1 - 38, wherein the cell culture matrix includes a coating on the surface of the polymer mesh material, and the coating is configured to promote the adhesion of adherent cells.

[0242] Aspect 40 relates to the cell culture matrix of Aspect 39, wherein cells adhere to the coating.

[0243] Aspect 41 relates to the cell culture matrix of Aspect 39 or Aspect 40, wherein the coating is a biological or synthetic bioactive molecule configured to promote cell adhesion to the cell culture matrix.

[0244] Aspect 42 relates to the cell culture matrix of any one of Aspects 39 - 41, wherein the coating is at least one of a hydrogel, collagen, a bioactive molecule or peptide, and a biological protein.

[0245] Aspect 43 relates to a cell culture substrate of any one of aspects 39 - 42, wherein the functionalized surface is plasma-treated.

[0246] Aspect 44 relates to a cell culture substrate of any one of aspects 1 - 43, wherein the cells include at least one of the following: adherent cells, suspension cells, and loosely adherent cells, which adhere to the woven mesh.

[0247] Aspect 45 relates to a bioreactor system, which includes: a cell culture container including at least one reservoir; and a cell culture substrate disposed in the at least one reservoir, the cell culture substrate including a woven substrate having a plurality of interwoven fibers, and the surface of the fibers being configured for cell adhesion.

[0248] Aspect 46 relates to the system of aspect 45, wherein the woven substrate includes a uniform arrangement of a plurality of interwoven fibers.

[0249] Aspect 47 relates to the system of aspect 45 or aspect 46, wherein the woven substrate includes a plurality of openings disposed between the plurality of fibers.

[0250] Aspect 48 relates to the system of any one of aspects 45 - 47, wherein the plurality of fibers include polymer fibers.

[0251] Aspect 49 relates to the system of aspect 48, wherein the polymer fibers include at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0252] Aspect 50 relates to the system of any one of aspects 45 - 49, wherein the cell culture substrate includes a plurality of woven substrates.

[0253] Aspect 51 relates to the system of aspect 50, wherein each of the plurality of substrates includes a first side, a second side opposite the first side, and a thickness separating the first side and the second side, wherein the plurality of openings pass through the thickness of the substrate.

[0254] Aspect 52 relates to the system of aspect 50 or aspect 51, wherein the substrates among the plurality of substrates are arranged adjacent to each other such that one of the first side and the second side of a substrate is adjacent to the other of the first side or the second side of an adjacent substrate.

[0255] Aspect 53 relates to the system of any one of aspects 50 - 52, wherein at least a portion of the plurality of substrates is not separated by a spacer material or a barrier.

[0256] Aspect 54 relates to the system of any one of aspects 50 - 53, wherein at least a portion of the plurality of substrates are in physical contact with each other.

[0257] Aspect 55 relates to a system of any one of aspects 45 - 54, wherein the cell culture vessel includes at least one port configured to supply material to or remove material from the at least one reservoir through the at least one port.

[0258] Aspect 56 relates to the system of 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.

[0259] Aspect 57 relates to the system of aspect 56, wherein the material includes at least one of a culture medium, a cell, or a cell product.

[0260] Aspect 58 relates to a cell culture system, comprising: a bioreactor vessel; and a cell culture matrix disposed in the bioreactor vessel and configured to culture cells; wherein the cell culture matrix includes a substrate, the substrate including 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, and wherein the plurality of openings are configured to allow at least one of a cell culture medium, a cell, or a cell product to flow through the thickness of the substrate.

[0261] Aspect 59 relates to the cell culture system of aspect 58, wherein the substrate includes at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0262] Aspect 60 relates to the cell culture system of aspect 58 or aspect 59, wherein the substrate includes at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh.

[0263] Aspect 61 relates to the cell culture system of aspect 60, wherein the substrate includes a woven mesh containing one or more fibers.

[0264] Aspect 62 relates to the cell culture system of aspect 61, wherein the one or more fibers include a cross-sectional shape that is at least one of flat, circular, rectangular, or polygonal.

[0265] Aspect 63 relates to the cell culture system of aspect 62 or aspect 62, wherein the one or more fibers include at least one of a monofilament fiber and a multifilament fiber.

[0266] Aspect 64 relates to the cell culture system of any one of aspects 61 - 63, wherein the one or more fibers comprise a first fiber having a first fiber diameter, and the first fiber diameter is 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.

[0267] Aspect 65 relates to the cell culture system of aspect 64, wherein the one or more fibers further comprise a second fiber having a second fiber diameter, and the second fiber diameter is 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.

[0268] Aspect 66 relates to the cell culture system of aspect 65, wherein the second fiber diameter is different from the first fiber diameter.

[0269] Aspect 67 relates to the cell culture system of any one of aspects 58 - 64, wherein the plurality of openings include 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.

[0270] Aspect 68 relates 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 wherein the fiber diameter is about 270 μm to about 276 μm and the opening diameter is about 785 μm to about 795 μm.

[0271] Aspect 69 relates 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 wherein the fiber diameter is about 215 μm to about 225 μm and the opening diameter is about 515 μm to about 530 μm.

[0272] Aspect 70 relates 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 wherein the fiber diameter is about 150 μm to about 165 μm and the opening diameter is about 235 μm to about 255 μm.

[0273] Aspect 71 relates to the cell culture system of any one of aspects 67 - 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.

[0274] Aspect 72 relates to a cell culture system according to any one of aspects 58 - 71, wherein the plurality of openings include openings having a square, rectangular, rhombic, oblong, circular, or oval shape.

[0275] Aspect 73 relates to a cell culture system according to any one of aspects 58 - 72, wherein the plurality of openings are arranged in a regular pattern.

[0276] Aspect 74 relates to a cell culture system according to any one of aspects 58 - 73, wherein the cell culture substrate includes a single - layer substrate.

[0277] Aspect 75 relates to a cell culture system according to any one of aspects 58 - 74, wherein the cell culture substrate includes a multi - layer substrate, the multi - layer substrate including at least a first substrate layer and a second substrate layer, wherein the first substrate layer includes a first side and a second side opposite the first side, and the second substrate layer includes a third side and a fourth side opposite the third side, and the second side faces the third side.

[0278] Aspect 76 relates to the cell culture system of aspect 75, wherein the multi - layer substrate is configured such that the first substrate layer has a predetermined arrangement relative to the second substrate layer.

[0279] Aspect 77 relates to the cell culture system of aspect 76, wherein the multi - layer substrate is configured such that the intersections of the fibers on the first substrate layer face the openings in the second substrate layer.

[0280] Aspect 78 relates to the cell culture system of aspect 76 or aspect 77, wherein the openings in the first substrate layer and the openings in the second substrate layer at least partially overlap.

[0281] Aspect 79 relates to the cell culture system of aspect 78, wherein the openings in the first substrate layer and the openings in the second substrate layer are aligned.

[0282] Aspect 80 relates to the cell culture system of aspect 75, wherein the multi - layer substrate is configured such that the first substrate layer has a random arrangement relative to the second substrate layer.

[0283] Aspect 81 relates to a cell culture system according to any one of aspects 58 - 80, wherein the cell culture substrate is disposed in a bioreactor vessel such that the overall flow direction of the culture medium through the bioreactor vessel is parallel or perpendicular to the first side and the second side.

[0284] Aspect 82 relates to a cell culture system according to any one of aspects 58 - 81, wherein the cell culture substrate includes a plurality of substrates randomly packed into the bioreactor vessel.

[0285] Aspect 83 relates to a cell culture system of any one of aspects 58 - 82, wherein the bioreactor vessel is a packed bed bioreactor.

[0286] Aspect 84 relates to a cell culture system of any one of aspects 58 - 83, wherein the bioreactor vessel includes: a culture space disposed within the bioreactor vessel and containing a cell culture substrate, and one or more openings configured to provide fluid to the culture space or remove fluid from the culture space.

[0287] Aspect 85 relates to the cell culture system of aspect 84, wherein the one or more openings include an inlet and an outlet, the inlet being configured to provide fluid to the interior of the culture space, and the outlet being configured to remove fluid from the culture space of the bioreactor vessel.

[0288] Aspect 86 relates to the cell culture system of aspect 85, wherein the bioreactor vessel includes: a first end containing the inlet, a second end opposite the first end and containing the outlet, and a culture space disposed between the first end and the second end.

[0289] Aspect 87 relates to the cell culture system of aspect 86, wherein the cell culture substrate has a shape corresponding to the shape of the culture space.

[0290] Aspect 88 relates to a cell culture system of any one of aspects 58 - 87, wherein the cell culture substrate includes a polymeric mesh material configured as a cylindrical roll.

[0291] Aspect 89 relates to the cell culture system of aspect 88, wherein the central longitudinal axis of the cylindrical roll is parallel to the flow direction of the culture medium.

[0292] Aspect 90 relates to the cell culture system of aspect 88 or aspect 89, wherein the cylindrical roll is configured to expand into the shape of the culture space in the bioreactor vessel by unrolling of the cylindrical roll.

[0293] Aspect 91 relates to a cell culture system of any one of aspects 88 - 90, wherein the cylindrical roll is configured to be inserted into the culture space when the cylindrical roll is in a contracted state and, when disposed within the culture space, to expand within the culture space.

[0294] Aspect 92 relates to a cell culture system of any one of aspects 88 - 91, wherein the cylindrical roll and the culture space are configured such that the frictional force between the polymeric mesh material and the culture space wall holds the polymeric mesh material in place within the culture space.

[0295] Aspect 93 relates to the cell culture system of aspect 91, wherein the cylindrical roll is configured to be inserted into the culture space through an opening in the bioreactor vessel.

[0296] Aspect 94 relates to the cell culture system of aspect 93, wherein the opening is one of the inlet and outlet of the bioreactor vessel.

[0297] Aspect 95 relates to the cell culture system of any one of aspects 88 - 94, wherein the bioreactor vessel includes a substrate support member located within the culture space, and the substrate support member is configured to direct, align, or secure the cell culture substrate within the culture space.

[0298] Aspect 96 relates to the cell culture system of aspect 95, wherein the substrate support member includes a support member extending from one of the first or second ends towards the other of the first or second ends, and a 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.

[0299] Aspect 97 relates to the cell culture system of any one of aspects 58 - 96, wherein the bioreactor vessel is configured to rotate about the central longitudinal axis of the bioreactor vessel during cell culture.

[0300] Aspect 98 relates to the cell culture system of aspect 97, wherein during cell culture, the central longitudinal axis is perpendicular to the direction of gravity.

[0301] Aspect 99 relates to the cell culture system of aspect 97 or aspect 98, wherein the cell culture system is configured such that during rotation of the bioreactor vessel, the substrate moves through the cell culture fluid.

[0302] Aspect 100 relates to the cell culture system of any one of aspects 97 - 99, wherein the cell culture system further includes a rotation device operatively connected to the bioreactor vessel and configured to rotate the bioreactor vessel about the central longitudinal axis.

[0303] Aspect 101 relates to the cell culture system of any one of aspects 58 - 100, wherein the cell culture substrate includes a plurality of substrates, and the plurality of substrates includes woven meshes having different geometric structures, and the different geometric structures differ in at least one of fiber diameter, opening diameter, or opening geometry.

[0304] Aspect 102 relates to the cell culture system of aspect 101, wherein the woven meshes having different geometric structures are arranged within the bioreactor vessel in a predetermined arrangement based on desired flow characteristics within the bioreactor vessel.

[0305] Aspect 103 relates to the cell culture system of aspect 102, wherein the desired flow characteristics include at least one of the following: uniform perfusion of the liquid medium through the cell culture substrate, and cell growth distributed across the entire cell culture substrate.

[0306] Aspect 104 relates to the cell culture system of aspect 102 or aspect 103, wherein the woven mesh with different geometric structures includes a first mesh with a first geometric structure and a second mesh with a second geometric structure, and wherein the predetermined arrangement includes: relative to the overall flow direction, the first mesh is upstream of the second mesh.

[0307] Aspect 105 relates to the cell culture system of aspect 104, wherein the predetermined arrangement includes that a stack of the first meshes is arranged upstream of a stack of the second meshes.

[0308] Aspect 106 relates to the cell culture system of aspect 104 or aspect 105, wherein the predetermined arrangement includes that stacks of the first meshes and stacks of the second meshes are arranged alternately along the overall flow direction.

[0309] Aspect 107 relates to the cell culture system of any one of aspects 58 - 106, and further includes a device for harvesting adherent cells or cell by-products.

[0310] Aspect 108 relates to the cell culture system of aspect 107, wherein the cell by-products include at least one of proteins, antibodies, viruses, viral vectors, virus-like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0311] Aspect 109 relates to the cell culture system of any one of aspects 58 - 108, wherein the substrate includes a functionalized surface, and the functionalized surface is physically or chemically modified to improve the adhesion of adherent cells to the polymer mesh material.

[0312] Aspect 110 relates to the cell culture system of any one of aspects 58 - 109, wherein the cell culture substrate includes a surface configured to adsorb or absorb components in the culture medium onto the mesh surface.

[0313] Aspect 111 relates to the cell culture system of any one of aspects 58 - 110, wherein the cell culture substrate includes a coating on the surface of the polymer mesh material, and the coating is configured to promote the adhesion of adherent cells.

[0314] Aspect 112 relates to the cell culture system of aspect 111, wherein cells adhere to the coating.

[0315] Aspect 113 relates to the cell culture system of aspect 111 or aspect 112, wherein the coating is a biological or synthetic bioactive molecule configured to promote cell adhesion to the cell culture substrate.

[0316] Aspect 114 relates to the cell culture system of any one of aspects 111 - 113, wherein the coating is a hydrogel, collagen, At least one of a bioactive molecule or peptide, and a biological protein.

[0317] Aspect 115 relates to the cell culture system of any one of aspects 110 - 113, wherein the functionalized surface is plasma-treated.

[0318] Aspect 116 relates to the cell culture system of any one of aspects 58 - 115, wherein the cells include at least one of the following: adherent cells, suspension cells, and loosely adherent cells, which adhere to the woven mesh.

[0319] Aspect 117 relates to the cell culture system of any one of aspects 58 - 116, which further includes a culture medium regulating container configured to supply a culture medium to the inlet of the bioreactor container.

[0320] Aspect 118 relates to a bioreactor system, which includes: a cell culture container including a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture substrate disposed in the at least one reservoir, the cell culture substrate including a plurality of woven substrates, each woven substrate including a plurality of interwoven fibers, and the surface of the fibers being configured for cell adhesion, wherein the bioreactor system is configured to allow a material to flow through the at least one reservoir in a flow direction from the first end to the second end, and wherein the substrates in the plurality of woven substrates are stacked such that each woven substrate is substantially parallel to each of the other woven substrates and substantially perpendicular to the flow direction.

[0321] Aspect 119 relates to the system of aspect 118, wherein each of the substrates includes 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 passing through the thickness of the substrate.

[0322] Aspect 120 relates to the system of aspect 118 or aspect 119, wherein the substrate includes at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0323] Aspect 121 relates to the system of any one of aspects 118 - 120, wherein the plurality of interwoven fibers includes a first fiber having a first fiber diameter, and the first fiber diameter is 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.

[0324] Aspect 122 relates to the system of aspect 121, wherein the plurality of interwoven fibers further comprises a second fiber having a second fiber diameter, and the second fiber diameter is 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.

[0325] Aspect 123 relates to the system of aspect 122, wherein the second fiber diameter is equal to or less than the first fiber diameter.

[0326] Aspect 124 relates to the system of any one of aspects 119 - 123, wherein the plurality of openings include 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.

[0327] Aspect 125 relates to the system of any one of aspects 119 - 124, 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.

[0328] Aspect 126 relates to the system of any one of aspects 119 - 125, wherein the plurality of openings are arranged in a regular pattern.

[0329] Aspect 127 relates to the system of any one of aspects 118 - 126, wherein the cell culture matrix comprises a plurality of substrates, and the plurality of substrates comprises a woven mesh having different geometric structures, wherein the different geometric structures are different in at least one of the fiber diameter, the opening diameter, or the opening geometry.

[0330] Aspect 128 relates to the system of aspect 127, wherein the woven meshes having different geometric structures are arranged in a predetermined arrangement based on desired flow characteristics within a bioreactor vessel.

[0331] Aspect 129 relates to the system of any one of aspects 118 - 128, wherein the cell culture matrix is configured to culture and / or harvest at least one of cells, proteins, antibodies, viruses, viral vectors, virus - like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0332] Aspect 130 relates to the system of any one of aspects 118 - 129, wherein the substrate comprises a functionalized surface that is physically or chemically modified to improve the adhesion of adherent cells to the polymer mesh material.

[0333] Aspect 131 relates to a system of any one of aspects 118 - 130, wherein the plurality of interwoven fibers are arranged in an ordered, non - random arrangement relative to each other of the plurality of interwoven fibers.

[0334] Aspect 132 relates to a system of any one of aspects 118 - 131, wherein at least a portion of the plurality of substrates is not separated by spacer material or a barrier.

[0335] Aspect 133 relates to a system of any one of aspects 118 - 132, wherein at least a portion of the plurality of substrates are in physical contact with each other.

[0336] Aspect 134 relates to a method of culturing cells in a bioreactor system of any one of aspects 118 - 133, the method comprising: inoculating cells on a cell culture substrate; culturing the cells on the cell culture substrate; and harvesting the product of the cell culture, wherein a plurality of openings in the substrate are configured to allow at least one of cell culture medium, cells, or cell products to flow through the thickness of the substrate.

[0337] Aspect 135 relates to the method of aspect 134, wherein the inoculating comprises adhering the cells to the substrate.

[0338] Aspect 136 relates to the method of aspect 135, wherein the inoculating comprises directly injecting a cell inoculum into the cell culture substrate.

[0339] Aspect 137 relates to the method of any one of aspects 134 - 136, further comprising: after injecting the cell inoculum, perfusing cell culture medium through the culture chamber.

[0340] Aspect 138 relates to the method of any one of aspects 134 - 137, further comprising: providing a culture medium conditioning container fluidly connected to the bioreactor container and supplying cell culture medium from the culture medium conditioning container to the bioreactor container.

[0341] Aspect 139 relates to the method of aspect 138, wherein during or after culturing, at least a portion of the culture medium is recovered from the bioreactor container and returned to the culture medium conditioning container.

[0342] Aspect 140 relates to the method of any one of aspects 134 - 139, further comprising: controlling the flow of cell culture medium to the cell culture chamber, wherein the cell culture medium comprises at least one of cells, cell culture nutrients, or oxygen.

[0343] Aspect 141 relates to a bioreactor system comprising: a cell culture vessel including a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture substrate disposed in the at least one reservoir, the cell culture substrate including a plurality of woven substrates, each woven substrate including a plurality of interwoven fibers, and the surface of the fibers being configured for cell adhesion, wherein the bioreactor system is configured to cause material to flow through the at least one reservoir in a flow direction from the first end to the second end, and wherein the substrates in the plurality of woven substrates are stacked such that each woven substrate is substantially parallel to each of the other woven substrates and substantially parallel to the flow direction.

[0344] Aspect 142 relates to the system of aspect 141, wherein each substrate of the substrates includes 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 through the thickness of the substrate.

[0345] Aspect 143 relates to the system of aspect 141 or aspect 119, wherein the substrate includes at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0346] Aspect 144 relates to the system of any one of aspects 141 - 143, wherein the plurality of interwoven fibers includes a first fiber having a first fiber diameter, the first fiber diameter being from about 50 μm to about 1000 μm, from about 50 μm to about 600 μm, from about 50 μm to about 400 μm, from about 100 μm to about 325 μm, or from about 150 μm to about 275 μm.

[0347] Aspect 145 relates to the system of aspect 144, wherein the plurality of interwoven fibers further includes a second fiber having a second fiber diameter, the second fiber diameter being from about 50 μm to about 1000 μm, from about 50 μm to about 600 μm, from about 50 μm to about 400 μm, from about 100 μm to about 325 μm, or from about 150 μm to about 275 μm.

[0348] Aspect 146 relates to the system of aspect 145, wherein the second fiber diameter is equal to or less than the first fiber diameter.

[0349] Aspect 147 relates to the system of any one of aspects 142 - 146, wherein the plurality of openings includes an opening diameter of from about 100 μm to about 1000 μm, from about 200 μm to about 900 μm, or from about 225 μm to about 800 μm.

[0350] Aspect 148 relates to a system of any one of aspects 142 - 147, wherein the ratio of the opening 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.

[0351] Aspect 149 relates to a system of any one of aspects 142 - 148, wherein the plurality of openings are arranged in a regular pattern.

[0352] Aspect 150 relates to a system of any one of aspects 141 - 149, wherein the cell culture substrate comprises a plurality of substrates, the plurality of substrates comprising woven meshes having different geometries, wherein the different geometries differ in at least one of fiber diameter, opening diameter, or opening geometry.

[0353] Aspect 151 relates to the system of aspect 150, wherein the woven meshes having different geometries are arranged in a predetermined arrangement based on desired flow characteristics within a bioreactor vessel.

[0354] Aspect 152 relates to a system of any one of aspects 141 - 151, wherein the cell culture substrate is configured to culture and / or harvest at least one of cells, proteins, antibodies, viruses, viral vectors, virus - like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0355] Aspect 153 relates to a system of any one of aspects 141 - 152, wherein the substrate comprises a functionalized surface that is physically or chemically modified to improve the adhesion of adherent cells to the polymeric mesh material.

[0356] Aspect 154 relates to a system of any one of aspects 141 - 153, wherein the plurality of interwoven fibers are arranged in an ordered, non - random arrangement relative to each other interwoven fiber of the plurality of interwoven fibers.

[0357] Aspect 155 relates to a system of any one of aspects 141 - 154, wherein at least a portion of the plurality of substrates is not separated by a spacer material or a barrier.

[0358] Aspect 156 relates to a system of any one of aspects 141 - 155, wherein at least a portion of the plurality of substrates are in physical contact with each other.

[0359] Aspect 157 relates to a method of culturing cells in a bioreactor system of any one of aspects 141 - 156, the method comprising: inoculating cells on a cell culture substrate; culturing the cells on the cell culture substrate; and harvesting the product of the cell culture, wherein the plurality of openings in the substrate are configured to allow at least one of cell culture medium, cells, or cell products to flow through the thickness of the substrate.

[0360] Aspect 158 relates to the method of aspect 157, wherein the seeding includes adhering cells to a substrate.

[0361] Aspect 159 relates to the method of aspect 158, wherein the seeding includes directly injecting a cell inoculum into a cell culture substrate.

[0362] Aspect 160 relates to the method of any one of aspects 157 - 159, further comprising: after injecting the cell inoculum, perfusing a cell culture medium through a culture chamber.

[0363] Aspect 161 relates to the method of any one of aspects 157 - 160, further comprising: providing a culture medium conditioning container fluidly connected to a bioreactor container and supplying a cell culture medium from the culture medium conditioning container to the bioreactor container.

[0364] Aspect 162 relates to the method of aspect 161, wherein during or after culturing, at least a portion of the culture medium is recovered from the bioreactor container and returned to the culture medium conditioning container.

[0365] Aspect 163 relates to the method of any one of aspects 157 - 162, further comprising: controlling the flow of a cell culture medium to a cell culture chamber, wherein the cell culture medium includes at least one of cells, cell culture nutrients, or oxygen.

[0366] Aspect 164 relates to a bioreactor system comprising: a cell culture container including a first end, a second end, and at least one reservoir between the first end and the second end; and a cell culture substrate disposed in the at least one reservoir, the cell culture substrate including a woven substrate comprising a plurality of interwoven fibers, and a surface of the fibers being configured for cell adhesion, and wherein the woven substrate is disposed in the at least one reservoir in a wound configuration to provide a cylindrical cell culture substrate, and a surface of the woven substrate is parallel to a longitudinal axis of the cylindrical cell culture substrate.

[0367] Aspect 165 relates to the system of aspect 164, wherein the woven substrate is disposed in the at least one reservoir as a cylindrical substrate that at least partially surrounds a central longitudinal axis of the bioreactor container.

[0368] Aspect 166 relates to the system of aspect 164 or aspect 265, wherein the bioreactor system is configured to allow a material to flow through the at least one reservoir in a flow direction from the first end to the second end.

[0369] Aspect 167 relates to the system of aspect 166, wherein a central longitudinal axis of the cylindrical substrate is parallel to a flow direction of the culture medium.

[0370] Aspect 168 relates to a system of any of aspects 164 - 167, wherein the cylindrical substrate comprises a wound woven substrate that is configured to expand into contact with the wall of the at least one reservoir by unwinding the wound woven substrate.

[0371] Aspect 169 relates to a system of any of aspects 164 - 168, wherein the wound woven substrate is configured to expand into the shape of the interior of the at least one reservoir within a cell culture vessel.

[0372] Aspect 170 relates to the system of aspect 169, wherein the wound woven substrate is configured to be inserted into a culture space when the wound woven substrate is in a contracted wound state and to expand within the reservoir when disposed within the reservoir.

[0373] Aspect 171 relates to the system of aspect 169 or aspect 170, wherein the wound woven substrate and the reservoir are configured such that the frictional force between the woven substrate and the reservoir wall substantially holds the woven substrate in place within the reservoir.

[0374] Aspect 172 relates to a system of any of aspects 169 - 171, wherein the wound woven substrate is configured to be inserted into the reservoir through an opening in the cell culture vessel.

[0375] Aspect 173 relates to the system of aspect 172, wherein the opening is one of an inlet and an outlet of the cell culture vessel.

[0376] Aspect 174 relates to a system of any of aspects 164 - 173, wherein the cell culture vessel includes a substrate support member located within the reservoir, the substrate support member being configured to guide, align, or secure the woven substrate within the culture space.

[0377] Aspect 175 relates to the system of aspect 174, wherein the substrate support member includes a support member extending from one of a first or second end towards the other of the first or second end, wherein the wound woven substrate is configured to surround at least a portion of the periphery of the support member such that the support member is parallel to the central longitudinal axis of the wound woven substrate.

[0378] Aspect 176 relates to a system of any of aspects 164 - 175, wherein during cell culture, the central longitudinal axis is perpendicular to the direction of gravity.

[0379] Aspect 177 relates to a system of any of aspects 164 - 175, wherein at least one of the reservoir and the cell culture substrate is configured to rotate about the central longitudinal axis of the bioreactor vessel during cell culture.

[0380] Aspect 178 relates to the system of aspect 177, wherein the bioreactor system is configured to move a substrate through the cell culture fluid during rotation of the cell culture vessel.

[0381] Aspect 179 relates to the system of aspect 177 or aspect 178, wherein the bioreactor system further includes a rotating device operatively connected to the cell culture vessel and configured to rotate the cell culture vessel about a central longitudinal axis.

[0382] Aspect 180 relates to the system of any one of aspects 164 - 179, wherein the cylindrical cell culture substrate includes a woven cell culture substrate and does not have any other solid material between adjacent surfaces of the cell culture substrate.

[0383] Aspect 181 relates to a method of culturing cells in a bioreactor, the method comprising: providing a bioreactor vessel including: a cell culture chamber within the bioreactor vessel, and a cell culture substrate disposed in the cell culture chamber and configured for culturing cells thereon, the cell culture substrate including a substrate including 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; inoculating cells on the cell culture substrate; culturing cells on the cell culture substrate; and harvesting the product of the cell culture, wherein the plurality of openings in the substrate are configured to allow at least one of cell culture medium, cells, or cell products to flow through the thickness of the substrate.

[0384] Aspect 182 relates to the method of aspect 181, wherein the substrate includes at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh.

[0385] Aspect 183 relates to the method of aspect 181 or aspect 182, wherein the substrate includes a polymer material.

[0386] Aspect 184 relates 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.

[0387] Aspect 185 relates to the method of any one of aspects 181 - 184, wherein the inoculating includes adhering cells to the substrate.

[0388] Aspect 186 relates to the method of any one of aspects 181 - 185, wherein the inoculating includes directly injecting a cell inoculum into the cell culture substrate.

[0389] Aspect 187 relates to the method of aspect 186, wherein the cell inoculum is injected through a cell inoculum injection port in a bioreactor vessel.

[0390] Aspect 188 relates to the method of aspect 186 or aspect 187, wherein the volume of the cell inoculum is approximately equal to the void volume of the cell culture chamber.

[0391] Aspect 189 relates to the method of any one of aspects 186 - 188, further comprising: after injecting the cell inoculum, perfusing cell culture medium through the culture chamber.

[0392] Aspect 190 relates to the method of any one of aspects 181 - 189, further comprising: during culturing, supplying at least one of cell culture medium and oxygen to the cells.

[0393] Aspect 191 relates to the method of aspect 190, wherein supplying cell culture medium comprises: flowing the cell culture medium through the cell culture chamber and over the substrate.

[0394] Aspect 192 relates to the method of aspect 190 or aspect 191, wherein supplying cell culture medium comprises: providing a culture medium conditioning container fluidly connected to the bioreactor vessel and supplying the cell culture medium from the culture medium conditioning container to the bioreactor vessel.

[0395] Aspect 193 relates to the method of aspect 192, wherein during or after culturing, at least a portion of the culture medium is recovered from the bioreactor vessel and returned to the culture medium conditioning container.

[0396] Aspect 194 relates to the method of any one of aspects 181 - 193, further comprising: controlling the flow of cell culture medium to the cell culture chamber, wherein the cell culture medium comprises at least one of cells, cell culture nutrients, or oxygen.

[0397] Aspect 195 relates to the method of any one of aspects 181 - 194, further comprising: analyzing the cell culture medium, cells, and / or cell products within or output from the bioreactor vessel.

[0398] Aspect 196 relates to the method of aspect 195, wherein the analysis comprises: 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 wherein pH2, pO2, and [glucose]2 are measured at the outlet of the cell culture chamber or the bioreactor vessel.

[0399] Aspect 197 relates to the method of Aspect 195 or Aspect 196, wherein the flow of the cell culture medium to the cell culture chamber is controlled based at least in part on the results of analyzing the cell culture medium, cells, and / or cell products.

[0400] Aspect 198 relates to the method of any one of Aspects 196 - 197, wherein if pH2 ≥ pH 2最小 、pO2 ≥ pO 2最小 and [glucose]2 ≥ [glucose] 2最小 at least one of which is satisfied, the perfusion flow rate of the cell culture medium reaching the cell culture chamber continues at the current rate, where pH 2最小 、pO 2最小 and [glucose] 2最小 are predetermined based on the design of the cell culture system.

[0401] Aspect 199 relates to the method of any one of Aspects 196 - 198, wherein if the current flow rate is less than or equal to the predetermined maximum flow rate of the cell culture system, the perfusion flow rate is increased.

[0402] Aspect 200 relates to the method of any one of Aspects 196 - 199, wherein if the current flow rate is not less than or equal to the predetermined maximum flow rate of the cell culture system, the controller of the cell culture system re - evaluates at least one of the following: pH 2最小 、pO 2最小 and [glucose] 2最小 ; pH1, pO1 and [glucose]1; and the height of the bioreactor vessel.

[0403] Aspect 201 relates to the method of any one of Aspects 181 - 200, wherein after culturing for at least about 24 hours, at least about 48 hours, or at least about 72 hours, the cells have a viability of more than about 90% or more than about 95%.

[0404] Aspect 202 relates to the method of any one of Aspects 181 - 201, wherein the cells include at least one of the following: adherent cells, suspension cells, and loosely adherent cells that adhere to the cell culture matrix.

[0405] Aspect 203 relates to the method of any one of Aspects 181 - 202, wherein the products of the cell culture include at least one of cells, proteins, antibodies, viruses, viral vectors, virus - like particles (VLPs), microvesicles, exosomes, and polysaccharides.

[0406] Aspect 204 relates to the method of aspect 203, wherein the product of cell culture comprises cells, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of which are viable.

[0407] Aspect 205 relates to a cell culture substrate, which comprises: a woven substrate, which comprises a plurality of interwoven fibers and a plurality of openings disposed between the plurality of fibers, wherein each of the fibers comprises a surface configured for cell adhesion.

[0408] Aspect 206 relates to the substrate of aspect 205, wherein the surface of the fiber is configured for cells to adhere thereto releasably.

[0409] Aspect 207 relates to the substrate of aspect 205 or aspect 206, wherein the plurality of fibers comprise polymer fibers.

[0410] Aspect 208 relates to the substrate 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.

[0411] Aspect 209 relates to the substrate of any one of aspects 205-208, wherein the cell culture substrate further comprises a plurality of woven substrates.

[0412] Aspect 210 relates to the substrate of aspect 209, wherein each of the plurality of substrates comprises a first side, a second side opposite to the first side, and a thickness separating the first side and the second side, wherein the plurality of openings pass through the thickness of the substrate.

[0413] Aspect 211 relates to the substrate of aspect 209 or aspect 210, wherein the substrates among the plurality of substrates are arranged adjacent to each other such that one of the first side and the second side of a substrate is adjacent to the other of the first side or the second side of an adjacent substrate.

[0414] Aspect 212 relates to the substrate of any one of aspects 209-211, wherein at least a portion of the plurality of substrates is not separated by a spacer material or a barrier.

[0415] Aspect 213 relates to the substrate of any one of aspects 205-212, wherein at least a portion of the plurality of substrates are in physical contact with each other.

[0416] Definitions

[0417] "Fully synthetic" or "total synthesis" means that a cell culture product (such as a microcarrier or the surface of a culture vessel) consists entirely of synthetic source materials and does not contain any materials derived from or of animal origin. The disclosed fully synthetic cell culture products eliminate the risk of xenogeneic contamination.

[0418] The terms "comprises", "comprising", or the like mean including but not limited to, i.e., inclusive and not exclusive.

[0419] According to embodiments herein, a "user" means a person who uses the systems, methods, products, or kits disclosed herein, including a person who cultures cells to harvest cells or cell products, or a person who uses the cultured and / or harvested cells or cell products.

[0420] The term "about" used to describe numerical values and their ranges, such as amounts, concentrations, volumes, process temperatures, process times, yields, flow rates, pressures, viscosities, etc., of components in a composition, or numerical values and their ranges of component dimensions, etc., in connection with embodiments of the present disclosure, refers to variations in the quantity that can occur, for example, in typical measurements and processing steps for preparing materials, compositions, complexes, concentrates, component parts, manufactured articles, or applied formulations; in inadvertent errors in these procedures; in differences in the manufacture, source, or purity of starting materials or components used to carry out the methods; and in similar considerations. The term "about" also includes amounts that differ due to the aging of a composition or formulation having a particular initial concentration or mixture, and amounts that differ due to the mixing or processing of a composition or formulation having a particular initial concentration or mixture.

[0421] "Optional" or "optionally" means that the subsequent described event or circumstance may or may not occur, and the description includes both the case where the event or circumstance occurs and the case where the event or circumstance does not occur.

[0422] Unless otherwise specified, the indefinite articles "a" or "an" and their corresponding definite article "the" as used herein mean at least one, or one or more.

[0423] Abbreviations well known to those of ordinary skill in the art may be used (e.g., "h" or "hrs" for hours; "g" or "gm" for grams; "mL" for milliliters; "rt" for room temperature; "nm" for nanometers, and similar abbreviations).

[0424] The specific and preferred numerical values and their ranges disclosed in terms of components, ingredients, additives, dimensions, conditions, and the like are for illustration only, and they do not exclude other defined numerical values or other numerical values within the defined ranges. The systems, kits, and methods of the present disclosure may include any of the numerical values described herein or any combination of the various numerical values, specific numerical values, more specific numerical values, and preferred numerical values, including explicit or implicit intermediate numerical values and intermediate ranges.

[0425] Unless otherwise stated, no method described herein is to be construed as requiring that its steps be performed in a specific order. Accordingly, when a method claim does not actually recite its steps as following a certain order or when it is not otherwise specifically indicated in the claims or the specification that the steps are limited to a specific order, no particular order is intended to be implied.

[0426] 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 disclosed embodiments. Since those skilled in the art can make various improvements, combinations, sub-combinations, and changes to the disclosed embodiments by combining the spirit and essence of the embodiments, the disclosed embodiments should be considered to include all of the content within the scope of the appended claims and their equivalents.

Claims

1. A cell culture substrate, comprising: A multi-layer substrate, each substrate layer in the multi-layer 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 layer and passing through the thickness of the substrate layer, Wherein the plurality of openings are configured to allow at least one of a cell culture medium, cells, or cell products to flow through the thickness of the substrate layer, Wherein the plurality of openings are arranged in a regular pattern, Wherein the multi-layer substrate comprises a woven web containing one or more fibers, and the surface of the fibers is configured for cell adhesion, Wherein the cell culture substrate has structural rigidity, Wherein the one or more fibers comprise at least one of monofilament fibers and multifilament fibers, Wherein the multi-layer substrate comprises a plurality of substrate layers arranged in a stack, Wherein the plurality of substrate layers are in physical contact with each other, and Wherein the plurality of substrate layers are not separated by a spacer material or a barrier.

2. The cell culture substrate according to claim 1, wherein, The multi-layer substrate comprises at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

3. The cell culture substrate according to claim 1, wherein, The one or more fibers comprise a fiber diameter that is 50 µm to 1000 µm.

4. The cell culture matrix according to claim 1, wherein, The one or more fibers comprise a fiber diameter that is 50 µm to 600 µm.

5. The cell culture matrix according to claim 1, wherein, The one or more fibers comprise a fiber diameter that is 50 µm to 400 µm.

6. The cell culture substrate according to claim 1, wherein, The one or more fibers comprise a fiber diameter that is 100 µm to 325 µm.

7. The cell culture matrix according to claim 1, wherein, The one or more fibers comprise a fiber diameter that is 150 µm to 275 µm.

8. The cell culture substrate according to claim 1, wherein, The plurality of openings comprise 100 µm to 1000 µm.

9. The cell culture matrix according to claim 1, wherein, The plurality of openings comprise an opening diameter of 200 µm to 900 µm.

10. The cell culture substrate according to claim 1, wherein, The plurality of openings comprise an opening diameter of 225 µm to 800 µm.

11. The cell culture substrate according to claim 1, wherein, The one or more fibers comprise a fiber diameter of 250 µm to 300 µm, and the plurality of openings comprise an opening diameter of 750 µm to 800 µm.

12. The cell culture matrix according to claim 1, wherein, The one or more fibers comprise a fiber diameter of 270 µm to 276 µm, and the plurality of openings comprise an opening diameter of 785 µm to 795.

13. The cell culture substrate according to claim 1, wherein, The one or more fibers comprise a fiber diameter of 200 µm to 230 µm, and the plurality of openings comprise an opening diameter of 500 µm to 550 µm.

14. The cell culture matrix according to claim 1, wherein, The one or more fibers comprise a fiber diameter of 215 µm to 225, and the plurality of openings comprise an opening diameter of 515 µm to 530 µm.

15. The cell culture substrate according to claim 1, wherein, The one or more fibers comprise a fiber diameter of 125 µm to 175 µm, and the plurality of openings comprise an opening diameter of 225 µm to 275 µm.

16. The cell culture substrate according to claim 1, wherein, The one or more fibers comprise a fiber diameter of 150 µm to 165 µm, and the plurality of openings comprise an opening diameter of 235 µm to 255 µm.

17. The cell culture substrate according to claim 1, wherein, - The one or more ratios include a fiber diameter, the plurality of openings include an opening diameter, and the ratio of the opening diameter to the fiber diameter is from 1.0 to 3.

5.

18. The cell culture matrix according to claim 1, wherein, - The one or more ratios include a fiber diameter, the plurality of openings include an opening diameter, and the ratio of the opening diameter to the fiber diameter is from 1.25 to 3.

25.

19. The cell culture matrix according to claim 1, wherein, - The one or more ratios include a fiber diameter, the plurality of openings include an opening diameter, and the ratio of the opening diameter to the fiber diameter is from 1.4 to 3.

0.

20. The cell culture substrate according to claim 1, wherein, - The one or more ratios include a fiber diameter, the plurality of openings include an opening diameter, and the ratio of the opening diameter to the fiber diameter is from 1.5 to 2.

9.

21. The cell culture matrix according to claim 1, wherein, - The one or more ratios include a fiber diameter, the plurality of openings include an opening diameter, and the ratio of the opening diameter to the fiber diameter is from 1.5 to 2.

4.

22. The cell culture matrix according to claim 1, wherein, - The one or more ratios include a fiber diameter, the plurality of openings include an opening diameter, and the ratio of the opening diameter to the fiber diameter is from 2.4 to 2.

9.

23. The cell culture substrate according to claim 1, wherein, The multi-layer substrate includes at least a first substrate layer and a second substrate layer, wherein the first substrate layer has a predetermined arrangement relative to the second substrate layer.

24. The cell culture matrix according to claim 23, wherein, The multi-layer substrate is configured such that the intersections of the fibers on the first substrate layer face the openings in the second substrate layer.

25. The cell culture substrate according to claim 23, wherein, The openings in the first substrate layer and the openings in the second substrate layer at least partially overlap.

26. The cell culture substrate according to claim 25, wherein, The openings in the first substrate layer and the openings in the second substrate layer are aligned.

27. The cell culture matrix according to claim 23, wherein, The multi-layer substrate is configured such that the first substrate layer has a random arrangement relative to the second substrate layer.

28. The cell culture matrix according to claim 1, wherein, The first side and the second side of one substrate layer in the plurality of substrate layers are substantially parallel to the first side and the second side of the other substrate layers in the stacked arrangement.

29. The cell culture substrate according to claim 1, wherein, The multi-layer substrate is in the configuration of a cylindrical roll.

30. The cell culture matrix according to claim 1, wherein, The multi-layer substrate includes a plurality of substrate layers, and the plurality of substrate layers include woven meshes having different geometries, wherein the different geometries are different in at least one of fiber diameter, opening diameter, or opening geometry.

31. The cell culture substrate according to claim 30, wherein, The woven meshes having different geometries are arranged in a predetermined arrangement based on desired flow characteristics through the cell culture substrate.

32. The cell culture matrix according to claim 31, wherein, The desired flow characteristics include at least one of the following: uniform perfusion of the liquid culture medium through the cell culture substrate, and cell growth distributed throughout the cell culture substrate.

33. The cell culture substrate according to claim 1, wherein, The cell culture substrate is configured for culturing and / or harvesting at least one of cells and viruses.

34. The cell culture matrix according to claim 1, wherein, The cell culture substrate is configured for culturing and / or harvesting at least one of proteins and polysaccharides.

35. The cell culture substrate according to claim 1, wherein, The cell culture substrate is configured for culturing and / or harvesting antibodies.

36. The cell culture matrix according to claim 1, wherein, The cell culture substrate is configured for culturing and / or harvesting at least one of viral vectors, virus-like particles (VLPs), microvesicles, and exosomes.

37. The cell culture matrix according to claim 1, wherein, The multi-layer substrate includes a functionalized surface, which is physically or chemically modified to improve the adhesion of adherent cells to the polymer mesh material.

38. The cell culture matrix according to claim 1, wherein, The cell culture substrate includes a surface configured to adsorb or absorb components in the culture medium onto the mesh surface.

39. The cell culture substrate according to claim 1, wherein, The cell culture substrate includes a coating on the surface of the polymer mesh material, and the coating is configured to promote the adhesion of adherent cells.

40. The cell culture matrix according to claim 39, wherein, The coating is a biological or synthetic bioactive molecule configured to promote cell adhesion to the cell culture substrate.

41. The cell culture substrate according to claim 39, wherein, The coating is at least one of a hydrogel, collagen, Matrigel®, a bioactive molecule or peptide, and a biological protein.

42. The cell culture substrate according to claim 39, wherein, The functionalized surface is treated with plasma.

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