Systems and methods for coating bioreactor substrates

Abstract

A method for coating a cell culture substrate in situ in a bioreactor is provided, comprising providing a bioreactor vessel having a cell culture chamber having an inlet for introducing fluid into the cell culture chamber and an outlet for introducing fluid out of the cell culture chamber, and a cell substrate disposed in the cell culture chamber for culturing cells. The method also includes providing a coating solution for coating the cell substrate, introducing the coating solution into the cell culture chamber so that the coating solution contacts the cell substrate, and removing excess coating solution from the cell culture chamber. After removing the coating solution, the coated cell substrate remains in the cell culture chamber.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Patent Application No. 63 / 425,467, filed November 15, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE DISCLOSURE The present disclosure relates generally to systems and methods for coating substrates in cell culture bioreactors. In particular, the disclosure relates to in situ coating of substrates in perfusion bioreactor vessels. [Background technology]

[0003] The bioprocessing industry performs large-scale cultivation of cells for the production of hormones, enzymes, antibodies, vaccines, and cell therapies. The cell and gene therapy market is growing rapidly, with promising therapies advancing rapidly toward clinical trials and commercialization. However, a single cell therapy dose can require billions of cells or trillions of viruses. Therefore, the ability to provide large quantities of cell product in a short period of time is critical for clinical success.

[0004] A significant portion of cells used in bioprocessing are anchorage-dependent, meaning they require a surface to adhere to in order to grow and function. Adherent cell culture dominates the production of viral vectors for gene and engineered cell therapy. This is because the cells used for viral vector production are primarily anchorage-dependent. Viral vectors are commonly used to deliver genetic material to cells and tissues, resulting in the correction of genetic defects, enhanced cell and tissue function, or improved production of cellular products, ultimately leading to potential curative therapies. Adherent cell culture also dominates the scale-up of stem cells for regenerative medicine. This is because stem cells, such as induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs), are also inherently anchorage-dependent. Stem cells hold great potential for cell therapy, tissue engineering, and regenerative medicine, as well as pharmaceutical and biotechnological applications. There is a strong need for a reliable and efficient platform for scaling up adherent cell culture.

[0005] Many materials are used for cell culture substrates in bioreactors. One of these is polyethylene terephthalate (PET), which is primarily used for viral vector and vaccine production, but its usefulness in other applications for adherent cell culture is limited. For example, many existing fixed-bed bioreactors have not been used for stem cell culture. Therefore, there is a strong need to develop fixed-bed cell culture substrates with functionalized or coated surfaces that can support the culture of a wider range of cell types.

[0006] Mammalian cells are used to produce therapeutic proteins, monoclonal antibodies, viral vectors, and even cultivated meat. Furthermore, in tissue engineering and regenerative medicine, billions of stem cells are used to create tissue-engineered constructs or to replace cells lost or damaged in degenerative diseases. While suspension cell cultures are widely used to produce proteins and antibodies, adherent cell cultures dominate the production of viral vectors for gene and engineered cell therapy and stem cells for regenerative medicine. Viral vectors are commonly used to deliver genetic material to cells and tissues, resulting in the correction of genetic defects, enhanced cell and tissue function, or improved production of cellular products, ultimately leading to potential curative therapies.

[0007] Stem cells hold great potential for cell therapy, tissue engineering, and regenerative medicine, as well as pharmaceutical and biotechnological applications. However, most cells used for viral vector production are anchorage-dependent, as are stem cells such as induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs). Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), are capable of indefinite self-renewal and can differentiate into derivatives of all three primary germ layers. These cells hold great potential for clinical applications, regenerative medicine, tissue engineering, drug screening, and early developmental biology research. Large numbers of hPSCs are required for cell therapy and regenerative medicine applications. This necessitates technologies that enable efficient scale-up and expansion of hPSCs.

[0008] Culture of hPSCs requires a controlled culture environment to ensure cell attachment, survival, proliferation, self-renewal, and maintenance of pluripotency and genomic stability. Human PSCs have been cultured on naturally derived extracellular matrices (e.g., Corning® Matrigel® Matrix, Life Technologies' Geltrex®), recombinant proteins (e.g., Vitronectin, Laminin-511, Laminin-521), and synthetic surfaces (Corning® Synthemax®).

[0009] A need exists for a cell culture bioreactor system and a method of using such a system that allows the end user to modify the bioreactor by coating the cell culture substrate in situ within the bioreactor to suit an intended application or cell type and to achieve optimal efficiency and performance for that application. Summary of the Invention

[0010] According to embodiments, a method for in situ coating of a cell culture substrate in a bioreactor is provided. The method includes providing a bioreactor vessel having a cell culture chamber therein. The cell culture chamber includes an inlet for allowing fluid to enter the cell culture chamber and an outlet for allowing fluid to exit the cell culture chamber. The bioreactor vessel also includes a cell substrate disposed within the cell culture chamber for culturing cells on the cell substrate. The method includes providing a coating solution for coating the cell substrate, introducing the coating solution into the cell culture chamber through the inlet such that the coating solution contacts and coats the cell substrate, and removing excess coating solution from the cell culture chamber via the outlet or inlet. After removing the coating solution, the coated cell substrate remains in the cell culture chamber.

[0011] According to embodiments, a method of culturing cells in a bioreactor is provided, the method comprising coating a cell substrate in a bioreactor as described herein, seeding cells onto the coated cell substrate, culturing the cells on the coated cell substrate, and harvesting a product of the culturing of the cells.

[0012] According to an embodiment, a system for culturing adherent cells in a bioreactor is provided. The system includes a bioreactor vessel having a cell culture chamber therein. The cell culture chamber includes an inlet for introducing fluid into the cell culture chamber and an outlet for introducing fluid out of the cell culture chamber. A cell culture substrate also includes a cell substrate disposed within the cell culture chamber for culturing cells on the cell substrate. A recirculation loop is also provided that can supply fluid to the bioreactor vessel via the inlet and remove fluid from the bioreactor vessel via the outlet. The system further includes a coating solution container fluidly connected to the cell culture chamber for holding a substrate coating solution. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram of a fixed-bed bioreactor system for coating a cell substrate within a bioreactor, according to an embodiment. [Figure 2] FIG. 2 is a schematic diagram of the bioreactor system of FIG. 1 during a step of coating a cell substrate, according to an embodiment. [Figure 3] FIG. 3 is a schematic diagram of the bioreactor system of FIGS. 1 and 2 after coating a cell substrate, according to an embodiment. [Figure 4] FIG. 1 is a schematic diagram of a cell culture system, according to one or more embodiments. [Figure 5] 1 shows a flowchart of a process for coating a cell substrate in a bioreactor for culturing cells, according to one or more embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0014] Various embodiments of the present disclosure will now be described in detail with reference to the drawings, where available. References to various embodiments do not limit the scope of the invention, which is limited only by the claims appended hereto. Additionally, any examples described herein are not limiting and merely describe some of the many possible embodiments of the claimed invention.

[0015] The surface chemistry of the adherent cell culture substrate may need to be modified to provide the desired cell adhesion properties. Such modifications can be achieved through chemical treatment of the substrate's polymeric material or by grafting cell adhesion molecules onto the substrate surface. Alternatively, the substrate may be coated with a thin layer of a biocompatible hydrogel that exhibits cell adhesion properties, including, for example, collagen or Matrigel®. A variety of coatings may be used, including, for example, extracellular matrix proteins, fibronectin, collagen, hydrogel solutions, polymer solutions, and recombinant proteins. As will be appreciated by those skilled in the art, any suitable coating may be used. Alternatively, the surface of the cell substrate may be rendered cell adhesive through treatment processes using various types of plasma, process gases, and / or chemicals known in the art. However, in one or more embodiments, the cell substrate may provide an efficient cell growth surface without surface treatment. For simplicity and flexibility in bioreactor manufacturing, it may be advantageous to provide an uncoated cell culture bioreactor, allowing the end user to determine the type of coating to apply to the cell substrate in the bioreactor depending on their cell type or intended application. Accordingly, the embodiments provided herein advantageously allow a user to coat a substrate in situ inside a bioreactor. Thus, by providing a pre-packaged bioreactor with a cell substrate and allowing the cell substrate to be coated in place, a flexible and easy-to-use solution is provided while minimizing the risk of contamination or assembly errors that may occur if the end user must remove and repackage the substrate into the bioreactor after coating.

[0016] Embodiments of the present disclosure relate to systems and methods for in situ coating of cell substrates in fixed-bed bioreactors, as well as systems and methods for culturing cells in such bioreactors. These fixed-bed bioreactors can be used to seed, culture, and / or expand various types of cells, including mesenchymal stem cells, cancer cells, T cells, fibroblasts, and myoblasts, among others. Prior to culturing adherent cells on the cell substrate, it may be desirable to coat the cell substrate with a coating to improve performance for a particular cell type or application. For example, an adhesion-promoting compound may be applied to the cell growth surface to promote adhesion and subsequent contact, such as expansion, of cells, such as, for example, human mesenchymal stem cells (hMSCs).

[0017] Embodiments of the present disclosure include fixed-bed bioreactor systems for cell culture. According to aspects of such embodiments, the bioreactor system may be a closed system in which the contents of the bioreactor system are not directly exposed to the atmosphere to prevent contamination. The bioreactor system may be automated. In aspects of the embodiments, the system may include cell culture medium and / or a coating solution for coating a cell substrate within the bioreactor. The bioreactor system may include a fluid flow path including a path from an inlet of the bioreactor vessel, through a cell culture chamber containing the substrate, and out of the bioreactor via an outlet. The fluid flow path may also include one or more medium conditioning vessels fluidly connected to the cell culture chamber and which may be integrated with the bioreactor vessel or separate from the bioreactor vessel. The system may further include a coating solution source fluidly connected to the fluid flow path and configured to inject a coating solution into the fluid flow path such that the coating solution extends into the cell culture space. The system may include one or more pumps for circulating culture medium through the fluid flow path and / or for injecting the coating solution into the cell culture space.

[0018] In embodiments, the system includes a controller for controlling operation of the system, including one or more pumps. The controller may include a computer system including a processor. The controller, in embodiments, is configured to control one or more pumps to circulate a fluid (e.g., cell culture medium or coating solution) at a flow rate through the fluid flow path. The controller, according to aspects of the embodiment, controls the one or more pumps to transfer cell culture medium, nutrients, and / or cells from a source to the fluid flow path and to the bioreactor. The system may include a graphical user interface and a memory, the memory being in communication with and readable by the processor and including instructions. When the instructions are executed by the processor, the processor may receive instructions, for example, to coat a cell substrate in a bioreactor. In response to the instructions to coat the bioreactor, the processor may perform a series of steps to coat the bioreactor and then receive instructions, for example, to introduce cells into the bioreactor. In response to the instructions to introduce cells, the processor may perform a series of steps to introduce cells into the bioreactor from a cell inlet source.

[0019] FIG. 1 illustrates a cell culture system 100 according to an embodiment of the present disclosure. The cell culture system 100 includes a cell culture vessel 102 having an internal reservoir containing a cell culture space 104 in which adherent cells can be seeded, cultured, transfected, differentiated, and / or harvested. A fixed-bed cell substrate 106 is disposed within the cell culture space 104. The fixed-bed 106 is made from a cell substrate 108, as described herein. According to an aspect of the embodiment, the cell substrate 108 is a porous material having a predetermined structure, such as openings 109 or an ordered array of pores and rigid filaments. The structure and arrangement of the cell substrate 108 and the fixed bed 106 are such that the fixed bed 106 allows for uniform fluid flow therethrough, which improves cell seeding, nutrient distribution, substrate coating, and cell recovery. The cell culture system 100 may include an inlet distribution plate 114 and an outlet distribution plate 116 to help evenly distribute medium, cells, coating solutions, and other fluids throughout the fixed bed. The cell culture system 100 also includes an inlet 110 and an outlet 112 for allowing fluid to enter and exit the cell culture space 104, respectively. The inlet 110 and outlet 112 are connected to fluid channels 118a, 118b for providing a path for fluid flow into and out of the cell culture vessel 102. The fluid channels 118a, 118b may form a perfusion channel for recirculating fluid in a loop through the bioreactor, as discussed further below.

[0020] According to embodiments, the cell culture system 100 includes a coating solution container 120 that is fluidly connected to the cell culture vessel 102 and capable of supplying a coating solution 122 contained therein to the cell culture space 104. The coating solution container 120 may be connected to the fluid flow path 118a or 118b via a coating passage 124, or may be directly connected to the cell culture vessel 102 via a separate inlet outside the fluid flow paths 118a, 118b. As shown in FIG. 2 , the cell culture system 100 is capable of supplying the coating solution 112 from the coating solution container 120 to the cell culture space 104 containing the cell substrate 108. After the coating solution 122 fills the cell culture space 104 and contacts the cell substrate 108, the coating solution 122 may remain in contact with the cell substrate 108 for a predetermined time or until application of the coating to the cell substrate 108 is completed (e.g., by some physical or chemical reaction; by some external stimulus such as heating, cooling, or radiation). After coating is deemed complete, coating solution 122 can be removed from cell culture space 104, as shown in Figure 3, leaving a coated cell substrate created from coating 123 on cell substrate 108. Removal of coating solution 122 from cell culture space can be accompanied by washing with a washing solution (not shown) either while excess coating solution is being removed or at a time after removal is complete. The result is a bioreactor with a cell substrate suitable for the desired cell culture application.

[0021] FIG. 4 illustrates a cell culture system 400 according to one or more embodiments. The system 400 includes a bioreactor 402 containing a fixed-bed cell substrate according to one or more embodiments disclosed herein. The bioreactor 402 can be fluidly connected to a medium conditioning vessel 404, and the system can supply the cell culture medium 406 in the conditioning vessel 404 to the bioreactor 402. The medium conditioning vessel 404 can include sensor and control components found in typical bioreactors used in the bioprocessing industry for suspension batch, fed-batch, or perfusion culture. These include, but are not limited to, a DO oxygen sensor, a pH sensor, an oxygen generator / gas sparging unit, a temperature probe, and nutrient and base addition ports. The gas mixture supplied to the sparging unit can be controlled by gas flow controllers for N, O, and CO gases. The medium conditioning vessel 404 also houses an impeller for medium mixing. All of the medium parameters measured by the sensors listed above can be controlled by a medium conditioning control unit 418, which communicates with the medium conditioning vessel 404 and can measure and / or adjust the conditions of the cell culture medium 406 to desired levels. As shown in FIG. 4, the medium conditioning vessel 404 is provided as a vessel separate from the bioreactor vessel 402. This can have advantages in that the medium can be conditioned separately from where the cells are cultured and the conditioned medium can then be supplied to the cell culture space. However, in some embodiments, the medium conditioning can be performed within the bioreactor vessel 402.

[0022] Media from the media 406 conditioning vessel 404 is delivered to the bioreactor 402 via an inlet 408, which may also include an injection port for a cell inoculum to initiate cell seeding and cultivation. The bioreactor vessel 402 may also include one or more outlets 410 through which the cell culture media 406 exits the vessel 402. Additionally, cells or cell products may be discharged through the outlets 410. One or more sensors 412 may be provided in the line to analyze the contents of the outflow from the bioreactor 402. In some embodiments, the system 400 includes a flow control unit 414 for controlling the flow to the bioreactor 402. For example, the flow control unit 414 may receive a signal from one or more sensors 412 (e.g., an O sensor) and, based on the signal, adjust the flow rate to the bioreactor 402 by sending a signal to a pump 416 (e.g., a peristaltic pump) upstream of the inlet 408 to the bioreactor 402. Therefore, based on one or a combination of the factors measured by the sensor 412, the pump 416 can control the flow to the bioreactor 402 to achieve the desired cell culture conditions.

[0023] The medium perfusion rate is controlled by a signal processing unit 414, which collects and compares sensor signals from sensors located in the medium conditioning vessel 404 and the fixed-bed bioreactor outlet 410. Due to the packed-flow nature of the medium perfusion through the fixed-bed bioreactor 402, nutrient, pH, and oxygen gradients develop along the fixed bed. The bioreactor perfusion flow rate can be automatically controlled by a flow control unit 414 operably connected to a peristaltic pump 416. Examples of additional control and system components can be found in U.S. Patent Application Publication No. 2020 / 0248124 A1, which is incorporated herein by reference.

[0024] The cell culture system 400 also includes a coating solution container 420 fluidly connected to the bioreactor vessel 402 and capable of supplying a coating solution contained therein to the bioreactor vessel 402. The coating solution is selected to improve performance for a given cell culture application. A pump 422 can supply the coating solution directly to the bioreactor vessel 402 or to a fluid flow path connected to the bioreactor vessel 402. The controller 414 can send a signal to activate the pump 422 when it is time to supply the coating solution to the cell substrate within the bioreactor. After the coating solution fills the bioreactor system 402 and contacts the cell substrate, the coating solution can remain in contact with the cell substrate for a predetermined time or until application of the coating to the cell substrate is complete (e.g., by some physical or chemical reaction; by some external stimulus such as heating, cooling, or radiation). After the coating is deemed complete, the coating solution can be removed from the cell culture space. For example, the controller 414 can send a signal to a pump to provide a washing solution to the reactor 402 to remove excess coating solution and / or to assist in removing excess coating solution. The washing solution can contain a medium or fluid, such as phosphate buffered saline (PBS) or other cell culture medium.

[0025] Traditional large-scale cell culture bioreactors have used different types of packed-bed bioreactors. These packed beds typically contain a porous matrix to retain adherent or suspended cells and support their growth and proliferation. Because the packed bed matrix provides a high surface area-to-volume ratio, cell densities can be higher than in other systems. However, packed beds often function as depth filters, where cells become physically trapped or entangled in the fibers of the substrate. Therefore, due to the linear flow of cell inoculum through the packed bed, cells are subject to uneven distribution inside the packed bed, leading to variations in cell density throughout the depth or width of the packed bed. For example, cell density may be higher in the inlet region of the bioreactor and significantly lower closer to the outlet of the bioreactor. This uneven distribution of cells inside the packed bed can significantly hinder the scalability and predictability of such bioreactors in bioprocess manufacturing and can lead to reduced efficiency in terms of cell growth or viral vector production per unit surface area or volume of the packed bed.

[0026] Another problem encountered in packed-bed bioreactors disclosed in the prior art is the channeling effect. Due to the random nature of the packed nonwoven fibers, the local fiber density at any given cross-section of the packed bed is not uniform. Regions of low fiber density (high bed permeability) experience moderately fast flow, while regions of high fiber density (low bed permeability) experience much slower flow. The resulting uneven medium perfusion across the packed bed creates a channeling effect, which manifests as gradients of important nutrients and metabolites that adversely affect overall cell culture and bioreactor performance. Cells located in regions of low medium perfusion become starved and very frequently die from nutrient deprivation or metabolite toxicity. Cell recovery is yet another problem encountered when bioreactors packed with nonwoven fibrous scaffolds are used. Because the packed bed acts as a depth filter, cells released at the end of the cell culture process are trapped inside the packed bed, resulting in very low cell recovery rates. This significantly limits the use of such bioreactors in bioprocesses where viable cells are the product. Therefore, non-uniformity leads to regions with different exposure to flow and shear, effectively reducing the available cell culture area, causing non-uniform cultures, and hindering transfection efficiency and cell release.

[0027] To address these and other problems with existing cell culture solutions, embodiments of the present disclosure provide cell growth substrates, matrices of such substrates, and / or packed-bed systems using such substrates that enable efficient and high-yield cell culture and cell product (e.g., proteins, antibodies, viral particles) production for anchorage-dependent cells. Embodiments include porous cell culture substrates made from an ordered and regular array of porous substrate materials that enable uniform cell seeding and medium / nutrient perfusion, as well as efficient cell harvest. Embodiments also enable scalable cell culture solutions with substrates and bioreactors that can seed and grow cells and / or harvest cell products from process development scale to full production size scale without sacrificing uniform performance of the embodiments. For example, in some embodiments, bioreactors can be configured to scale the surface area (VG / cm) of the substrate across production scales. 2 ) can be easily scaled from process development scale to production scale with equivalent viral genomes per batch. The recoverability and scalability of embodiments herein enable their use in efficient seed trains to grow cell populations at multiple scales on the same cell substrate. Additionally, embodiments herein provide cell culture substrates with high surface areas that, in combination with other features described, enable high-yield cell culture solutions. In some embodiments, for example, the cell culture substrates and / or bioreactors discussed herein can grow up to 10 per batch. 16 ~10 pieces 18 It is possible to produce 100 viral genomes (VG).

[0028] In one embodiment, the fixed-bed substrate provides a structurally defined surface area for adherent cells to attach and grow, forming a highly uniform, multiply interconnected fluidic network with good mechanical strength when assembled within a fixed-bed or other bioreactor. In certain embodiments, a mechanically stable, non-degradable woven mesh can be used as a substrate to support adherent cell production. The cell substrates disclosed herein support the attachment and growth of anchorage-dependent cells in a high-volume density format. Uniform cell seeding of such substrates and efficient recovery of cells or other bioreactor products are achievable. Additionally, embodiments of the present disclosure can provide uniform cell distribution during the inoculation step, support cell culture to achieve a confluent monolayer or multilayer of adherent cells on the disclosed substrate, and avoid the formation of large and / or uncontrollable 3D cellular aggregates with limited nutrient diffusion and increased metabolite concentrations. Therefore, the substrate eliminates diffusion limitations during bioreactor operation. Additionally, the substrate allows for easy and efficient cell recovery from the bioreactor. The structurally defined substrate of one or more embodiments allows for complete and consistent cell recovery from the fixed bed of the bioreactor.

[0029] Also provided, according to some embodiments, is a method of cell culture using a bioreactor having a substrate for bioprocessing the production of a therapeutic protein, antibody, viral vaccine, or viral vector.

[0030] In contrast to existing cell culture substrates (i.e., nonwoven substrates of randomly ordered fibers) used in cell culture bioreactors, embodiments of the present disclosure include cell culture substrates with a defined and ordered structure. The defined and ordered structure enables consistent and predictable cell culture results. Additionally, the substrate has an open porous structure that prevents cell entrapment and allows uniform flow through the fixed bed. This structure enables improved cell seeding, nutrient delivery, cell growth, and cell recovery. According to one or more specific embodiments, the substrate is formed of a substrate material having a thin, sheet-like structure with first and second sides 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. Additionally, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings is of a size and geometry that allows cells to adhere to the surface of the substrate material as if it were an approximately two-dimensional (2D) surface, while also allowing adequate fluid flow around the substrate material and through the openings. In some embodiments, the substrate is a polymer-based material and can be formed as a molded polymer sheet; a polymer sheet with openings through its thickness; multiple filaments fused into a mesh-like layer; a 3D printed substrate; or multiple filaments woven into a mesh layer. The physical structure of the substrate provides a high surface-to-volume ratio for culturing anchorage-dependent cells. According to various embodiments, the substrate can be positioned or packed into a bioreactor in certain ways as discussed herein for uniform cell seeding and growth, uniform medium perfusion, and efficient cell recovery.

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

[0032] Additionally, embodiments disclosed herein enable not only cell attachment and growth on cell culture substrates, but also viable recovery of cultured cells. The inability to recover viable cells is a significant drawback of current platforms, leading to difficulties in establishing and maintaining sufficient numbers of cells for production capacity. According to one aspect of embodiments of the present disclosure, viable cells can be recovered from cell culture substrates, including 80% to 100% viable, or about 85% to about 99% viable, or about 90% to about 99% viable. For example, the recovered cells may be at least 80% viable, at least 85% viable, at least 90% viable, at least 91% viable, at least 92% viable, at least 93% viable, at least 94% viable, at least 95% viable, at least 96% viable, at least 97% viable, at least 98% viable, or at least 99% viable. Cells can be released from the cell culture substrate using, for example, trypsin, trypsin, or accutase.

[0033] According to embodiments, the cell culture substrate can be a woven mesh layer made from a first plurality of fibers extending in a first direction and a second plurality of fibers extending in a second direction. The woven fibers of the substrate form a plurality of openings, which can be defined by one or more widths or diameters. The size and shape of the openings can vary based on the type of weave (e.g., the number, shape, and size of the filaments; the angle between intersecting filaments; etc.). On a macroscale, the woven mesh can be characterized as a two-dimensional sheet or layer. However, close inspection of the woven mesh reveals a three-dimensional structure due to the ascending and descending intersecting fibers of the mesh. Without wishing to be bound by theory, it is believed that the three-dimensional structure of the substrate is advantageous for providing a large surface area for culturing adherent cells, and that the structural rigidity of the mesh can provide a consistent, predictable cell culture substrate structure that allows uniform fluid flow.

[0034] In one or more embodiments, the fibers can have diameters ranging from about 10 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, about 10 μm to about 300 μm, about 20 μm to about 250 μm, or about 20 μm to about 170 μm, or about 150 μm to about 300 μm. At the microscale level, due to the scale of the fibers relative to cells (e.g., the fiber diameter is larger than that of cells), the surface of the monofilament fiber presents an approximation of a 2D surface for adherent cells to attach and grow on. The fibers can be woven into a mesh with openings ranging from about 10 μm x 10 μm to about 1000 μm x 1000 μm. In some embodiments, the openings can have diameters of about 50 μm to about 1000 μm, about 100 μm to about 750 μm, about 125 μm to about 600 μm, about 150 μm to about 500 μm, about 200 μm to about 400 μm, about 10 μm to about 200 μm, about 20 μm to about 150 μm, or about 200 μm to about 300 μm. These ranges of filament diameter and opening diameter are exemplary of some embodiments but are not intended to limit the possible feature sizes of meshes according to all embodiments. The combination of fiber diameter and opening diameter is selected to provide efficient and uniform fluid flow through the substrate, for example, when the cell culture substrate includes several adjacent mesh layers (e.g., a stack of individual layers or a rolled mesh layer).

[0035] Factors such as fiber diameter, opening diameter, and weave type / pattern determine the surface area available for cell attachment and growth. Additionally, when a cell culture substrate includes stacks, rolls, or other arrangements of overlapping substrates, the packing density of the cell culture substrate affects the surface area of ​​the fixed-bed substrate. Packing density can vary depending on the packing thickness of the substrate material (e.g., the space required for a layer of 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 determined by dividing the total height of the stack by the number of layers in the stack. Packing thickness varies based on fiber diameter and weave, but can also vary based on the alignment of adjacent layers within the stack. For example, due to the three-dimensional nature of woven layers, there is a degree of interlocking or overlap that allows adjacent layers to accommodate each other based on their alignment. In one alignment, adjacent layers can fit closely together, while in a second alignment, adjacent layers may have zero overlap, such as when the bottommost point of the top layer is in direct contact with the topmost point of the bottom layer. In certain applications, it may be desirable to provide a cell culture substrate with a low density packing of layers (e.g., when higher permeability is a priority) or a higher packing density (e.g., when maximizing substrate surface area is a priority). According to one or more embodiments, the packing thickness can be about 10 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, about 10 μm to about 300 μm, or about 20 μm to about 250 μm.

[0036] The above structural factors can determine the surface area of ​​the cell culture substrate, whether it is a single layer of cell culture substrate or a cell culture substrate having multiple layers of substrate. For example, in certain embodiments, a single layer of woven mesh substrate having a circular shape and diameter of 6 cm has a surface area of ​​approximately 68 cm. 2As used herein, "effective surface area" is the total surface area of ​​fibers in a portion of a substrate material available for cell attachment and growth. Unless otherwise specified, references to "surface area" refer to this effective surface area. According to one or more embodiments, a single woven mesh substrate layer with a 6 cm diameter has an effective surface area of ​​approximately 50 cm. 2 ~about 90cm 2 , approx. 53cm 2 ~Approx. 81cm 2 , about 68cm 2 , about 75cm 2 , or approximately 81 cm 2 These ranges of effective surface area are provided by way of example only, and some embodiments may have different effective surface areas. Cell culture substrates can also be characterized in terms of porosity, as discussed in the Examples herein.

[0037] The substrate mesh can be made from monofilament or multifilament fibers of polymeric materials compatible with cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. Mesh substrates can have different patterns or weaves, including, for example, knitted, warp knitted, or woven (e.g., plain weave, twill weave, Dutch weave, five-needle weave) patterns or weaves.

[0038] By using a structurally defined culture substrate with sufficient rigidity, uniformity of high flow resistance across the substrate or fixed bed is achieved. According to various embodiments, the substrate can be deployed in a monolayer or multilayer format. This flexibility eliminates diffusion limitations and provides uniform delivery of nutrients and oxygen to cells attached to the substrate. In addition, the open substrate lacks any cell confinement regions in fixed-bed configurations, allowing for complete cell recovery with high viability at the end of the culture. The substrate also delivers the packing uniformity associated with fixed beds, enabling direct scalability from process development units to large-scale industrial bioprocessing units. The ability to directly recover cells from fixed beds eliminates the need to resuspend the substrate in a stirred or mechanically shaken vessel, which adds complexity and can impose harmful shear stress on the cells. Furthermore, the high packing density of the cell culture substrate results in high bioprocess productivity in manageable volumes at an industrial scale.

[0039] Embodiments of the present disclosure include cell substrates that are multi-layer substrates. The multi-layer substrate includes a first mesh substrate layer and a second mesh substrate layer stacked on top of each other. The number of substrates in the stack can be adjusted to match the required density or number of cells (or cell products). However, embodiments are not limited to this configuration, and various configurations can be used for the cell substrate. For example, the cell substrate can be a roll of cell substrate material or a small piece of substrate material fixed to the reactor.

[0040] The geometry of the mesh substrate layers is designed to allow efficient and uniform flow through one or more substrate layers. Additionally, the structure of the cell substrate can accommodate fluid flow through the substrate in multiple orientations. For example, the direction of bulk fluid flow can be perpendicular to the major side surfaces of the first and second substrate layers, or the cell substrate can be oriented relative to the flow so that the sides of the substrate layers are parallel to the direction of bulk flow. In addition to fluid flow perpendicular or parallel to the first and second sides of the mesh layers, the substrate can be arranged with multiple substrate pieces at intermediate angles or in a random arrangement relative to the fluid flow. This flexibility in orientation is made possible by the essentially isotropic flow behavior of woven substrates. In contrast, substrates for adherent cells in existing bioreactors do not exhibit this behavior; instead, fixed beds of substrates create preferential flow channels and tend to have substrate materials with anisotropic permeability. The flexibility of the cell substrates of the present disclosure allows for use in a variety of applications and bioreactor or vessel designs, while enabling better and more uniform permeability throughout the bioreactor vessel.

[0041] As discussed herein, the cell substrate can be used within a bioreactor vessel according to one or more embodiments. For example, the substrate can be used in a fixed-bed bioreactor configuration or other configuration 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 what may be considered a two-dimensional culture surface configuration, such as within a flat-bottom culture dish, in which one or more layers of the substrate are laid flat to provide a culture substrate for the cells. Due to contamination concerns, the vessel can be a disposable vessel that can be discarded after use.

[0042] According to one or more embodiments, a cell culture system is provided in which a cell culture substrate is used within a culture chamber of a bioreactor vessel. Within the cell culture chamber is a fixed-bed cell substrate made from a stack of cell substrate layers. The cell substrate layers are stacked so that a first or second side of one substrate layer faces a first or second side of an adjacent substrate layer. The bioreactor vessel has an inlet at one end for the input of medium, cells, and / or nutrients into the culture chamber and an outlet at the opposite end for the removal of medium, cells, or cell products from the culture chamber. By allowing the substrate layers to be stacked in this manner, the system can be easily scaled up without adversely affecting cell attachment and growth due to the defined structure and efficient fluid flow through the stacked substrates. While the vessel may generally be described as having an inlet and an outlet, some embodiments may use one or both of the inlet and outlet to flow medium, cells, or other contents both into and out of the culture chamber. For example, an inlet may be used to allow medium or cells to enter a culture chamber during a cell seeding, perfusion, or culturing step, but may also be used to remove one or more of medium, cells, or cell products through the inlet during a harvesting step. Thus, the terms "inlet" and "outlet" are not intended to limit the function of these openings.

[0043] In one or more embodiments, the flow resistance and volumetric density of a fixed bed can be controlled by interleaving substrate layers of different geometries. In particular, mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the fluid flow resistance in a fixed-bed format. By interleaving meshes of different sizes and geometries, flow resistance can be controlled or varied in one or more specific portions of a bioreactor. This allows for better uniformity of liquid perfusion in the fixed bed. Various combinations of meshes of different sizes allow for different profiles of cell growth surface volumetric density and flow resistance. For example, a fixed bed with zones of varying volumetric cell densities (e.g., a series of zones creating a density pattern such as low / high / low / high) can be assembled by interleaving meshes of different sizes.

[0044] According to embodiments, the direction of bulk fluid flow through the bioreactor is from the inlet to the outlet, and in aspects of the embodiments, the first and second major sides of the cell substrate layer are perpendicular to the direction of bulk flow. In contrast, aspects of some embodiments include a bioreactor vessel and a stack of cell substrates within a culture space having first and second sides that are parallel to the direction of bulk flow. Thus, the cell substrates of the presently disclosed embodiments can be used in either configuration. In any of these examples, the cell substrates are sized and shaped to fill the interior space defined by the culture chamber, such that the culture space within each vessel is filled to a cell growth surface area to maximize efficiency in terms of cells per unit volume. The cell culture space of the system may be fed by a single inlet and have a single outlet, or may have multiple inlets and / or multiple outlets. However, according to various embodiments herein, a distribution plate may be used to distribute medium, cells, or nutrients across the cross-section of the fixed bed, thereby helping to improve the uniformity of fluid flow through the fixed bed. Thus, multiple inlets represent a way in which a distribution plate can provide multiple holes across the fixed bed cross section to create a more uniform flow.

[0045] In some embodiments, the fixed bed is arranged so that the cell substrate is formed into a cylindrical roll. For example, a sheet of cell substrate material (e.g., one or more mesh substrates) is rolled into a cylinder about the central longitudinal axis of the cell culture space. The cylindrical roll has a width along a dimension perpendicular to the central longitudinal axis and a height along a direction parallel to the central longitudinal axis. In one or more embodiments, the cylindrical roll is designed to fit within the bioreactor vessel so that the central longitudinal axis is parallel to the direction of bulk fluid flow through the bioreactor or culture chamber containing the cylindrical roll. The bioreactor system may further include a central support member around which the cell substrate is positioned. According to some embodiments, the central support member can serve purely for physical support and / or alignment of the cell substrate, but can also serve other functions. For example, the central support member can include one or more openings along the length of the fixed bed for supplying medium to the cell substrate. In other embodiments, the central support member can include one or more attachment sites for holding one or more portions of the cell culture substrate to an interior portion of the cylindrical roll. These attachment sites may be hooks, fasteners, posts, clamps, or other means of attaching the mesh sheet to the central support member.

[0046] One or more embodiments of the present disclosure provide a cell inoculation step that differs from conventional methods. In conventional methods, a packed bed with a conventional substrate is filled with culture medium, and the concentrated inoculum is injected into a medium circulation loop. The cell suspension is pumped through the bioreactor at an increased flow rate to reduce cell seeding non-uniformity through entrapment on the conventional fixed-bed substrate. In such conventional methods, pumping of cells at a high flow rate in the circulation loop continues, possibly for several hours, until the majority of the cells are entrapped within the packed-bed bioreactor. However, due to the non-uniform deep-bed filtration nature of conventional packed-bed bioreactors, the cells are unevenly distributed inside the packed bed, with higher cell densities in the inlet region of the bioreactor and lower cell densities in the outlet region of the bioreactor.

[0047] In contrast, according to an embodiment of the present disclosure, a volume of cell inoculum equal to the void volume of the culture chamber in the bioreactor is injected directly into the packed bed through a cell inoculum injection port at the inlet 408 of the bioreactor 402 ( FIG. 4 ). The cell suspension is then uniformly distributed inside the packed bed due to the uniform and continuous fluid pathways present in the cell culture substrate described herein. To prevent cell settling due to gravity during the initial seeding stage, medium perfusion can be initiated immediately after inoculum injection. The perfusion flow rate is maintained below a preprogrammed threshold to balance gravity and avoid washing cells out of the packed-bed bioreactor. Therefore, during the initial cell attachment stage, cells gently move around inside the packed bed, achieving uniform cell distribution and attachment on the available substrate surface.

[0048] Embodiments include a method for coating a cell substrate in situ within a bioreactor vessel. For example, system 400 of FIG. 4 can operate according to process steps according to one or more embodiments. As shown in FIG. 5, this method 500 can include providing a cell culture bioreactor (S502), providing a coating solution (S504), and flowing the coating solution into a cell culture space of the bioreactor (S506). Insertion of the coating solution into the cell culture space S506 can be followed by an incubation step S507, during which the coating solution remains in the cell culture space and coats the substrate. This incubation step S507 can include applying some additional stimulus, such as heating, cooling, or application of some type of radiation, to assist the coating solution in forming a coating on the cell substrate. During incubation step S507, the coating solution can remain stagnant within the cell culture space or can be perfused through the cell culture space, either in a loop or as a one-time perfusion through the reactor. The incubation period S507 can also include a reaction occurring to form a coating on the cell substrate from the coating solution. Method 500 can further include removing the coating solution from the bioreactor (S508) after the coating has formed on the cell substrate. Removing the coating solution S508 can occur after a predetermined period deemed sufficient for the coating given the process parameters. Optionally, a washing step S509 can be performed during or after removing step S508. For example, the coating solution can be forced out of the bioreactor by injection of a washing solution that flushes the bioreactor, or a washing solution can be introduced into the bioreactor after excess coating solution has been removed. Next, the cell culture process can include cell seeding and attachment (S510) and cell culture medium, followed by a culture process that can include cell expansion, transduction, cell differentiation, production of viral vectors or other cell products, and harvesting.

[0049] The cell culture substrate can be arranged in a number of configurations within the culture chamber, depending on the desired system. For example, in one or more embodiments, the system includes one or more layers of substrate having a width that spans the width of the defined cell culture space within the culture chamber. Multiple layers of substrate can be stacked in this manner to a predetermined height. As discussed above, the substrate layers can be arranged such that a first side and a second side of one or more layers are perpendicular to the direction of bulk flow of culture medium through the defined culture space within the culture chamber, or the first side and a second side of one or more layers can be parallel to the direction of bulk flow. In one or more embodiments, the cell culture substrate includes one or more substrate layers in a first orientation relative to the bulk flow and one or more other layers in a second orientation that is different from the first orientation. For example, the various layers can have first and second sides that are parallel or perpendicular to the direction of bulk flow, or at an angle therebetween.

[0050] In one or more embodiments, the cell culture system includes multiple separate pieces of cell culture substrate in a packed bed configuration, where the length and / or width of the substrate pieces are small relative to the culture chamber. As used herein, a substrate piece is considered to have a length and / or width that is small relative to the culture chamber when the length and / or width of the substrate piece is about 50% or less of the length and / or width of the culture space. Thus, the cell culture system can include multiple substrate pieces packed into the culture space in a desired arrangement. The arrangement of the substrate pieces can be random or semi-random, or can have a predetermined order or alignment, such as pieces oriented in a substantially similar orientation (e.g., horizontal, vertical, or at an angle of 0° to 90° relative to the direction of bulk flow).

[0051] As used herein, "defined culture space" refers to the space within a culture chamber occupied by a cell culture substrate and in which cell seeding and / or culturing occurs. The defined culture space can fill substantially the entire culture chamber or can occupy a portion of the space within the culture chamber. As used herein, "bulk flow direction" is defined as the direction of bulk mass flow of fluid or culture medium through or across the cell culture substrate during culturing of cells and / or during flow of culture medium into or out of the culture chamber.

[0052] In one or more embodiments, the cell culture substrate is secured within the culture chamber by a securing mechanism. The securing mechanism may secure a portion of the cell culture substrate to a wall of the culture chamber surrounding the substrate or to a wall of the chamber at one end of the culture chamber. In some embodiments, the securing mechanism attaches a portion of the cell culture substrate to a member extending through the culture chamber, such as a member extending parallel to the longitudinal axis of the culture chamber or a member extending perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture substrate may be contained within the culture chamber without being fixedly attached to the walls of the chamber or bioreactor vessel. For example, the substrate may be contained by the boundaries of the culture chamber or other structural members within the chamber such that the substrate is held within a predetermined area of ​​the bioreactor vessel without being fixedly attached to those boundaries or structural members.

[0053] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. The culture chamber can accommodate a cell culture substrate and a substrate according to one or more of the embodiments described herein. In the roller bottle configuration, the bioreactor vessel can be operably attached to a means for moving the bioreactor vessel about a central longitudinal axis of the vessel. For example, the bioreactor vessel can be rotated about the central longitudinal axis. 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 back and forth in a rotational direction). During operation, the rotation of the bioreactor vessel causes movement of cells and / or fluid within the chamber. This movement can be considered relative to the walls of the chamber. For example, as the bioreactor vessel rotates about its central longitudinal axis, gravity can cause fluid, culture medium, and / or unadhered cells to leave toward the lower portion of the chamber. However, in one or more embodiments, the cell culture substrate is essentially fixed relative to the vessel and therefore rotates with the vessel. In one or more other embodiments, the cell culture substrate is unattached and can move freely relative to the vessel as desired as the vessel rotates. Cells may adhere to the cell culture substrate, while the movement of the vessel allows the cells to receive both cell culture medium or liquid, as well as exposure to oxygen or other gases within the culture chamber.

[0054] By using cell culture substrates according to embodiments of the present disclosure, such as substrates comprising woven or mesh substrates, roller bottle containers are provided with increased surface area available for adherent cells to attach, grow, and function. In particular, using a woven mesh substrate of monofilament polymer material within a roller bottle, the surface area can be increased by approximately 2.4 to approximately 4.8 times, or approximately 10 times, compared to a standard roller bottle. As discussed herein, each monofilament strand of the mesh substrate can present itself as a 2D surface for adherent cells to attach. In addition, multiple layers of mesh can be placed within the roller bottle, resulting in an increase in the total available surface area in the range of approximately 2 to 20 times that of a standard roller bottle. Therefore, existing roller bottle facilities and processes, including cell seeding, medium exchange, and cell harvesting, can be modified with the addition of the improved cell culture substrates disclosed herein with minimal impact on existing operational infrastructure and processing steps.

[0055] 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, medium, or cells can be supplied to or removed from the chamber. A single port in the vessel can function as both an inlet and an outlet, or multiple ports can be provided for dedicated inlets and outlets.

[0056] The packed-bed cell culture substrate of one or more embodiments can consist of a woven cell culture mesh substrate without any other form of cell culture substrate disposed within or interspersed with the cell culture substrate. That is, the woven cell culture mesh substrate of the disclosed embodiments is an effective cell culture substrate that does not require the type of irregular, non-woven substrate used in existing solutions. This allows for simplified design and construction of cell culture systems while providing a high-density cell culture substrate with other advantages discussed herein related to flow uniformity, recovery, etc.

[0057] As discussed herein, the provided cell culture substrates and bioreactor systems offer many advantages. For example, embodiments of the present disclosure can support the production of any of several viral vectors, such as AAV (all serotypes) and lentivirus, and can be applied for in vivo and ex vivo gene therapy applications. Uniform cell seeding and distribution maximizes viral vector yield per vessel, and the design allows for the recovery of viable cells, which may be useful for seed trains consisting of multiple expansion periods using the same platform. In addition, embodiments herein are scalable from process development scale to production scale, ultimately saving development time and costs. The methods and systems disclosed herein also enable automation and control of the cell culture process to maximize vector yield and improve reproducibility. Finally, viral vector production level scale (e.g., 10 per batch) can be scaled up to 100 cells per batch. 16 ~10 pieces 18 The number of vessels required to reach 100 AAV volumes (µg) can be significantly reduced compared to other cell culture solutions.

[0058] Embodiments are not limited to container rotation about a central longitudinal axis. For example, the container may rotate about an axis that is not centrally located relative to the container. In addition, the axis of rotation may be a horizontal axis or a vertical axis.

[0059] Example Implementation Below are descriptions of various aspects of implementations of the disclosed subject matter. Each aspect may include one or more of various features, characteristics, or advantages of the disclosed subject matter. The implementations are intended to illustrate some aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible implementations.

[0060] Aspect 1 is directed to a method of coating a cell culture substrate in situ in a bioreactor, the method including: providing a bioreactor vessel comprising: a cell culture chamber within the bioreactor vessel, the cell culture chamber including an inlet for allowing fluid to enter the cell culture chamber and an outlet for allowing fluid to exit the cell culture chamber; and a cell substrate disposed within the cell culture chamber and configured to culture cells thereon; providing a coating solution for coating the cell substrate; introducing the coating solution into the cell culture chamber through the inlet such that the coating solution contacts and coats the cell substrate; and removing excess coating solution from the cell culture chamber via the outlet or inlet, wherein the coated cell substrate remains in the cell culture chamber after removing the coating solution.

[0061] Aspect 2 relates to the method of aspect 1, further comprising incubating the coating solution in the cell culture chamber before removing the coating solution.

[0062] Aspect 3 relates to the method of aspect 1 or 2, further comprising washing the cell culture chamber with a washing solution during or after removal of the coating solution.

[0063] Aspect 4 relates to a method according to aspects 1 to 3, wherein providing a coating solution comprises preparing a coating solution suitable for a particular cell culture application.

[0064] Aspect 5 relates to a method according to aspects 1-4, wherein the coating solution comprises a material for enhancing cell attachment and / or growth on the cell substrate.

[0065] Aspect 6 relates to the method of any one of aspects 1 to 6, wherein the coating solution comprises at least one of an extracellular matrix protein, fibronectin, collagen, a hydrogel solution, a polymer solution, and a recombinant protein.

[0066] Example 7 relates to a method according to Examples 1-6, wherein introducing the coating solution comprises perfusing the coating solution through the bioreactor such that the coating solution enters the cell culture chamber via an inlet, flows through the cell substrate, and exits via an outlet.

[0067] Aspect 8 relates to a method according to aspects 1-7, further comprising controlling the flow rate of the coating solution through the bioreactor to promote coating adhesion and / or uniformity on the cell substrate.

[0068] Example 9 relates to the method of Example 8, wherein controlling the flow rate comprises pulsing the coating solution or reversing the direction of flow of the coating solution.

[0069] Aspect 10 relates to a method according to any one of aspects 1 to 9, wherein the cell substrate comprises 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 and passing through the thickness of the cell substrate.

[0070] Example 11 relates to a method according to Examples 1-10, wherein the cell substrate comprises at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh sheet.

[0071] Aspect 12 relates to a method according to aspects 1 to 11, wherein the cell substrate comprises a polymeric material.

[0072] Example 13 relates to the method of example 12, wherein the polymeric material is at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0073] Aspect 14 relates to a method according to aspects 1-13, wherein the bioreactor is configured to provide uniform fluid flow through the cell substrate and / or cell culture chamber.

[0074] Embodiment 15 relates to a method of culturing cells in a bioreactor, the method comprising coating a cell substrate in the bioreactor of any of embodiments 1 to 14, seeding cells onto the coated cell substrate, culturing the cells on the coated cell substrate, and harvesting a product of the culturing of the cells.

[0075] Example 16 relates to the method of example 15, wherein the coated cell substrate comprises a uniform structure configured to allow the flow of at least one of cell culture medium, cells, or cell products through the cell culture substrate.

[0076] Example 17 relates to a method according to example 15 or 16, wherein the seeding comprises attaching the cells to a coated cell substrate.

[0077] Example 18 relates to a method according to Examples 15-17, wherein seeding comprises injecting a cell inoculum directly into a cell culture chamber in which the coated cell substrate is disposed.

[0078] Example 19 relates to a method according to Example 18, wherein the cell inoculum is injected through an inlet of the bioreactor or through a cell inoculum injection port in the bioreactor vessel.

[0079] Aspect 20 relates to the method of aspect 18 or the claims, further comprising perfusing cell culture medium through the culture chamber after injecting the cell inoculum.

[0080] Aspect 21 relates to a system for culturing adherent cells in a bioreactor, the system comprising: a bioreactor vessel comprising: a cell culture chamber within the bioreactor vessel, the cell culture chamber having an inlet for flowing fluid into the cell culture chamber and an outlet for flowing fluid out of the cell culture chamber; a cell substrate disposed within the cell culture chamber and configured to culture cells thereon; a recirculation loop configured to supply fluid to the bioreactor vessel via the inlet and remove fluid from the bioreactor vessel via the outlet; and a coating solution container fluidly connected to the cell culture chamber and configured to hold a substrate coating solution.

[0081] Example 22 relates to the system of Example 21, further comprising one or more pumps for circulating at least one of the fluid through the recirculation loop and the substrate coating solution into the cell culture chamber.

[0082] Aspect 23 relates to the system of aspect 21 or 22, further comprising a controller for controlling the flow of the fluid or cell coating solution.

[0083] Aspect 24 relates to the system of aspect 23, wherein controlling the flow comprises controlling a flow rate or a direction of the flow.

[0084] Aspect 25 relates to the system of Aspect 23 or 24, wherein the controller is configured to control the one or more pumps.

[0085] Aspect 26 relates to the system of aspects 23 to 25, wherein the controller comprises a processor and a memory, the memory including instructions and in communication with and readable by the processor.

[0086] Example 27 relates to the system of Example 26, wherein the instructions, when executed by the processor, cause the controller to receive a signal to coat the cell substrate in the cell culture chamber with a cell coating solution.

[0087] definition "Fully synthetic" or "fully synthetic" refers to a cell culture article, such as a microcarrier or culture vessel surface, that is composed entirely of synthetic-source materials and does not contain any animal-derived or animal-origin materials. The disclosed fully synthetic cell culture articles eliminate the risk of xenocontamination.

[0088] The words "include," "includes," or similar terms are meant to be non-exclusive, i.e., inclusive and not exclusive.

[0089] "User" refers to anyone who uses a system, method, article, or kit disclosed herein, including anyone who is culturing cells to harvest cells or cell products, or anyone who is using cells or cell products that have been cultured and / or harvested in accordance with embodiments herein.

[0090] When used to describe embodiments of the present disclosure, the term "about" refers to, for example, the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, viscosity, and similar values ​​and ranges of components in a composition, or to modify the dimensions of components and similar values ​​and ranges, and refers to variations in the numerical amount that may occur, for example, through typical measuring and handling procedures used to prepare a material, composition, composite, concentrate, component, article of manufacture, or formulation for use; through inadvertent errors in these procedures; through differences in the manufacture, source, or purity of starting materials or components used to carry out the method; and through similar considerations. The term "about" also encompasses amounts that differ due to aging of a composition or formulation having a particular initial concentration or mixture, and amounts that differ due to mixing or processing of a composition or formulation having a particular initial concentration or mixture.

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

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

[0093] Abbreviations known to those skilled in the art may be used (e.g., "h" or "hrs" for hours or hours, "g" or "gm" for gram(s), "mL" for milliliter, and "rt" for room temperature, "nm" for nanometer, and similar abbreviations).

[0094] Specific preferred values ​​disclosed for components, ingredients, additives, dimensions, conditions, and similar aspects, as well as ranges thereof, are for illustrative purposes only and do not exclude other defined values ​​or other values ​​within the defined ranges. The systems, kits, and methods of the present disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values ​​set forth herein, including any stated or implied intermediate values ​​and ranges.

[0095] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps are to be followed, or the claim or description specifically states that the steps are to be limited to a particular order, no particular order is intended to be inferred.

[0096] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope or spirit of the disclosed embodiments. Since modifications, combinations, subcombinations, and variations of the disclosed embodiments incorporating the spirit and content of the embodiments may occur to those skilled in the art, the disclosed embodiments should be construed as including all within the scope of the appended claims and their equivalents.

Claims

1. 1. A method for in situ coating of a cell culture substrate in a bioreactor, comprising:

1. A bioreactor vessel comprising: a cell culture chamber within the bioreactor vessel, the cell culture chamber having an inlet for allowing fluids to enter the cell culture chamber and an outlet for allowing fluids to exit the cell culture chamber; providing a bioreactor vessel comprising: a cell substrate disposed within the cell culture chamber and configured to culture cells thereon; providing a coating solution for coating the cell substrate; introducing the coating solution into the cell culture chamber through the inlet such that the coating solution contacts and coats the cell substrate; removing excess coating solution from the cell culture chamber via the outlet or the inlet; The method, wherein after removing the coating solution, a coated cell substrate remains within the cell culture chamber.

2. 10. The method of claim 1, further comprising incubating the coating solution in the cell culture chamber before removing the coating solution.

3. 3. The method of claim 1 or 2, further comprising rinsing the cell culture chamber with a washing solution during or after removing the coating solution.

4. The method of any one of claims 1 to 3, wherein providing the coating solution comprises preparing a coating solution suitable for a particular cell culture application.

5. The method of any one of claims 1 to 4, wherein the coating solution comprises a material to enhance cell attachment and / or growth on the cell substrate.

6. The method of any one of claims 1 to 5, wherein the coating solution comprises at least one of an extracellular matrix protein, fibronectin, collagen, a hydrogel solution, a polymer solution, and a recombinant protein.

7. 7. The method of any one of claims 1 to 6, wherein charging the coating solution comprises perfusing the coating solution through the bioreactor such that the coating solution enters the cell culture chamber via the inlet, flows through the cell substrate, and exits through the outlet.

8. 8. The method of any one of claims 1 to 7, further comprising controlling the flow rate of the coating solution through the bioreactor to promote coating adhesion and / or uniformity of the cell substrate.

9. The method of claim 8 , wherein controlling the flow rate comprises pulsing the coating solution or reversing the direction of flow of the coating solution.

10. 10. The method of any one of claims 1-9, wherein the cell substrate comprises 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 and through the thickness of the cell substrate.

11. 11. The method of any one of claims 1-10, wherein the cell substrate comprises at least one of a molded polymer lattice sheet, a 3D printed lattice sheet, and a woven mesh sheet.

12. The method of any one of claims 1 to 11, wherein the cell substrate comprises a polymeric material.

13. 13. The method of claim 12, wherein the polymeric material is at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

14. The method of any one of claims 1 to 13, wherein the bioreactor is configured to provide uniform fluid flow through the cell substrate and / or cell culture chamber.

15. 1. A method for culturing cells in a bioreactor, comprising: Coating a cell substrate in a bioreactor according to any one of claims 1 to 14; seeding cells onto the coated cell substrate; culturing the cells on the coated cell substrate; and recovering a product of said culturing of said cells.

16. 16. The method of claim 15, wherein the coated cell substrate comprises a uniform structure configured to allow the flow of at least one of cell culture medium, cells, or cell products through the cell culture substrate.

17. 17. The method of claim 15 or 16, wherein said seeding comprises allowing said cells to attach to said coated cell substrate.

18. 18. The method of any one of claims 15 to 17, wherein the seeding comprises injecting a cell inoculum directly into the cell culture chamber on which the coated cell substrate is disposed.

19. 20. The method of claim 18, wherein the cell inoculum is injected through the inlet of the bioreactor or through a cell inoculum injection port in the bioreactor vessel.

20. 20. The method of claim 18 or 19, further comprising perfusing cell culture medium through the culture chamber after injecting the cell inoculum.

21. 1. A system for culturing adherent cells in a bioreactor, comprising:

1. A bioreactor vessel comprising: a cell culture chamber within the bioreactor vessel, the cell culture chamber having an inlet for allowing fluids to enter the cell culture chamber and an outlet for allowing fluids to exit the cell culture chamber; a cell substrate disposed within the cell culture chamber and configured to culture cells thereon; and a recirculation loop configured to supply fluid to the bioreactor vessel via the inlet and remove fluid from the bioreactor vessel via the outlet; a coating solution container fluidly connected to the cell culture chamber and configured to hold a substrate coating solution.

22. 22. The system of claim 21, further comprising one or more pumps for circulating at least one of the fluid through the recirculation loop and the substrate coating solution into the cell culture chamber.

23. 23. The system of claim 21 or 22, further comprising a controller for controlling the flow of the fluid or the cell coating solution.

24. 24. The system of claim 23, wherein the control of the flow comprises controlling a flow rate or a direction of flow.

25. 25. The system of claim 23 or 24, wherein the controller is configured to control the one or more pumps.

26. The system of any one of claims 23 to 25, wherein the controller comprises a processor and a memory, the memory containing instructions and in communication with and readable by the processor.

27. 27. The system of claim 26, wherein the instructions, when executed by the processor, cause the controller to receive a signal to coat the cell substrate in the cell culture chamber with the cell coating solution.

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