Systems and methods for coating bioreactor substrates

Abstract

There is provided a method of in situ coating a cell culture substrate in a bioreactor, the method comprising providing a bioreactor vessel having: a 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; and a cell substrate disposed in the cell culture chamber for culturing cells. The method comprises providing a coating solution for coating a cell substrate; inputting the coating solution into the cell culture chamber such that the coating solution contacts the cell substrate; and removing excess coating solution from the cell culture chamber. And after the coating solution is removed, the coated cell substrate is left in the cell culture room.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 425,467, filed on Nov. 15, 2022, under 35 U.S.C. § 119. The content of the U.S. Provisional Application is hereby incorporated by reference in its entirety and made a part of this application. Technical Field

[0003] The present disclosure generally relates to systems and methods for coating substrates in cell - culture bioreactors. Specifically, the present disclosure relates to in - situ coating of substrates in perfusion - type bioreactor vessels. Background Art

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

[0005] A large portion of the cells used in bioprocessing are adherent - dependent cells, meaning that the cells need to attach to a surface to grow and function. Adherent cell culture dominates the production of viral vectors for gene and modified cell therapies. This is because most of the cells used for viral vector production are adherent - dependent. Viral vectors are commonly used to deliver genetic material into cells and tissues, enabling the correction of genetic defects, enhancement of cell and tissue function, or improvement of cell product production, ultimately leading to potential curative treatments. Adherent cell culture is also the primary method for scaling up stem cells for regenerative medicine. This is because stem cells such as induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) are inherently adherent - dependent. Stem cells have broad prospects in cell therapy, tissue engineering, and regenerative medicine, as well as in pharmaceutical and biotechnological applications. There is an urgent need for reliable and efficient platforms to scale up adherent cell culture.

[0006] There are many materials for cell culture substrates in bioreactors. One of them is polyethylene terephthalate (PET), which is mainly used for viral vector and vaccine production, but its role in other applications of adherent cell culture is limited. For example, many existing fixed - bed bioreactors have not been used for stem cell culture. Therefore, there is an urgent need to develop a fixed - bed cell culture substrate with a functionalized or coated surface to support the culture of a wider range of cell types.

[0007] Mammalian cells are used to produce therapeutic proteins, monoclonal antibodies, viral vectors, and even cultured meat. Additionally, in tissue engineering and regenerative medicine, billions of stem cells are used to fabricate tissue engineering constructs or to replenish cells lost or damaged in degenerative diseases. Although suspension cell culture is widely used for protein and antibody production, adherent cell culture dominates in the production of viral vectors for gene and modified cell therapies and stem cells for regenerative medicine. Viral vectors are commonly used to deliver genetic material into cells and tissues, enabling the correction of genetic defects, enhancement of cell and tissue function, or improvement of cell product generation, ultimately leading to potential curative treatments.

[0008] Stem cells have broad prospects in cell therapy, tissue engineering, regenerative medicine, and pharmaceutical and biotechnological applications. However, most cells used for viral vector production are adherent-dependent; similarly, stem cells such as induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) are inherently adherent-dependent. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the ability to self-renew indefinitely and can differentiate into all derivatives of the three primary germ layers. These cells have great potential in clinical applications, regenerative medicine, tissue engineering, drug screening, and early developmental biology research. Cell therapy and regenerative medicine applications require large quantities of hPSCs. This necessitates technologies capable of efficiently scaling up and expanding hPSCs.

[0009] The culture of hPSCs requires a controlled culture environment to ensure cell attachment, survival, proliferation, self-renewal, and maintenance of cell pluripotency and genomic stability. Human PSCs are cultured on extracellular matrices of natural origin (such as Matrigel, Life Technologies' ), recombinant proteins (such as vitronectin, laminin-511, laminin-521), and synthetic surfaces ( ).

[0010] There is a need for cell culture bioreactor systems and methods of using such systems that enable end users to modify a bioreactor by in-situ coating the cell culture substrate in the bioreactor to make it suitable for its intended application or cell type and to achieve optimal efficiency and performance in said application. SUMMARY OF THE INVENTION

[0011] According to an embodiment, a method for in-situ coating 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 flow into the cell culture chamber and an outlet for allowing fluid to flow out of the cell culture chamber. The bioreactor vessel further includes a cell substrate disposed in the cell culture chamber for culturing cells thereon. 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 the cell substrate to coat the cell substrate; and removing excess coating solution from the cell culture chamber via the outlet or the inlet. After removing the coating solution, the coated cell substrate remains in the cell culture chamber.

[0012] According to an embodiment, a method for culturing cells in a bioreactor is provided. The method includes coating a cell substrate within the bioreactor as described herein; inoculating cells onto the coated cell substrate; culturing cells on the coated cell substrate; and harvesting the product of the cell culture.

[0013] 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 allowing fluid to flow into the cell culture chamber and an outlet for allowing fluid to flow out of the cell culture chamber. The cell culture substrate further includes a cell substrate disposed in the cell culture chamber for culturing cells thereon. A recirculation loop is also provided which 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 the substrate coating solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a schematic diagram of a fixed-bed bioreactor system for coating a cell substrate in a bioreactor according to an embodiment.

[0015] Figure 2 is during the step of coating the cell substrate according to an embodiment Figure 1 of the bioreactor system.

[0016] Figure 3 is after coating the cell substrate according to an embodiment Figure 1 and 2 of the bioreactor system.

[0017] Figure 4 is a schematic representation of a cell culture system according to one or more embodiments.

[0018] Figure 5 Shows a process flow diagram for coating a cell substrate in a bioreactor to culture cells, according to one or more embodiments. Detailed Description

[0019] Various embodiments of the present disclosure will be described in detail with reference to the accompanying drawings, if any. The various embodiments mentioned do not limit the scope of the invention, which is limited only by the scope of the appended claims. Additionally, any examples set forth in this specification are not limiting, but merely represent a portion of the many possible embodiments of the claimed invention.

[0020] It may be necessary to alter the surface chemistry of the adherent cell culture substrate to provide the desired cell attachment properties. These alterations can be achieved by chemically treating the polymeric material of the substrate or by grafting cell attachment molecules onto the substrate surface. Alternatively, a thin layer of a biocompatible hydrogel that exhibits cell attachment properties, such as collagen or A variety of coatings can be used, including, for example, extracellular matrix proteins, fibronectin, collagen, hydrogel solutions, polymer solutions, and recombinant proteins. Those skilled in the art will understand that any suitable coating can be used. Alternatively, the surface of the cell substrate can be made to have cell attachment properties by treatment methods using various types of plasmas, process gases, and / or chemicals known in the art. However, in one or more embodiments, the cell substrate can provide an efficient cell growth surface without surface treatment. To simplify the manufacturing process of the bioreactor and increase its flexibility, it may be advantageous to provide an uncoated cell culture bioreactor, such that the end user can decide on the type of coating to apply to the cell substrate within the bioreactor, depending on the cell type or application of interest. Thus, the embodiments provided herein facilitate in-situ coating of the substrate within the bioreactor by the user. Accordingly, by providing a bioreactor that is pre-loaded with a cell substrate and capable of coating the cell substrate in place, a solution is provided that is flexible and easy to use, while minimizing the risk of contamination or assembly errors that can occur when the end user must remove the substrate and reload it into the bioreactor after coating the substrate.

[0021] Embodiments of the present disclosure relate to systems and methods for in-situ coating of a cell substrate within a fixed-bed bioreactor, and systems and methods for culturing cells within such a bioreactor. These fixed-bed bioreactors can be used for inoculating, culturing, and / or expanding various types of cells, including mesenchymal stem cells, cancer cells, T cells, fibroblasts, and myoblasts, among others. Before culturing adherent cells on a cell substrate, it is preferable to coat the cell substrate with a coating to improve the performance for a particular cell type or application. For example, a compound that promotes attachment can be applied to the cell growth surface to facilitate contact and subsequent expansion of cells such as human mesenchymal stem cells (hMSCs).

[0022] Embodiments of the present disclosure include a fixed-bed bioreactor system for cell culture. In accordance with aspects of these embodiments, the bioreactor system can be a closed system, where the contents of the bioreactor system are not directly exposed to the atmosphere to prevent contamination. The bioreactor system can be automated. In aspects of the embodiments, the system can include a cell culture medium and / or a coating solution for coating the cell substrate within the bioreactor. The bioreactor system can include a fluid flow path that includes 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 can also include one or more medium conditioning vessels that are fluidly connected to the cell culture chamber and can be integral with or separate from the bioreactor vessel. The system can further include a coating solution source that is fluidly connected to the fluid flow path and is configured to inject the coating solution into the fluid flow path so that the coating solution flows into the cell culture space. The system can include one or more pumps for circulating the medium through the fluid flow path and / or for injecting the coating solution into the cell culture space.

[0023] In an embodiment, the system includes a controller for controlling the operation of the system including the one or more pumps. The controller may include a computer system that includes a processor. In an embodiment, the controller is configured to control the one or more pumps to cause a fluid (such as a cell culture medium or a coating solution) to circulate through a fluid flow path at a certain flow rate. In accordance with aspects of the embodiments, the controller controls the one or more pumps to transfer cell culture medium, nutrients, and / or cells from a source to the fluid flow path and into a bioreactor. The system may include a graphical user interface and a memory that communicate with and are readable by the processor and contain instructions. When the processor executes the instructions, the processor receives instructions such as to coat a cell substrate in the bioreactor. In response to the instruction to coat the bioreactor, the processor may execute a series of steps to coat the bioreactor and may then receive instructions such as to load cells into the bioreactor. In response to the instruction to load cells, the processor may execute a series of steps to load cells from a cell inlet source into the bioreactor, for example.

[0024] Figure 1 FIG. 4 shows 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 that contains 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 in the cell culture space 104. The fixed bed 106 is made of a cell substrate 108 as described herein. In accordance with aspects of the embodiments, 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 enables uniform fluid flow therethrough, thereby improving cell seeding, nutrient distribution, substrate coating, and cell harvesting. The cell culture system 100 may include an inlet distribution plate 114 and an outlet distribution plate 116 to assist in the uniform distribution of culture medium, cells, coating solution, and other fluids throughout the fixed bed. The cell culture system 100 further includes an inlet 110 and an outlet 112 for fluid inflow and outflow from the cell culture space 104, respectively. The inlet 110 and the outlet 112 are connected to fluid flow paths 118a, 118b for providing a passage for fluid inflow and outflow from the cell culture vessel 102. The fluid flow paths 118a, 118b may form a perfusion flow path that causes the fluid to recirculate through the bioreactor in a loop, as will be discussed further below.

[0025] According to an embodiment, the cell culture system 100 includes a coating solution container 120 that is fluidly connected to the cell culture container 102 and is capable of supplying the 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 the coating passage 124, or may be directly connected to the cell culture container 102 via a separate inlet outside the fluid flow paths 118a and 118b. As Figure 2 shown, the cell culture system 100 may supply the coating solution 112 from the coating solution container 120 to the cell culture space 104, which contains 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 be brought into contact with the cell substrate 108 for a predetermined time, or until the 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 the coating is considered complete, the coating solution 122 may be removed from the cell culture space 104, as Figure 3 shown, leaving a coated cell substrate made of the coating 123 on the cell substrate 108. The removal of the coating solution 122 from the cell culture space may be accompanied by washing with a washing solution (not shown), which may be performed while removing the excess coating solution, or at some time after the removal is completed. Thus, a bioreactor with a cell substrate suitable for the desired cell culture application is provided.

[0026] Figure 4 Shows a cell culture system 400 according to one or more embodiments. The system 400 includes a bioreactor 402 for accommodating a fixed bed cell substrate of one or more embodiments disclosed herein. The bioreactor 402 may be fluidly connected to a culture medium conditioning container 404, and the system is capable of supplying the cell culture medium 406 in the conditioning container 404 to the bioreactor 402. The culture medium conditioning container 404 may include sensors and control components present in typical bioreactors in the bioprocessing industry for suspension batch culture, fed-batch culture, or perfusion culture. These components include, but are not limited to, a DO oxygen sensor, a pH sensor, an oxygenator / gas bubbling unit, a temperature probe, and nutrient addition ports and base addition ports. The gas mixture supplied to the bubbling unit may be controlled by a gas flow controller for N2, O2, and CO2 gases. The culture medium conditioning container 404 also contains an impeller for mixing the culture medium. All culture medium parameters measured by the above-listed sensors may be controlled by a culture medium conditioning control unit 418 that communicates with the culture medium conditioning container 404 and is capable of measuring the conditions of the cell culture medium 406 and / or adjusting the conditions of the cell culture medium to a desired level. As Figure 4As shown, the provided culture medium conditioning container 404 is a container separate from the bioreactor container 402. The advantage of this is that it enables the separation of culture medium conditioning from the cell culture area, and then the conditioned culture medium is supplied to the cell culture space. However, in some embodiments, the culture medium conditioning can be performed within the bioreactor container 402.

[0027] The culture medium from the culture medium conditioning container 404 is delivered to the bioreactor 402 via the inlet 408. The bioreactor may also include an injection port for inoculating the cell inoculum and initiating 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. Additionally, cells or cell products may also be output through the outlet 410. To analyze the contents flowing out of the bioreactor 402, one or more sensors 412 may be provided in the pipeline. 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 one or more sensors 412 (such as an O2 sensor), and based on the signals, regulate the flow rate into the bioreactor 402 by sending signals to a pump 416 (such as a peristaltic pump) located 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 the desired cell culture conditions.

[0028] The culture medium perfusion rate is controlled by the signal processing unit 414, which collects and compares sensor signals from the culture medium conditioning container 404 and sensors located at the outlet 410 of the fixed bed bioreactor. Due to the pack flow nature of the culture medium perfusion through the fixed bed bioreactor 402, nutrient, pH, and oxygen gradients are formed along the fixed bed. The perfusion flow rate of the bioreactor can be automatically controlled by the flow control unit 414 operably connected to the peristaltic pump 416. Examples of other control and system components can be found in U.S. Patent Application Publication No. US2020 / 0248124A1, which is incorporated herein by reference.

[0029] Cell culture system 400 also includes a coating solution container 420, which is fluidly connected to the bioreactor container 402 and capable of supplying the coating solution contained therein to the bioreactor container 402. The coating solution is selected to improve the performance of a given cell culture application. Pump 422 can directly supply the coating solution to the bioreactor container 402 or to a fluid flow path connected to the bioreactor container 402. When it is time to supply the coating solution to the cell substrate in the bioreactor, the controller 414 can send a signal to activate the pump 422. After the coating solution fills the bioreactor system 402 and contacts the cell substrate, the coating solution can be in contact with the cell substrate for a predetermined time or until the application of the coating to the cell substrate is complete (e.g., through some physical or chemical reaction; through some external stimulus, such as heating, cooling, or radiation). After the coating is considered complete, the coating solution can be removed from the cell culture space. For example, the controller 414 can send a signal to the pump to remove the excess coating solution and / or supply a wash solution to the reactor 402 to assist in removing the excess coating solution. The wash solution can contain a culture medium or fluid, such as phosphate buffered saline (PBS) or other cell culture media.

[0030] In conventional large-scale cell culture bioreactors, different types of packed bed bioreactors are 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 to volume ratio, so the cell density can be higher than that in other systems. However, the packed bed typically acts as a deep filter, where cells are physically trapped or entangled in the fibers of the substrate. Therefore, due to the linear flow of the cell inoculum through the packed bed, the cells experience non-uniform distribution within the packed bed, resulting in variations 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, while it is significantly lower closer to the outlet of the bioreactor. This non-uniform distribution of cells inside the packed bed severely hinders the scalability and predictability of such bioreactors in bioprocess manufacturing and may even lead to reduced cell growth or viral vector production efficiency per unit surface area or volume of the packed bed.

[0031] Another problem encountered by packed-bed bioreactors disclosed in the prior art is the channeling effect. Due to the randomness of packing non-woven fibers, the local fiber density at any given cross-section of the packed bed is non-uniform. The culture medium flows faster in regions of low fiber density (higher bed permeability) and much slower in regions of high fiber density (lower bed permeability). The resulting non-uniform perfusion of the culture medium across the packed bed creates the channeling effect, which itself manifests as distinct gradients of nutrients and metabolites, thereby having a negative impact on overall cell culture and bioreactor performance. Cells located in low culture medium perfusion regions will starve and often die due to lack of nutrients or metabolite poisoning. Cell harvesting is another problem encountered when using bioreactors with packed non-woven fiber scaffolds. Since the packed bed acts as a depth filter, the cells released at the end of the cell culture process are trapped 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 regions experiencing different flow rates and shear forces, effectively reducing the available cell culture area, causing uneven culturing, and interfering with transfection efficiency and cell release.

[0032] To address these and other problems of existing cell culture solutions, embodiments of the present disclosure provide cell growth substrates, matrices of such substrates, and / or packed bed systems using such substrates, which are capable of achieving efficient and high-yield cell culture of adhesion-dependent cells and production of cell products (such as proteins, antibodies, virus particles). Embodiments include porous cell culture substrates made of an ordered and regular array of porous substrate materials, which are 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 substrate and bioreactor can perform cell seeding and growth and / or cell product harvesting 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 product scale, having a comparable number of viral genomes per unit substrate surface area (VG / cm 2 ) across the entire production scale. The harvestability and scalability of the embodiments herein enable these embodiments to be used for efficient seed train amplification culture to grow cell populations at multiple scales on the same cell substrate. Additionally, the embodiments herein provide a cell culture substrate that has a high surface area and, in combination with the other features described, is capable of achieving high-yield cell culture solutions. In some embodiments, for example, the cell culture substrates and / or bioreactors discussed herein can produce 10 16 to 10 18 viral genomes (VG) per batch.

[0033] In one embodiment, a fixed bed substrate is provided that has a structurally defined surface area for adherent cell attachment and proliferation, has good mechanical strength, and forms a highly uniform multi-interconnected fluid network when assembled in 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 substrate disclosed herein supports the attachment and proliferation of adherent-dependent cells in a high volume density. Such a substrate enables uniform cell seeding and efficient harvesting of cells or other products in a bioreactor. Additionally, embodiments of the present disclosure also support cell culture to provide a uniform cell distribution during the seeding step and achieve confluent monolayer or multilayer adherent cells on the disclosed substrate, and can avoid the formation of large and / or uncontrolled 3D cell aggregates where nutrient diffusion is limited and metabolite concentrations increase. Thus, the substrate eliminates diffusion limitations during bioreactor operation. Additionally, the substrate is also capable of simply and efficiently harvesting cells from the bioreactor. The structurally defined substrate of one or more embodiments is capable of completely recovering cells from the fixed bed of the bioreactor and continuously harvesting cells.

[0034] According to some embodiments, a method of cell culture using a bioreactor with the substrate is also provided for bioprocessing to produce therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0035] Compared to existing cell culture substrates used in cell culture bioreactors (i.e., non-woven substrates formed by randomly ordered fibers), embodiments of the present disclosure include cell culture substrates having a defined and ordered structure. The defined and ordered structure allows for obtaining consistent and predictable cell culture results. Additionally, the substrate has an open porous structure that prevents cell entrapment and enables cells to flow uniformly through a fixed bed. This configuration can improve cell seeding, nutrient delivery, cell growth, and cell harvesting. According to one or more specific embodiments, the substrate is formed from a substrate material having a flake-like configuration, with a first side and a second side separated by a relatively small thickness such that the thickness of the flake is small relative to the width and / or length of the first and second sides of the substrate. Additionally, a plurality of pores or openings are formed through the thickness of the substrate. The substrate material between the openings has a certain size and geometry, thereby allowing 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 some embodiments, the substrate is a polymer-based material and can be formed as a molded polymer flake; a polymer flake with openings punched through the thickness; a plurality of filaments fused into a network layer; a 3D-printed substrate; or a plurality of filaments woven into a network layer. The physical structure of the substrate has a high surface area to volume ratio for culturing adherent-dependent cells. According to various embodiments, the substrate can be arranged or filled in a bioreactor in certain ways as discussed herein for uniform cell seeding and growth, uniform medium perfusion, and efficient cell harvesting.

[0036] Embodiments of the present disclosure can implement a viral vector platform with practical dimensions, which can produce viral genomes at a scale of 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 yield is about 10 15 to about 10 18 or more viral genomes per batch. For example, in some embodiments, the viral genome yield can be about 10 15 to about 10 16 viral genomes or batches per batch, or about 10 16 to about 10 19 viral genomes per batch, or about 10 16 to 10 18 viral genomes per batch, or about 10 17 to about 10 19 viral genomes per batch, or about 10 18from about 0 to about 10 19 viral genomes, or about 10 18 or more viral genomes per batch.

[0037] In addition, the embodiments disclosed herein are capable of not only allowing cells to attach and grow on a cell culture substrate, but also harvesting live cultured cells. The inability to harvest live cells is a significant drawback of current platforms and it results in difficulty in constructing and maintaining a sufficient number of cells to reach production capacity. According to one aspect of the embodiments of the present disclosure, live cells can be harvested from the cell culture substrate, including between 80% and 100% live cells, or between about 85% and about 99% live cells, or between about 90% and about 99% live cells. For example, in the harvested cells, at least 80% are live cells, at least 85% are live cells, at least 90% are live cells, at least 91% are live cells, at least 92% are live cells, at least 93% are live cells, at least 94% are live cells, at least 95% are live cells, at least 96% are live cells, at least 97% are live cells, at least 98% are live cells, or at least 99% are live cells. Cells can be released from the cell culture substrate using, for example, trypsin, TrypLE or Accutase.

[0038] According to an embodiment, the cell culture substrate can be a woven mesh layer made of 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 that 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.). The woven mesh is characterized in that, macroscopically, it is a two-dimensional sheet or layer. However, upon closer inspection of the woven mesh, a three-dimensional structure is found due to the interlacing and undulating of the fibers in the mesh. Without wishing to be bound by theory, it is believed that the three-dimensional structure of the substrate is advantageous because it provides a larger surface area for culturing adherent cells and the structural rigidity of the mesh can provide a consistent and predictable cell culture substrate structure that enables uniform fluid flow.

[0039] In one or more embodiments, the diameter of the fibers can be in the following ranges: from about 10 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; from about 10 μm to about 300 μm; from about 20 μm to about 250 μm; from about 20 μm to about 170 μm; or from about 150 μm to about 300 μm. At the microscopic level, due to the scale difference between the fibers and the cells (e.g., the fiber diameter is larger than the cell), the surface of the monofilament fiber presents as an approximately 2D surface for adherent cells to attach and proliferate. The fibers can be woven into a mesh with openings ranging from about 10 μm × 10 μm to about 1000 μm × 1000 μm. In some embodiments, the diameter 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; 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 examples of some embodiments, but are not intended to limit the possible characteristic dimensions of the mesh according to all embodiments. The combination of fiber diameter and opening diameter is selected to enable efficient and uniform fluid flow through the substrate when, for example, the cell culture substrate includes multiple adjacent mesh layers (e.g., a stack or roll of mesh layers of each layer).

[0040] Factors such as fiber diameter, aperture diameter, and weave type / pattern will determine the surface area available for cell attachment and growth. Additionally, when the cell culture substrate comprises a stacked, wound, or other overlapping substrate arrangement, the packing density of the cell culture substrate will affect the surface area of the fixed-bed substrate. The packing density can vary with the packing thickness of the substrate material (e.g., the space required for the substrate layer). For example, if a stack of cell culture substrates has a certain height, each layer of the stack can be considered to have a certain packing thickness, which 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 interlock or overlap to some extent based on their arrangement relative to each other. In a first arrangement, adjacent layers can fit closely together, but in a second arrangement, adjacent layers can be non-overlapping, such as when the lowest point of the upper layer is in direct contact with the highest point of the lower layer. For certain applications, it may be desirable to provide a cell culture substrate with a lower layer packing density (e.g., when higher permeability is preferred) or a higher packing density (e.g., when maximizing the substrate surface area is preferred). According to one or more embodiments, the packing thickness can be from about 10 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; from about 10 μm to about 300 μm; or from about 20 μm to about 250 μm.

[0041] The above structural factors can determine the surface area of the cell culture substrate, whether it is a single-layer cell culture substrate or a cell culture substrate with multiple layers of substrates. For example, in one particular embodiment, a single-layer 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 attachment and growth. Unless otherwise specified, the "surface area" referred to is this effective surface area. According to one or more embodiments, the effective surface area of a single woven mesh substrate layer with a 6 cm diameter can be 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 effective surface area ranges are provided by way of example only, and some embodiments can have different effective surface areas. The cell culture substrate can also be characterized by porosity, which will be discussed in the examples herein.

[0042] The substrate mesh can be made of monofilament or multifilament fibers of a polymer material compatible in cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide. The mesh substrate can have different patterns or weaves, including, for example, knitting, warp knitting, or weaving (such as plain weave, twill weave, Dutch weave, five-harness satin weave).

[0043] By using a culture substrate defined by a structure with sufficient rigidity, high flow resistance uniformity throughout the substrate or fixed bed is achieved. According to various embodiments, the substrate can be deployed in a single-layer or multi-layer form. This flexibility eliminates diffusion limitations and uniformly delivers nutrients and oxygen to the cells attached to the substrate. Additionally, the open substrate has no cell-trapping zones in the fixed-bed configuration, allowing for the complete harvest of highly viable cells at the end of the culture. This substrate also achieves filling uniformity of the fixed bed and enables direct scale-up from process development units to large-scale industrial bioprocessing units. The ability to directly harvest cells from the fixed bed eliminates the need to resuspend the substrate in a stirred or mechanically agitated vessel, which increases complexity and may impose shear stress harmful to the cells. Furthermore, the high packing density of the cell culture substrate can generate high bioprocess productivity in an industrially manageable volume.

[0044] Embodiments of the present disclosure include a cell substrate in the form of a multi-layer substrate. 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 meet the desired cell (or cell product) density or quantity. However, the embodiments are not limited to this configuration, and the cell substrate can adopt various configurations. For example, the cell substrate can be a roll of cell substrate material or a small piece of substrate material fixed in a reactor.

[0045] The geometry of the reticulated basal layer is designed to allow fluids to flow efficiently and uniformly through one or more basal layers. Additionally, the structure of the cell substrate can accommodate fluid flow through the substrate in multiple orientations. For example, the flow direction of the bulk fluid can be perpendicular to the major side surfaces of the first and second basal layers, or the cell substrate can be oriented relative to the flow such that the side surfaces of the basal layer are parallel to the bulk flow direction. In addition to fluid flow perpendicular or parallel to the first and second sides of the mesh layer, the substrate can also be arranged at intermediate angles or even randomly relative to the fluid flow. This flexibility in orientation is enabled by the substantially isotropic flow behavior of the woven substrate. In contrast, substrates for adherent cells in existing bioreactors do not exhibit this behavior, but rather their fixed beds tend to form preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the cell substrates of the present disclosure enables their use in a variety of applications and bioreactor or vessel designs, while achieving better and more uniform permeability throughout the bioreactor vessel.

[0046] As discussed herein, according to one or more embodiments, the cell substrate can be used within a bioreactor vessel. For example, the substrate can be used in a fixed bed bioreactor configuration or in other configurations within a three-dimensional culture chamber. However, the embodiments are not limited to three-dimensional culture spaces, and the use of the substrate in what is considered a two-dimensional culture surface configuration can be contemplated, where one or more layers of the substrate are laid flat, for example, within a tissue culture dish, to provide a culture substrate for cells. Due to contamination concerns, the vessel can be a disposable vessel that can be discarded after use.

[0047] According to one or more embodiments, a cell culture system is provided where the cell culture substrate is used in the culture chamber of a bioreactor vessel. There is a fixed bed cell substrate in the cell culture chamber, which is made by stacking cell substrate layers. The cell substrate layers are stacked such that the first side or the second side of a substrate layer faces the first side or the second side of an adjacent substrate layer. The bioreactor vessel has an inlet at one end for inputting culture medium, cells, and / or nutrients into the culture chamber and an outlet at the opposite end for removing culture medium, cells, or cell products from the culture chamber. By allowing the substrate layers to be stacked in this way, the system can be easily scaled up without negatively affecting cell attachment and proliferation because the stacked substrates have a defined structure and efficient fluid flow through. Although the vessel can generally be described as having an inlet and an outlet, some embodiments can use one or both of the inlet and the outlet to allow the inflow and outflow of culture medium, cells, or other contents into and out of the culture chamber. For example, the inlet can be used to allow the inflow of culture medium or cells into the culture chamber during the cell seeding, perfusion, or culture phase, but can also be used to remove one or more of the culture medium, cells, or cell products through the inlet during the harvest phase. Thus, the terms "inlet" and "outlet" are not intended to limit the functions of these openings.

[0048] In one or more embodiments, the flow resistance and volume density of the fixed bed can be controlled by staggering the stacking of substrate layers with different geometries. Specifically, the mesh size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the flow resistance of the fluid in the fixed bed form. By inserting meshes of different sizes and geometries, the flow resistance of one or more specific parts in the bioreactor can be controlled or changed. This enables more uniform liquid perfusion in the fixed bed. Different combinations of meshes of different sizes can obtain different distributions of the volume density of the cell growth surface and the flow resistance. For example, a fixed bed with regions having different volume cell densities (e.g., a series of regions producing a pattern of low density / high density / low density / high density, etc.) can be assembled by staggering the use of meshes of different sizes.

[0049] According to an embodiment, the overall flow direction of fluid through the bioreactor is from the inlet to the outlet direction, and in various aspects of the embodiment, the first major side and the second major side of the cell substrate layer are perpendicular to the overall flow direction. In contrast, some aspects of some embodiments include a bioreactor vessel and a stack of cell substrates within the culture space, which have a first side and a second side parallel to the overall flow direction. Thus, the cell substrates of the embodiments of the present disclosure can be used in either configuration. In any of these instances, the size and shape of the cell substrate are configured to fill the internal space defined by the culture chamber such that the culture space in each vessel is filled with a cell growth surface, thereby maximizing efficiency in terms of the number of cells per unit volume. The cell culture space of the system can be fed through a single inlet and have a single outlet, or can have multiple inlets and / or multiple outlets. However, according to various embodiments herein, a distribution plate can be used to assist in distributing the culture medium, cells, or nutrients across the entire cross-section of the fixed bed and thereby improve the uniformity of fluid flow through the fixed bed. Thus, multiple inlets indicate how many holes to make in the distribution plate to create a more uniform flow across the entire cross-section of the fixed bed.

[0050] In some embodiments, the fixed bed is arranged such that the cell substrate is formed into a cylindrical reel. For example, a sheet of cell substrate material (such as one or more mesh substrates) is wound around the central longitudinal axis of the cell culture space to form a cylinder. The cylindrical reel 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 reel is designed to be within the bioreactor vessel such that the central longitudinal axis is parallel to the overall flow direction of fluid through the bioreactor or culture chamber that houses the cylindrical reel. The bioreactor system can also include a central support member around which the cell substrate is positioned. According to some embodiments, the central support member can be used only for the physical support and / or alignment of the cell substrate, but can also provide other functions. For example, the central support member can have one or more openings for supplying the culture medium to the cell substrate along the length of the fixed bed. 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 within the interior of the cylindrical reel. These attachment sites can be hooks, clasps, struts, clips, or other members for attaching the mesh to the central support member.

[0051] One or more embodiments of the present disclosure provide a cell seeding step that is different from conventional methods. In conventional methods, a culture medium is filled into a packed bed with a conventional substrate, and a concentrated inoculum is injected into the culture medium circulation loop. The cell suspension is pumped through the bioreactor at an increasing flow rate to reduce the non-uniformity of cell seeding caused by capturing cells on the conventional fixed-bed substrate. In such conventional methods, pumping cells at a higher flow rate in the circulation loop may continue for several hours until most cells are captured in the packed-bed bioreactor. However, due to the non-uniform deep-bed filtration nature of the conventional packed-bed bioreactor, cells are non-uniformly distributed inside the packed bed, where the cell density is higher at the inlet region of the bioreactor and lower at the outlet region of the bioreactor.

[0052] In contrast, according to an embodiment of the present disclosure, a cell inoculum having a volume equal to the void volume of the culture chamber in the bioreactor is directly injected into the packed bed through the cell inoculum injection port at the inlet 408 of the bioreactor 402 ( Figure 4 ). Then, due to the presence of uniform and continuous fluid channels in the cell culture substrate described herein, the cell suspension is uniformly distributed inside the packed bed. To prevent cell sedimentation caused by gravity during the initial seeding stage, medium perfusion can be started immediately after the inoculum is injected. The perfusion flow rate is maintained below a pre-programmed threshold to balance gravity and avoid washing cells out of the packed-bed bioreactor. Thus, during the initial cell attachment stage, the cells gently tumble inside the packed bed and achieve uniform cell distribution and attachment on the available substrate surface.

[0053] Embodiments include methods for in-situ coating a cell substrate within a bioreactor vessel. For example, Figure 4 the system 400 can be operated according to the method steps of one or more embodiments. As Figure 5As shown, method 500 may include providing a cell culture bioreactor (S502), providing a coating solution (S504), and flowing the coating solution into the cell culture space of the bioreactor (S506). After introducing the coating solution into the cell culture space in S506, an incubation step (S507) may be performed, during which the coating solution remains in the cell culture space to coat the substrate. This incubation step S507 may include adding some additional stimulus, such as heating, cooling, or applying some radiation, to help the coating solution form a coating on the cell substrate. During the incubation step S507, the coating solution may be static in the cell culture space, or may be perfused through the cell culture space in a loop or in a one-time perfusion through the reactor. The incubation period S507 may also include a reaction occurring to form a coating on the cell substrate by the coating solution. Method 500 may also include a step of removing the coating solution from the bioreactor after forming a coating on the cell substrate (S508). The step of removing the coating solution S508 may be performed after a predetermined period considered sufficient to obtain a coating according to process parameters. Optionally, a washing step S509 may be performed during or after the removal step S508. For example, the bioreactor may be rinsed by injecting a washing solution to force the coating solution out of the bioreactor, or the washing solution may be input into the bioreactor after the excess coating solution has been removed. Next, a cell culture process may be performed, including inoculating and attaching cells (S510) and a cell culture medium, followed by a culture process, which may include cell expansion, transfection, cell differentiation, production of viral vectors or other cell products, and harvesting.

[0054] Depending on the desired system, the cell culture substrate may be arranged in a variety of configurations within the culture chamber. For example, in one or more embodiments, the system includes one or more substrate layers whose width extends across the width of the cell culture space defined within the culture chamber. Multiple layers of the substrate may be stacked in this manner to a predetermined height. As discussed above, the substrate layers may 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 flowing through the culture space defined within the culture chamber, or the first and second sides of one or more layers may be parallel to the overall flow direction. In one or more embodiments, the cell culture substrate includes one or more substrate layers having a first orientation relative to the overall flow, and one or more other layers having a second orientation different from the first orientation. For example, each layer may have a first and a second side parallel to or perpendicular to the overall flow direction, or at an angle between the first and second sides.

[0055] In one or more embodiments, a cell culture system includes a plurality of discrete cell culture substrate blocks in a packed bed configuration, where the length and / or width of each substrate block is small relative to the culture chamber. As used herein, when the length and / or width of a substrate block is about 50% or less of the length and / or width of the culture space, each substrate block is considered to have a small length and / or width relative to the culture chamber. Thus, the cell culture system can include a plurality of substrate blocks filled in the culture space in a desired arrangement. The arrangement of the substrate blocks can be random or semi-random, or can have a predetermined order or arrangement, such as the substrate blocks being oriented in a generally similar orientation (e.g., horizontal, vertical, or at an angle between 0° and 90° relative to the overall flow direction).

[0056] As used herein, a "defined culture space" refers to the space within the culture chamber occupied by the cell culture substrate, and where cell seeding and / or culturing will occur. The defined culture space can substantially fill the entire culture chamber, or can occupy a portion of the space within the culture chamber. As used herein, an "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.

[0057] In one or more embodiments, the cell culture substrate is fastened within the culture chamber by a fixing mechanism. The fixing mechanism can fasten a portion of the cell culture substrate to the wall of the culture chamber surrounding the substrate, or to the wall at one end of the culture chamber. In some embodiments, the fixing mechanism adheres a portion of the cell culture substrate to a component extending through the culture chamber, such as a component extending parallel to the longitudinal axis of the culture chamber, or a component extending perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture substrate can be accommodated within the culture chamber without being fixedly attached to the wall of the culture chamber or bioreactor vessel. For example, the substrate can be accommodated within the boundaries of the culture chamber or within other structural components within the culture chamber to hold the substrate in a predetermined region of the bioreactor vessel without fixedly fastening the substrate to these boundaries or structural components.

[0058] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. A culture chamber is capable of containing a cell culture substrate and a substrate according to one or more embodiments described in the present disclosure. In a roller bottle configuration, the bioreactor vessel can be operably attached to a member 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., intermittently rotating in a single direction or alternating directions, or oscillating in a reciprocating 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 as 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 portion 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 unattached and can freely move to a desired angle relative to the vessel as the vessel rotates. The cells can attach to the cell culture substrate, and the movement of the vessel enables the cells to be exposed to the cell culture medium or liquid, as well as oxygen or other gases within the culture chamber.

[0059] By using a cell culture substrate according to an embodiment of the present disclosure, such as a substrate including a woven or reticulated substrate, the roller bottle container will have a larger surface area available for adherent cells to attach, proliferate, and function. Specifically, using a reticulated substrate woven from a monofilament polymer material within a roller bottle can increase the surface area to about 2.4 to about 4.8 times or about 10 times the surface area of a standard roller bottle. As discussed herein, each monofilament strand of the reticulated substrate itself can serve as a 2D surface for adherent cells to attach. Additionally, multiple layers of mesh can be arranged within the roller bottle, increasing the total available surface area range to about 2 to 20 times the surface area of a standard roller bottle. Thus, existing roller bottle facilities and processes, including cell seeding, medium replacement, and cell harvesting, can be altered by adding the improved cell culture substrates disclosed herein with minimal impact on existing operating infrastructure and processing steps.

[0060] The bioreactor vessel optionally includes one or more outlets capable of attaching to an inlet and / or outlet member. Liquid, culture medium, or cells can be supplied to or removed from the chamber through the one or more outlets. A single port in the vessel can serve as both an inlet and an outlet, or multiple ports can be provided as dedicated inlets and outlets.

[0061] The packed bed cell culture substrate of one or more embodiments may consist of a woven cell culture mesh substrate, and no other form of cell culture substrate is provided or dispersed in the cell culture substrate. That is, the woven cell culture mesh substrate of the embodiments of the present disclosure is an effective cell culture substrate and does not require the use of irregular non-woven substrates in existing solutions. This can simplify the design and construction of the cell culture system while providing a high-density cell culture substrate with other advantages discussed herein regarding flow uniformity, harvestability, etc.

[0062] As discussed herein, the cell culture substrate and bioreactor system have numerous advantages. For example, the embodiments of the present disclosure can support the production of any one of a variety of viral vectors such as AAV (all serotypes) and lentivirus, and can be used for in vivo and ex vivo gene therapy applications. Uniform cell seeding and distribution maximize the viral vector yield per container, and these designs can also harvest live cells, which is very useful for seed expansion cultures consisting of multiple amplification stages using the same platform. In addition, the embodiments herein can be scaled from process development scale to production scale, ultimately saving 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 with other cell culture solutions, the number of containers required to reach the viral vector production scale (e.g., 10 16 to 10 18 AAV VGs per batch) can be significantly reduced.

[0063] The embodiments are not limited to the container rotating about a central longitudinal axis. For example, the container can rotate about an axis that is not in a central position relative to the container. Additionally, the axis of rotation can be a horizontal axis or a vertical axis.

[0064] Exemplary Embodiments

[0065] The following is a description of various aspects of the embodiments 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 regarded as a comprehensive or exhaustive description of all possible embodiments.

[0066] Aspect 1 relates to a method for in-situ coating a cell culture substrate in a bioreactor, the method comprising: providing a bioreactor vessel comprising: a cell culture chamber within the bioreactor vessel, the cell culture chamber including an inlet for allowing fluid to flow into the cell culture chamber and an outlet for allowing fluid to flow out of the cell culture chamber; and a cell substrate disposed in the cell culture chamber and configured for culturing cells on the cell substrate; providing a coating solution for coating the cell substrate; introducing the coating solution into the cell culture chamber through the inlet so that the coating solution contacts the cell substrate, thereby coating the cell substrate; and removing excess coating solution from the cell culture chamber via the outlet or the inlet, wherein after removing the coating solution, the coated cell substrate remains in the cell culture chamber.

[0067] Aspect 2 relates to the method according to Aspect 1, the method further comprising incubating the coating solution in the cell culture chamber before removing the coating solution.

[0068] Aspect 3 relates to the method according to Aspect 1 or 2, the method further comprising washing the cell culture chamber with a washing solution during or after removing the coating solution.

[0069] Aspect 4 relates to the method according to Aspects 1 to 3, wherein providing the coating solution includes preparing a coating solution suitable for a specific cell culture application.

[0070] Aspect 5 relates to the method according to Aspects 1 to 4, wherein the coating solution includes materials for enhancing cell attachment and / or growth on the cell substrate.

[0071] Aspect 6 relates to the method according to Aspects 1 to 6, wherein the coating solution includes at least one of the following: extracellular matrix proteins, fibronectin, collagen, hydrogel solutions, polymer solutions, and recombinant proteins.

[0072] Aspect 7 relates to the method according to Aspects 1 to 6, wherein introducing the coating solution includes perfusing the coating solution through the bioreactor such that the coating solution flows into the cell culture chamber via the inlet, flows over the cell substrate, and exits through the outlet.

[0073] Aspect 8 relates to the method according to Aspects 1 to 7, the method further comprising controlling the flow rate of the coating solution through the bioreactor to promote coating adhesion and / or uniformity of the cell substrate.

[0074] Aspect 9 relates to the method according to Aspect 8, wherein controlling the flow rate includes pulse delivering the coating solution or reversing the flow direction of the coating solution.

[0075] Aspect 10 relates to the method according to Aspects 1 to 9, wherein the cell substrate 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 and passing through the thickness of the cell substrate.

[0076] Aspect 11 relates to the method according to Aspects 1 to 10, wherein the cell substrate includes at least one of the following: a molded polymer mesh sheet, a 3D printed mesh sheet, and a woven mesh sheet.

[0077] Aspect 12 relates to the method according to Aspects 1 to 11, wherein the cell substrate includes a polymer material.

[0078] Aspect 13 relates to the method according to Aspect 12, wherein the polymer material is at least one of the following: polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0079] Aspect 14 relates to the method according to Aspects 1 to 13, wherein the bioreactor is configured to provide a uniform fluid flow through the cell substrate and / or the cell culture chamber.

[0080] Aspect 15 relates to a method for culturing cells in a bioreactor, the method comprising: coating a cell substrate within the bioreactor according to any one of Aspects 1 to 14; inoculating cells onto the coated cell substrate; culturing the cells on the coated cell substrate; and harvesting the product of the cell culture.

[0081] Aspect 16 relates to the method according to Aspect 15, wherein the coated cell substrate includes a uniform structure configured to allow at least one of a cell culture medium, cells, or cell products to flow through the cell culture medium bottom.

[0082] Aspect 17 relates to the method according to Aspect 15 or Aspect 16, wherein the inoculation includes attaching the cells to the coated cell substrate.

[0083] Aspect 18 relates to the method according to Aspects 15 to 17, wherein the inoculation includes directly injecting a cell inoculum into the cell culture chamber provided with the coated cell substrate.

[0084] Aspect 19 relates to the method according to Aspect 18, wherein the cell inoculum is injected through an inlet of the bioreactor or through a cell inoculum injection port in the bioreactor vessel.

[0085] Aspect 20 relates to the method as described in aspect 18 or the claims, the method further comprising, after injecting the cell inoculum, perfusing cell culture medium through the culture chamber.

[0086] Aspect 21 relates to a system for culturing adherent cells in a bioreactor, the system comprising: a bioreactor vessel, the bioreactor vessel comprising: a cell culture chamber within the bioreactor vessel, the cell culture chamber comprising an inlet for allowing fluid to flow into the cell culture chamber and an outlet for allowing fluid to flow out of the cell culture chamber, and a cell substrate, the cell substrate being disposed in the cell culture chamber and configured for culturing cells on the cell substrate; a recirculation loop configured to supply fluid into the bioreactor vessel via the inlet and remove the fluid from the bioreactor vessel via the outlet; and a coating solution container fluidly connected to the cell culture chamber and configured for holding a substrate coating solution.

[0087] Aspect 22 relates to the system as described in aspect 21, the system further comprising one or more pumps for circulating at least one of the fluid flowing through the recirculation loop and the substrate coating solution into the cell culture chamber.

[0088] Aspect 23 relates to the system as described in aspect 21 or 22, the system further comprising a controller for controlling the flow of the fluid or the cell coating solution.

[0089] Aspect 24 relates to the system as described in aspect 23, wherein controlling the flow comprises controlling the flow rate or the flow direction.

[0090] Aspect 25 relates to the system as described in aspect 23 or 24, wherein the controller is configured to control the one or more pumps.

[0091] Aspect 26 relates to the system as described in aspects 23 to 25, wherein the controller comprises a processor and a memory, the memory containing instructions and communicating with the processor and being readable by the processor.

[0092] Aspect 27 relates to the system as described in aspect 26, wherein when the processor executes the instructions, the controller receives a signal to coat the cell substrate in the cell culture chamber with the cell coating solution.

[0093] Definitions

[0094] "Fully synthetic" or "completely synthetic" refers to a cell culture that is composed entirely of synthetic source materials and does not contain any animal-derived or animal-sourced materials, such as microcarriers or the surface of a culture vessel. The disclosed fully synthetic cell culture eliminates the risk of xenocontamination.

[0095] The terms "include", "includes", or similar terms mean including but not limited to, i.e., inclusive rather than exclusive.

[0096] "User" refers to a person who uses the systems, methods, articles, or kits disclosed herein, and includes a person who cultures cells to harvest cells or cell products, or who uses cells or cell products cultured and / or harvested according to the examples herein.

[0097] When describing embodiments of the present disclosure, the use of "about" to modify, for example, amounts, concentrations, volumes, processing temperatures, processing times, yields, flow rates, pressures, viscosities, and similar values and ranges thereof of components in a composition, or dimensions and similar values and ranges thereof of components, means that variations in the numerical amounts may occur, for example, due to the following reasons: typical measurement and processing procedures for preparing materials, compositions, composites, concentrates, components, articles, or using formulations; inadvertent errors in these procedures; differences in the manufacture, source, or purity of starting materials or components used to implement the methods; and similar considerations. The term "about" also encompasses 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 mixing or processing a composition or formulation having a particular initial concentration or mixture.

[0098] The term "optional" or "optionally" means that the subsequent described event or circumstance may or may not occur, and the description includes instances where the event or circumstance occurs and instances where the event or circumstance does not occur.

[0099] Unless otherwise specified, the indefinite article "a" or "an" and its corresponding definite article "the" as used herein mean at least one, or one or more.

[0100] Abbreviations well known to those of ordinary skill in the art may be used (e.g., "h" or "hrs" for hour, "g" or "gm" for gram, "mL" for milliliter, and "rt" for room temperature, "nm" for nanometer, and similar abbreviations).

[0101] The specific and preferred values and ranges thereof disclosed with respect to components, ingredients, additives, dimensions, conditions, and similar aspects are for illustration only; they do not exclude other defined values or other values within the defined ranges. The systems, kits, and methods of the present disclosure may include any value or any combination of the values, specific values, more specific values, and preferred values described herein, including intermediate values and ranges, whether explicit or implicit.

[0102] Unless explicitly stated otherwise, it is not intended that any method described herein be construed as requiring that its steps be performed in a specific order. Therefore, if a method claim does not actually recite the order in which its steps are to be followed or if the steps are not otherwise specifically stated in the claims or specification to be limited to a specific order, no specific order is intended to be implied.

[0103] It is obvious to those skilled in the art that various modifications and variations may be made without departing from the scope or spirit of the disclosed embodiments. Since modifications, combinations, sub-combinations and variations of the disclosed embodiments incorporating the spirit and purpose of the embodiments may be conceived by those skilled in the art, the disclosed embodiments should be interpreted as including all contents within the scope of the appended claims and their equivalents.

Claims

1. A method for in-situ coating a cell culture substrate in a bioreactor, the method comprising: Providing a bioreactor vessel, the bioreactor vessel comprising: A cell culture chamber within the bioreactor vessel, the cell culture chamber including an inlet for allowing fluid to flow into the cell culture chamber and an outlet for allowing fluid to flow out of the cell culture chamber, and A cell substrate, the cell substrate being disposed in the cell culture chamber and configured for culturing cells on the cell substrate; Providing a coating solution for coating the cell substrate; Inputting the coating solution into the cell culture chamber through the inlet so that the coating solution contacts the cell substrate, thereby coating the cell substrate; and Removing excess coating solution from the cell culture chamber via the outlet or the inlet, Wherein, after removing the coating solution, the coated cell substrate remains in the cell culture chamber.

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

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

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

5. The method according to any one of claims 1 to 4, wherein the coating solution includes materials for enhancing cell attachment and / or growth on the cell substrate.

6. The method according to any one of claims 1 to 5, wherein the coating solution includes at least one of the following: extracellular matrix proteins, fibronectin, collagen, hydrogel solutions, polymer solutions, and recombinant proteins.

7. The method according to any one of claims 1 to 6, wherein inputting the coating solution includes perfusing the coating solution through the bioreactor such that the coating solution flows into the cell culture chamber via the inlet, flows over the cell substrate, and flows out through the outlet.

8. The method according to any one of claims 1 to 7, the method 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 according to claim 8, wherein controlling the flow rate includes pulse delivering the coating solution or reversing the flow direction of the coating solution.

10. The method according to any one of claims 1 to 9, wherein the cell substrate 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 cell substrate and passing through the thickness of the cell substrate.

11. The method according to any one of claims 1 to 10, wherein the cell substrate includes at least one of the following: a molded polymer mesh sheet, a 3D printed mesh sheet, and a woven mesh sheet.

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

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

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

15. A method for culturing cells in a bioreactor, the method comprising: coating a cell substrate within the 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 harvesting the product of the cell culture.

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

17. The method according to claim 15 or claim 16, wherein the seeding comprises attaching the cells to the coated cell substrate.

18. The method according to any one of claims 15 to 17, wherein the seeding comprises directly injecting a cell inoculum into the cell culture chamber provided with the coated cell substrate.

19. The method according to claim 18, wherein the cell inoculum is injected into the bioreactor vessel through an inlet of the bioreactor or through a cell inoculum injection port.

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

21. 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 comprising an inlet for allowing fluid to flow into the cell culture chamber and an outlet for allowing fluid to flow out of the cell culture chamber, and a cell substrate, which is disposed in the cell culture chamber and is configured for culturing cells on the cell substrate; 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, which is in fluid connection with the cell culture chamber and is configured for containing a substrate coating solution.

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

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

24. The system according to claim 23, wherein said controlling the flow comprises controlling the flow rate or the flow direction.

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

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

27. The system according to claim 26, wherein, When said processor executes said instructions, said controller receives a signal to coat the cell substrate in said cell culture chamber with said cell coating solution.

Images