Systems and methods for differentiating stem cells within bioreactor

Abstract

The invention provides a method for in-situ differentiation of stem cells in a cell culture bioreactor using the bioreactor. The method includes providing a bioreactor vessel having a cell culture chamber with an inlet for flowing a fluid into the cell culture chamber and an outlet for flowing a fluid out of the cell culture chamber. The bioreactor vessel also includes a cell substrate disposed in the cell culture chamber for culturing cells on the cell substrate. The method comprises inoculating undifferentiated stem cells onto the cell substrate in the cell culture chamber, and perfusing the cell culture chamber with a differentiation medium to promote differentiation of the undifferentiated stem cells into specific cell lineages, thereby changing the undifferentiated stem cells into differentiated cells.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 425,339, filed on November 15, 2022, the content of which is hereby incorporated by reference in its entirety. Technical Field

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

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

[0005] Most cells used in bioprocessing are adherent - dependent, meaning that the cells need to adhere 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 the cells used for viral - vector production are mainly 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 also dominates the scaling - 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 adherent - dependent by nature. 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 these materials is polyethylene terephthalate (PET), which is mainly used in the production of viral vectors and vaccines, 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 that can support the culture of a wider range of cell types.

[0007] Mammalian cells are used for the production of 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 lost or damaged cells 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 production, ultimately leading to potential curative treatments.

[0008] Stem cells have broad prospects in cell therapy, tissue engineering and regenerative medicine, as well as pharmaceutical and biotechnological applications. However, cells used for viral vector production are mainly adherent-dependent; similarly, stem cells such as induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) are also adherent-dependent in nature. 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 numbers of hPSCs. This necessitates technologies capable of efficiently scaling up and expanding hPSCs. Additionally, for cell therapy and regenerative medicine applications, it is necessary to produce large numbers of cells differentiated into specific lineages. This requires technologies that can support the efficient and effective differentiation of hPSCs.

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

[0010] There is a need for a cell culture bioreactor system and methods of using such systems that not only allow the culture of terminally differentiated somatic cells but also the differentiation of hPSCs in a bioreactor. SUMMARY OF THE INVENTION

[0011] According to an embodiment, a method for differentiating cells in a bioreactor system is provided. The method includes: inoculating stem cells in a bioreactor having a cell culture chamber containing a cell substrate; and culturing the stem cells using a cell culture medium that supports the culture of undifferentiated stem cells. The method further includes perfusing a differentiation medium into the cell culture chamber to promote the differentiation of the stem cells into one or more lineages. The method further includes washing the cells with a washing solution after the differentiation is complete. The washing solution can be a fluid medium and / or a phosphate buffered solution (PBS). The method can further include additional culturing of the differentiated cells in the bioreactor. Optionally, the method further includes harvesting the differentiated cells or cell by-products. Harvesting can include perfusing a dissociation reagent into the cell culture chamber to release and remove the differentiated cells from the bioreactor. Removing the differentiated cells from the bioreactor can further include pressurizing the bioreactor to force the differentiated cells and any medium out of the bioreactor.

[0012] 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 on the cell substrate. The method includes: providing a coating solution for coating the cell substrate; inputting 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.

[0013] According to an embodiment, a method for culturing cells in a bioreactor is provided. The method includes coating a cell substrate within the bioreactor described herein; inoculating cells on the coated cell substrate; culturing the cells on the coated cell substrate; and harvesting the cell culture product.

[0014] 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 within the bioreactor vessel. 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 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 containing a substrate coating solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 FIG. is a schematic diagram of a fixed bed bioreactor system for coating a cell substrate in a bioreactor according to an embodiment.

[0016] Figure 2 FIG. is of the bioreactor system during the step of coating the cell substrate according to an embodiment Figure 1 FIG.

[0017] Figure 3 FIG. is after coating the cell substrate according to an embodiment Figure 1 and 2 FIG.

[0018] Figure 4 FIG. is a schematic diagram of a cell culture system according to one or more embodiments.

[0019] Figure 5 FIG. shows a process flow diagram for coating a cell substrate in a bioreactor for culturing cells according to one or more embodiments.

[0020] Figure 6 FIG. is a schematic diagram of a cell culture system for differentiating stem cells and culturing differentiated cells according to one or more embodiments.

[0021] Figure 7 FIG. shows a process flow diagram for inoculating, differentiating, and culturing stem cells in a bioreactor according to one or more embodiments. DETAILED DESCRIPTION

[0022] Various embodiments of the present disclosure will be described in detail with reference to the accompanying drawings (if any). The reference to various embodiments does 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 and merely illustrate some of the many possible embodiments of the claimed invention.

[0023] It may be necessary to modify the surface chemistry of the adherent cell culture substrate to provide the desired cell adhesion properties. Such modifications can be carried out by chemical treatment of the polymeric material of the substrate or by grafting cell adhesion molecules onto the substrate surface. Alternatively, the substrate can be coated with a thin layer of biocompatible hydrogel having cell adhesion properties, including, for example, collagen or A variety of coatings can be used, including, for example, extracellular matrix proteins, fibronectin, collagen, hydrogel solutions, polymer solutions, and recombinant proteins. As will be understood by those skilled in the art, any suitable coating can be used. Alternatively, the surface of the cell substrate can be made to have cell adhesion properties by treatment processes using various types of plasmas, process gases, and / or chemicals known in the industry. However, in one or more embodiments, the cell substrate is capable of providing an efficient cell growth surface without surface treatment. To simplify and provide flexibility in the manufacture of bioreactors, it is desirable to provide an uncoated cell culture bioreactor such that the end user can decide which type of coating to apply to the cell substrate in the bioreactor based on their cell type or application of interest. Thus, the embodiments provided herein conveniently allow the user to coat the substrate in situ inside the bioreactor. Accordingly, by providing a bioreactor pre-filled with a cell substrate and enabling the cell substrate to be appropriately coated, a flexible and easy-to-use solution is provided while minimizing the risk of contamination or assembly errors that may occur when the end user has to remove the substrate after coating and refill it into the bioreactor.

[0024] To provide additional flexibility to the user, embodiments of the present disclosure include systems and methods for in situ differentiation of stem cells in a bioreactor vessel. This allows the bioreactor system of the present disclosure to be used with undifferentiated cells (e.g., stem cells) that can be customized in the bioreactor itself according to the desired cell lineage and application.

[0025] 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 the cell substrate, it may be necessary to coat the cell substrate with a coating to improve performance for a particular cell type or application. For example, an adhesion-promoting compound can be applied to the cell growth surface to facilitate contact, such as adhesion and subsequent expansion, of cells such as human mesenchymal stem cells (hMSCs).

[0026] Embodiments of the present disclosure include a fixed-bed bioreactor system for cell culture. In accordance with aspects of such 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 a cell substrate within the bioreactor. The bioreactor system can include a fluid flow path that includes a path starting from an inlet of the bioreactor vessel, passing through a cell culture chamber containing the substrate, and exiting the bioreactor via an outlet. The fluid flow path can further include one or more medium conditioning vessels that are fluidly connected to the cell culture chamber and can be integrated with or separated 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 such that the coating solution enters the cell culture space. The system can include one or more pumps for circulating the medium through the fluid flow path and / or injecting the coating solution into the cell culture space.

[0027] In an embodiment, the system includes a controller for controlling the operation of the system including one or more pumps. The controller can include a computer system that includes a processor. In an embodiment, the controller is configured to control one or more pumps to circulate a fluid (e.g., cell culture medium or coating solution) through the fluid flow path at a certain flow rate. In accordance with aspects of the embodiments, the controller controls one or more pumps to deliver cell culture medium, nutrients, and / or cells from a source to the fluid flow path and into the bioreactor. The system can include a graphical user interface and a memory that communicates with and is readable by the processor and includes instructions. When the processor executes the instructions, the processor receives instructions such as to coat a cell substrate in the bioreactor. In response to an instruction to coat the bioreactor, the processor can perform a series of steps to coat the bioreactor and then can receive instructions such as to load cells into the bioreactor. In response to an instruction to load cells, the processor can perform a series of steps to load the cells from, for example, a cell inlet source into the bioreactor.

[0028] Figure 1FIG. 0 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 includes 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. As described herein, the fixed bed 106 is made of a cell substrate 108. According to an embodiment of the aspects, the cell substrate 108 is a porous material having a predetermined structure, such as an ordered array of openings 109 or pores and rigid filaments. The structure and arrangement of the cell substrate 108 and the fixed bed 106 enable the fixed bed 106 to allow fluid to flow uniformly therethrough, which improves 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 uniformly distributing the 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 allowing fluid to enter and exit the cell culture space 104, respectively. The inlet 110 and the outlet 112 are connected to fluid flow paths 118a and 118b, respectively, for providing fluid flow passages into and out of the cell culture vessel 102. The fluid flow paths 118a, 118b may form a perfusion flow path to recycle the fluid through the bioreactor in a circulating manner, as further discussed below.

[0029] According to an embodiment, the cell culture system 100 includes a coating solution container 120 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 other than the fluid flow paths 118a, 118b. As Figure 2 shown, the cell culture system 100 can supply 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 can be brought into contact with the cell substrate 108 for a predetermined time or until the coating of 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 can be removed from the cell culture space 104, as Figure 3As shown, a coated cell substrate made of the cell substrate 108 and the coating 123 thereon is left. While removing the coating solution 122 from the cell culture space, it can be washed with a washing solution (not shown), and the washing can be carried out while removing the excess coating solution or at some time after the removal is completed. Thus, a bioreactor having a cell substrate suitable for a desired cell culture application is provided.

[0030] Figure 4 A cell culture system 400 according to one or more embodiments is shown. The system 400 includes a bioreactor 402 that houses a fixed-bed cell substrate of one or more embodiments disclosed herein. The bioreactor 402 can be fluidly connected to a culture medium conditioning container 404, and the system is capable of supplying the cell culture medium 406 within the conditioning container 404 to the bioreactor 402. The culture medium conditioning container 404 can include sensors and control components that are present in typical bioreactors in the bioprocessing industry for suspension batch, fed-batch, or perfusion culture. These sensors and control 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 can be controlled by a gas flow controller for N2, O2, and CO2 gases. The culture medium conditioning container 404 also includes an impeller for culture medium mixing. All culture medium parameters measured by the above sensors can 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 it to a desired level. As Figure 4 shown, the culture medium conditioning container 404 is provided as a container separate from the bioreactor container 402. This can have the advantage that the culture medium can be conditioned in a place different from where the cells are cultured and then the conditioned culture medium can be supplied to the cell culture space. However, in some embodiments, the culture medium conditioning can be carried out in the bioreactor container 402.

[0031] The culture medium from the culture medium regulating container 404 is conveyed to the bioreactor 402 via the inlet 408, which may also include an injection port for the cell inoculum to inoculate and start culturing cells. 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 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 (e.g., an O2 sensor) and, based on the signals, adjust the flow rate into the bioreactor 402 by sending signals to a pump 416 (e.g., a peristaltic pump) upstream of the inlet 408 of the bioreactor 402. Thus, based on one or a combination of factors measured by the sensors 412, the pump 416 can control the flow rate into the bioreactor 402 to obtain the desired cell culture conditions.

[0032] The culture medium perfusion rate is controlled by the signal processing unit 414, which collects and compares sensor signals from the culture medium regulating 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 devices and system components can be found in U.S. Patent Application Publication No. US2020 / 0248124A1, which is incorporated herein by reference.

[0033] Cell culture system 400 further includes a coating solution container 420, which is fluidly connected to the bioreactor container 402 and is 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. The pump 422 can supply the coating solution directly to the bioreactor container 402 or to a fluid flow path connected to the bioreactor container 402. When it is necessary 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 brought into contact with the cell substrate for a predetermined time or until the coating is completed on the cell substrate (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 washing solution to the reactor 402 to help remove the excess coating solution. The washing solution can contain a culture medium or a fluid such as phosphate buffered saline (PBS) or other cell culture media.

[0034] 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 has a high surface area to volume ratio, so the cell density can be higher than in other systems. However, the packed bed generally acts as a depth filter, where cells are physically trapped or entangled on the fibers of the substrate. Thus, since the cell inoculum flows linearly through the packed bed, the distribution of cells inside the packed bed is non-uniform, resulting in a variation in cell density along the depth or width of the packed bed. For example, the cell density at the inlet region of the bioreactor may be higher, and the cell density closer to the outlet of the bioreactor is significantly lower. 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 a reduction in cell growth or virus vector production efficiency per unit surface area or volume of the packed bed.

[0035] Another problem encountered by packed bed bioreactors disclosed in the prior art is the channeling effect. Due to the randomness of the packed non-woven fibers, the local fiber density across any given cross-section of the packed bed is non-uniform. The culture medium flows faster in regions of low fiber density (high bed permeability) and slower in regions of high fiber density (low bed permeability). The resulting non-uniform culture medium perfusion across the packed bed creates a channeling effect, manifested as significant nutrient and metabolite gradients, which can negatively impact overall cell culture and bioreactor performance. Cells located in low culture medium perfusion regions starve and often die from lack of nutrients or metabolite poisoning. Cell harvesting is another problem encountered when using bioreactors packed with non-woven fiber scaffolds. Since the packed bed acts as a depth filter, cells released at the end of the cell culture process are trapped inside the packed bed and the cell recovery rate is very low. This severely limits the application of such bioreactors in bioprocesses where live cells are the product. Thus, the non-uniformity results in different regions being subject to different flow and shear forces, effectively reducing the available cell culture area, causing uneven culturing, and interfering with transfection efficiency and cell release.

[0036] To address these and other problems of existing cell culture protocols, embodiments of the present disclosure provide cell growth substrates, matrices of such substrates, and / or packed bed systems using such substrates, capable of performing highly efficient and high-yield cell culture of adherent-dependent cells and producing cell products (e.g., proteins, antibodies, virus particles). Embodiments include porous cell culture substrates made of an ordered and regular array of porous substrate materials, capable of enabling uniform cell seeding and culture medium / nutrient perfusion, as well as efficient cell harvesting. Embodiments are also capable of enabling scalable cell culture protocols, where the substrate and bioreactor can seed and grow cells and / or harvest cell products, from process development scale to full production scale, without sacrificing the uniform performance of the embodiments. For example, in some embodiments, the bioreactor can be easily scaled up from process development scale to product scale, with equivalent 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 trains to grow cell populations at multiple scales on the same cell substrate. Additionally, the embodiments herein provide a cell culture substrate with a high surface area, which in combination with the other features described, enables a high-yield cell culture protocol. In some embodiments, for example, the cell culture substrate and / or bioreactor discussed herein can produce 10 16 to 10 18 viral genomes (VG) per batch.

[0037] In one embodiment, the fixed bed substrate has a structurally defined surface area for adherent cell attachment and proliferation, which 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 the substrate to support adherent cell production. The cell substrates disclosed herein support the attachment and proliferation of adherent-dependent cells in a high volume density form. Such substrates can achieve uniform cell seeding and can also efficiently harvest cells or other products of the bioreactor. Additionally, the embodiments of the present disclosure support cell culture to provide a uniform cell distribution during the seeding step and to achieve confluent monolayer or multilayer of adherent cells on the disclosed substrates, and can avoid the formation of large and / or uncontrollable 3D cell aggregates with limited nutrient diffusion and increased metabolite concentration. Thus, the substrate eliminates diffusion limitations during the operation of the bioreactor. Additionally, the substrate enables easy and efficient harvesting of cells from the bioreactor. The structurally defined substrate of one or more embodiments enables complete cell recovery and continuous harvesting of cells from the fixed bed of the bioreactor.

[0038] According to some embodiments, there is also provided a method for cell culture using a bioreactor having a substrate for bioprocessing to produce therapeutic proteins, antibodies, viral vaccines or viral vectors.

[0039] Compared to existing cell culture substrates used in cell culture bioreactors (i.e., non-woven substrates with randomly arranged fibers), embodiments of the present disclosure include cell culture substrates having 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 for uniform flow through a fixed bed. This structure 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 sheet-like structure, where a first side and a second side of the sheet-like structure are 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 side and the second side of the substrate. Additionally, a plurality of pores or openings are formed along the thickness of the substrate. The size and geometry of the substrate material between the openings allow cells to adhere to the surface of the substrate material as if it approximated a two-dimensional (2D) surface, while also allowing sufficient fluid to flow around and through the openings in the substrate material. In some embodiments, the substrate is a polymer-based material and can be formed as a molded polymer sheet; a polymer sheet having openings punched along the thickness; multiple filaments fused into a mesh layer; a 3D printed substrate; or multiple filaments woven into a mesh layer. The physical structure of the substrate has a high surface-to-volume ratio for culturing adherent-dependent cells. According to various embodiments, the substrate can be arranged or packed in a bioreactor in certain ways as discussed herein to achieve uniform cell seeding and growth, uniform medium perfusion, and efficient cell harvesting.

[0040] Embodiments of the present disclosure can enable a full-scale virus vector platform that 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 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 18 to about 1019 a viral genome, or about 10 18 or more viral genomes per batch.

[0041] In addition, the embodiments disclosed herein can not only enable cells to attach and grow to the bottom of the cell culture medium, but also harvest live cultured cells. The inability to harvest live cells is a significant drawback of current platforms, and it makes it difficult to construct and maintain a sufficient number of cells to meet production capacity. According to aspects of the embodiments of the present disclosure, live cells can be harvested from the bottom of the cell culture medium, including live cells between 80% and 100%, or about 85% to about 99% live cells, or about 90% to about 99% live cells. For example, among 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 bottom of the cell culture medium using, for example, trypsin, TrypLE, or Accutase.

[0042] According to an embodiment, the bottom of the cell culture medium 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, 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.). The woven mesh can be characterized as a two-dimensional sheet or layer at the macroscopic scale. However, upon closer inspection of the woven mesh, it is found that due to the undulations of the cross fibers of the mesh, the mesh has a three-dimensional structure. Without being bound by theory, it is believed that the three-dimensional structure of the substrate is advantageous because it provides a large surface area for culturing adherent cells, and the structural rigidity of the mesh can provide a consistent and predictable structure for the bottom of the cell culture medium, which enables uniform fluid flow.

[0043] In one or more embodiments, the diameter of the fibers can be in the range of: 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; about 20 μm to about 170 μm; or about 150 μm to about 300 μm. At the microscale level, since the scale of the fibers is smaller than that of cells (e.g., the fiber diameter is larger than the cell), the surface of the monofilament fiber approximates a 2D surface for adherent cells to attach and proliferate. The fibers can be woven into a mesh with an opening range of about 10 μm × 10 μm to about 1000 μm × 1000 μm. In some embodiments, the diameter of the opening 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 sizes of the mesh according to all embodiments. The combination of fiber diameter and opening diameter is selected to achieve efficient and uniform fluid flow over the substrate when, for example, the cell culture substrate includes several adjacent mesh layers (e.g., a stack of individual layers or a rolled-up mesh layer).

[0044] 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 stack, roll, or other arrangement of overlapping substrates, 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 base layer). For example, if a stack of cell culture substrates has a certain height, it can be said that each layer of the stack has a packing thickness determined by dividing the total height of the stack by the number of layers in the stack. The packing thickness will vary based on fiber diameter and weave method, 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 the way they are arranged relative to each other. In a first arrangement, adjacent layers can fit closely together, but in a second arrangement, adjacent layers may not overlap, such as when the lowest point of the upper layer is in direct contact with the highest point of the lower layer. For certain applications, it may be desirable to provide cell culture substrates 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.

[0045] 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 a 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, reference to "surface area" refers to this effective surface area. According to one or more embodiments, the effective surface area of a single woven mesh base layer with a diameter of 6 cm 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 only as examples, and some embodiments can have different effective surface areas. The cell culture substrate can also be characterized by porosity, as discussed in the examples herein.

[0046] The substrate mesh can be made of monofilament or multifilament fibers of a polymer material compatible with 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 (e.g., plain weave, twill weave, Dutch weave, five-harness satin).

[0047] By using a structurally defined culture substrate with sufficient stiffness, high flow resistance uniformity across the entire 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 a fixed-bed configuration, allowing for complete harvest of cells at high viability at the end of the culture. The substrate also enables uniform packing of the fixed bed and allows for direct scale-up from a process development unit to a large-scale industrial bioprocessing unit. 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 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 a volume that is manageable at an industrial scale.

[0048] Embodiments of the present disclosure include a cell substrate that is 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 density or number of cells (or cell products). However, the embodiments are not limited to this configuration, and the cell substrate can take various configurations. For example, the cell substrate can be a roll of cell substrate material, or a small piece of substrate material fixed to a reactor.

[0049] The geometry of the reticulated substrate layer is designed to allow for 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 overall direction of fluid flow can be perpendicular to the major sides of the first and second substrate layers, or the cell substrate can be oriented relative to the flow such that the sides of the substrate layer are parallel to the overall flow direction. In addition to fluid flow perpendicular or parallel to the first and second sides of the reticulated layer, the substrate can also be arranged in multiple pieces at intermediate angles, or even randomly relative to the fluid flow. This directional flexibility is achieved through 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 create preferential flow channels and have substrate materials with anisotropic permeability. The flexibility of the cell substrates of the present disclosure allows them to be used in a variety of applications and bioreactor or vessel designs, while enabling better and more uniform permeability throughout the bioreactor vessel.

[0050] 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 it is contemplated that the substrate can be used in two-dimensional culture surface configurations, where one or more layers of the substrate are laid flat, such as 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.

[0051] 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. The culture chamber has a fixed-bed cell substrate made of a stack of cell substrate layers. The cell substrate layers are stacked such that the first or second side of a substrate layer faces the first or second side of an adjacent substrate layer. The bioreactor vessel 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 manner, the system can be easily scaled up without negatively impacting cell attachment and proliferation due to the well-defined structure and efficient fluid flow through the stacked substrate. 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 outlet to allow culture medium, cells, or other contents to flow into and out of the culture chamber. For example, the inlet can be used to allow culture medium or cells to flow into the culture chamber during cell seeding, perfusion, or culture phases, but can also be used to remove one or more of 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 function of those openings.

[0052] In one or more embodiments, the flow resistance and bulk density of a fixed bed can be controlled by staggering substrate layers of different geometries. Specifically, the pore size and geometry (e.g., fiber diameter, opening diameter, and / or opening geometry) define the fluid flow resistance of the fixed bed configuration. By staggering meshes of different sizes and geometries, the flow resistance of one or more specific portions of the bioreactor can be controlled or altered. This will result in more uniform liquid perfusion in the fixed bed. Various combinations of meshes of different sizes can achieve different distributions of the bulk density of the cell growth surface and flow resistance. For example, a fixed bed having regions of different volumetric cell densities (e.g., a series of regions producing low / high / low / high, etc. density patterns) can be assembled by staggering meshes of different sizes.

[0053] According to an embodiment, the overall flow direction of fluid through the bioreactor is in the direction from the inlet to the outlet, and, in aspects of the embodiment, the first and second major sides of the cell substrate layer are perpendicular to the overall flow direction. In contrast, aspects of some embodiments include a bioreactor vessel and a stack of cell substrates within the culture space having first and second sides 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 to provide a cell growth surface, thereby maximizing the efficiency of cells per unit volume. The cell culture space of the system can be supplied 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 the distribution of the culture medium, cells, or nutrients across the cross-section of the fixed bed, thereby improving the uniformity of fluid flow through the fixed bed. Thus, multiple inlets represent how a distribution plate can have multiple holes across the cross-section of the fixed bed to create a more uniform flow.

[0054] In some embodiments, the fixed bed is configured such that the cell substrate is formed as a cylindrical roll. For example, a sheet of cell substrate material (e.g., one or more mesh substrates) is rolled into a cylinder around 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 be within a bioreactor vessel such that the central longitudinal axis is parallel to the overall flow direction of fluid flowing through the bioreactor or culture chamber that houses the cylindrical roll. The bioreactor system may further comprise a central support member around which the cell substrate is positioned. According to some embodiments, the central support member may be used only for physical support and / or alignment of the cell substrate, but may also provide other functions. For example, the central support member may have one or more openings for supplying culture medium to the cell substrate along the length of the fixed bed. In other embodiments, the central support member may include one or more attachment sites for holding one or more portions of the cell culture substrate inside the cylindrical roll. These attachment sites may be hooks, clasps, posts, clips, or other devices for attaching the mesh to the central support member.

[0055] 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 increased flow rate to reduce non-uniform cell seeding caused by capturing cells on the conventional fixed bed substrate. In such conventional methods, pumping cells in the circulation loop at an increased flow rate 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 conventional packed bed bioreactors, 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.

[0056] In contrast, according to embodiments 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 due to gravity during the initial seeding stage, medium perfusion may be started immediately after the inoculum is injected. The perfusion flow rate is maintained below a preset 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.

[0057] The embodiments include a method for in-situ coating of a cell substrate within a bioreactor vessel. For example, Figure 4 system 400 can be operated in accordance with process steps according to one or more embodiments. As Figure 5 shown, method 500 can 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 injecting the coating solution into the cell culture space in S506, an incubation step (S507) can be performed, during which the coating solution remains in the cell culture space to coat the substrate. The incubation step S507 can 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 can be left standing in the cell culture space, or can be perfused through the cell culture space in a circulating form or in a single-pass perfusion through the reactor. The incubation period S507 can also include the reaction of the coating solution to form a coating on the cell substrate. Method 500 can further 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 can be performed after a predetermined period of time, which is considered sufficient to complete the coating according to process parameters. Optionally, a washing step S509 can be performed during or after the removal step S508. For example, the coating solution can be drained from the bioreactor by injecting a washing solution that flushes the bioreactor, or the washing solution can be introduced into the bioreactor after removing the excess coating solution. Next, a cell culture process can be performed, including inoculating and attaching the cells and the cell culture medium (S510), and then a culture process, which can include cell expansion, transfection, cell differentiation, production of viral vectors or other cell products, and harvesting.

[0058] Figure 6 shows a cell culture system 600 according to one or more embodiments. System 600 includes a bioreactor 602 that houses a fixed-bed cell substrate of one or more embodiments disclosed herein. Bioreactor 602 can be fluidly connected to a culture medium conditioning container 604, and the system is capable of supplying cell culture medium 606 within the conditioning container 604 to bioreactor 602. According to an embodiment, the culture medium 606 of the culture medium conditioning container can be a differentiation medium for supplying to undifferentiated cells at the start of the cell culture process. After differentiation, the culture medium 606 can be replaced with a culture medium suitable for culturing differentiated cells. Or, as Figure 6As shown, the system 600 can have two media conditioning containers 604 and 605: one for supplying the differentiation medium 607 and one for supplying the normal cell culture medium 606 used after differentiation. The media conditioning containers 604 and / or 605 can contain sensors and control components that are present in typical bioreactors used in the bioprocessing industry for suspension batch, fed-batch, or perfusion culture. These sensors and control components include but are not limited to DO oxygen sensors, pH sensors, oxygenators / gas bubbling units, temperature probes, and nutrient addition ports and base addition ports. The gas mixture supplied to the bubbling unit can be controlled by a gas flow controller for N2, O2, and CO2 gases. The media conditioning containers 604 and / or 605 also contain impellers for media mixing. All media parameters measured by the above sensors can be controlled by the media conditioning control unit 618, which communicates with the media conditioning containers 604 and / or 605 and is capable of measuring the conditions of the cell culture medium 606 and / or the differentiation medium 607 and adjusting them to the desired levels. As Figure 6 shown, the media conditioning containers 604 and 605 are provided as containers separate from the bioreactor container 602. This can have the advantage of being able to condition the media in a location different from where the cells are cultured and then supply the conditioned media to the cell culture space. However, in some embodiments, media conditioning can be performed in the bioreactor container 602.

[0059] The media 606 and 607 from the media conditioning containers 604 and 605 are delivered to the bioreactor 602 via the inlet 608, which can also include an injection port for the cell inoculum in order to inoculate and start culturing the cells. The bioreactor container 602 can also include one or more outlets 610 through which the cell culture media 606 and 607 leave the container 602. Additionally, cells or cell products can be output through the outlet 610. In order to analyze the contents flowing out of the bioreactor 602, one or more sensors 612 can be provided in the pipeline. In some embodiments, the system 600 includes a flow control unit 614 for controlling the flow rate into the bioreactor 602. For example, the flow control unit 614 can receive signals from one or more sensors 612 (e.g., an O2 sensor) and, based on the signals, adjust the flow rate into the bioreactor 602 by sending signals to a pump 616 (e.g., a peristaltic pump) upstream of the inlet 608 of the bioreactor 602. Thus, based on one or a combination of factors measured by the sensor 612, the pump 616 can control the flow rate into the bioreactor 602 to obtain the desired cell culture conditions.

[0060] The perfusion rate of the culture medium is controlled by a signal processing unit 614 that collects and compares sensor signals from a culture medium conditioning vessel 604 and sensors located at the outlet 610 of the fixed bed bioreactor. Due to the packed flow nature of the culture medium perfusion through the fixed bed bioreactor 602, nutrient, pH, and oxygen gradients form along the fixed bed. The perfusion flow rate of the bioreactor can be automatically controlled by a flow control unit 614 operably connected to a peristaltic pump 616. Examples of other control devices and system components can be found in U.S. Patent Application Publication No. US2020 / 0248124A1, which is incorporated herein by reference.

[0061] The cell culture system 600 also includes a coating solution container 620 that is fluidly connected to the bioreactor vessel 602 and is capable of supplying the coating solution contained therein to the bioreactor vessel 602. The coating solution is selected to enhance the performance of a given cell culture application. A pump 622 can supply the coating solution directly to the bioreactor vessel 602 or to a fluid flow path connected to the bioreactor vessel 602. When it is desired to supply the coating solution to the cell substrate in the bioreactor, the controller 614 can send a signal to activate the pump 622. After the coating solution fills the bioreactor system 602 and contacts the cell substrate, the coating solution can be brought into contact with the cell substrate for a predetermined time or until coating of the cell substrate is complete (e.g., by some physical or chemical reaction; by some external stimulus such as heating, cooling, or radiation). After coating is considered complete, the coating solution can be removed from the cell culture space. For example, the controller 614 can send a signal to the pump to remove excess coating solution and / or supply a wash solution to the reactor 602 to aid in removing the excess coating solution. The wash solution can contain a culture medium or a fluid such as phosphate buffered saline (PBS) or other cell culture media.

[0062] The system 600 further includes a cell inoculum 630. According to an embodiment, the cell inoculum includes undifferentiated stem cells that are injected into the bioreactor vessel 602 via an inlet 608. After injecting the undifferentiated cells, a differentiation medium 607 can be perfused into or through the bioreactor 602 to support the differentiation of the stem cells within the bioreactor into the desired differentiated cell line. After cell differentiation, a wash step can be performed using a wash solution. Finally, the differentiated cells can be further cultured using a cell culture medium 606.

[0063] An embodiment includes a method of in-situ differentiating stem cells within a bioreactor using a bioreactor system. For example, Figure 6 the system 600 in Figure 7As shown, method 700 may include providing a cell culture bioreactor (S702), inoculating undifferentiated stem cells onto or into a cell substrate within the cell culture space of the bioreactor (S704), and flowing a differentiation medium into the cell culture space of the bioreactor (S706). After injecting the differentiation medium into the cell culture space, the cells can differentiate into the desired cell line (S708). Optionally, a washing step S709 may be performed during or after the differentiation step S708. For example, the differentiation medium and any waste may be drained from the bioreactor by injecting a washing solution that flushes the bioreactor, or the washing solution may be introduced into the bioreactor after removing the excess differentiation medium and waste. Next, a cell culture process of the differentiated cells may be performed (S710). Finally, a harvesting step S712 may remove the differentiated cells and / or cell by-products for further downstream processing.

[0064] Depending on the desired system, the cell culture substrate may be arranged in the culture chamber in a variety of configurations. For example, in one or more embodiments, the system includes one or more layers of substrate that extend in width across the width of the cell culture space defined in the culture chamber. The multi-layer substrate may be stacked to a predetermined height in this manner. As described above, the substrate layer(s) 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 through the culture space defined in 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 that are in a first orientation relative to the overall flow and one or more other layers that are in a second orientation different from the first orientation. For example, the first and second sides of each layer may be parallel or perpendicular to the overall flow direction, or at an angle to the overall flow direction.

[0065] In one or more embodiments, the cell culture system includes a plurality of discrete cell culture substrate blocks in a packed bed configuration, where the length and / or width of the substrate blocks is small relative to the culture chamber. As used herein, the length and / or width of the substrate blocks is considered small relative to the culture chamber when the length and / or width of the substrate blocks is about 50% or less of the length and / or width of the culture space. Thus, the cell culture system may include a plurality of substrate blocks filled into the culture space in a desired arrangement. The arrangement of the substrate blocks may be random or semi-random, or may have a predetermined order or arrangement, such as the substrate blocks being oriented in a generally similar direction (e.g., horizontal, vertical, or at an angle of 0° to 90° relative to the overall flow direction).

[0066] As used herein, "defined culture space" refers to the space within the culture chamber occupied by the cell culture substrate, within which cell seeding and / or culturing is performed. The defined culture space may substantially fill the entire culture chamber, or may occupy a portion of the space within the culture chamber. As used herein, "overall flow direction" is defined as the direction of the overall mass flow of fluid or culture medium through or past the cell culture substrate during cell culturing and / or during the inflow or outflow of culture medium into or out of the culture chamber.

[0067] In one or more embodiments, the cell culture substrate is fixed within the culture chamber by a fixing mechanism. The fixing mechanism may fix a portion of the cell culture substrate to the culture chamber wall surrounding the substrate, or to the chamber wall at one end of the culture chamber. In some embodiments, the fixing mechanism adheres a portion of the cell culture substrate to a member passing through the culture chamber, such as a member parallel to the longitudinal axis of the culture chamber, or to a member perpendicular to the longitudinal axis. However, in one or more other embodiments, the cell culture substrate may be accommodated within the culture chamber but is not fixedly attached to the walls of the culture chamber or bioreactor vessel. For example, the substrate may be accommodated by the boundaries of the culture chamber or other structural members within the culture chamber such that the substrate is fixed within a predetermined area of the bioreactor vessel, rather than fixing the substrate to these boundaries or structural members.

[0068] One aspect of some embodiments provides a bioreactor vessel in a roller bottle configuration. The culture chamber is capable of accommodating a cell culture substrate and a substrate according to one or more embodiments described in the present disclosure. In the roller bottle configuration, the bioreactor vessel may be operably attached to means for moving the bioreactor vessel about the central longitudinal axis of the vessel. For example, the bioreactor vessel may rotate about the central longitudinal axis. The rotation may be continuous (e.g., continuously rotating in one direction), or discontinuous (e.g., intermittently rotating in a single direction or alternating directions, or oscillating in a back-and-forth rotational direction). In operation, the rotation of the bioreactor vessel causes cells and / or fluid to move within the chamber. This movement may be considered as movement relative to the chamber wall. For example, when the bioreactor vessel rotates about its central longitudinal axis, gravity may cause 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 may not be attached and may move freely relative to the vessel to the desired extent when the vessel rotates. Cells may adhere to the cell culture substrate, and the movement of the vessel allows the cells to come into contact with the cell culture medium or liquid, as well as oxygen or other gases within the culture chamber.

[0069] By using a cell culture substrate according to an embodiment of the present disclosure, such as a substrate including a woven or reticulated substrate, etc., a roller bottle container can be provided with a larger surface area for adherent cells to attach, proliferate, and function. Specifically, by using a woven reticulated substrate of a monofilament polymer material inside a roller bottle, the surface area can be increased by about 2.4 to about 4.8 times, or up to about 10 times, compared to a standard roller bottle. As described herein, each monofilament of the reticulated substrate can present a 2D surface for adherent cells to attach. Additionally, we can arrange multiple layers of mesh in the roller bottle, such that the total available surface area is increased to about 2 to 20 times that of a standard roller bottle. Thus, existing roller bottle facilities and processes, including cell seeding, medium exchange, and cell harvesting, can be modified by adding the improved cell culture substrate disclosed herein, with minimal impact on the existing operating infrastructure and processing steps.

[0070] The bioreactor container optionally includes one or more outlets capable of being attached to an inlet and / or outlet device. Through one or more outlets, liquids, media, or cells can be supplied to or removed from the chamber. A single port in the container can serve as both an inlet and an outlet simultaneously, or multiple ports can be provided as dedicated inlets and outlets.

[0071] The packed bed cell culture substrate in one or more embodiments can be composed of a woven cell culture reticulated substrate without the need to arrange or disperse any other form of cell culture substrate in the cell culture substrate. That is, the woven cell culture reticulated substrate of the embodiments of the present disclosure is an effective cell culture substrate without the need for the irregular non-woven substrate types used in existing solutions. This simplifies the design and construction of the cell culture system, while providing a high-density cell culture substrate and having other advantages related to flow uniformity, harvestability, etc., as discussed herein.

[0072] As described herein, the provided cell culture substrate and bioreactor system have numerous advantages. For example, embodiments of the present disclosure can support the production of any of a variety of viral vectors, such as AAV (all serotypes) and lentivirus, and can be applied to in vivo and in vitro gene therapy applications. Uniform cell seeding and distribution maximize the viral vector yield per container, and the design enables the harvesting of live cells, which can be very useful for seed cultures consisting of multiple amplification phases using the same platform. Additionally, the embodiments herein can be scaled up from the process development scale to the 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 to other cell culture protocols, reaching the viral vector production scale (e.g., 10 16 to 10 18The number of containers required for a given AAV VG can be significantly reduced.

[0073] 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.

[0074] Exemplary Embodiments

[0075] The following is a description of various aspects of the disclosed subject matter. Each aspect can 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.

[0076] Aspect 1 relates to a method of in situ differentiating stem cells in a cell culture bioreactor in the bioreactor, the method comprising: providing a bioreactor container comprising: a cell culture chamber within the bioreactor container, 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 disposed in the cell culture chamber and configured to culture cells thereon. The method further comprises inoculating undifferentiated stem cells onto the cell substrate in the cell culture chamber; and perfusing a differentiation medium into the cell culture chamber, the differentiation medium being configured to promote differentiation of the undifferentiated stem cells into a specific cell lineage, thereby converting the undifferentiated stem cells into differentiated cells.

[0077] Aspect 2 relates to the method according to aspect 1, further comprising, after perfusing the differentiation medium into the cell culture chamber, perfusing a dissociation reagent into the cell culture chamber, the dissociation reagent being configured to release the differentiated cells from the cell substrate.

[0078] Aspect 3 relates to the method according to aspect 2, further comprising a harvesting step, the harvesting step comprising removing the released differentiated cells from the cell culture chamber.

[0079] Aspect 4 relates to the method according to aspect 1, further comprising washing the cell culture chamber with a wash solution after perfusing the differentiation medium into the bioreactor.

[0080] Aspect 5 relates to the method according to aspect 4, wherein the wash solution comprises a fluid medium or a phosphate buffered solution (PBS).

[0081] Aspect 6 relates to the method according to aspect 2 or aspect 3, further comprising, after washing the cell culture chamber with a washing solution, perfusing a dissociation reagent into the cell culture chamber, the dissociation reagent being configured to release the differentiated cells from the cell substrate.

[0082] Aspect 7 relates to the method according to aspect 6, further comprising a harvesting step, the harvesting step comprising removing the released differentiated cells from the cell culture chamber.

[0083] Aspect 8 relates to the method according to aspects 1 to 7, further comprising: providing a coating solution for coating the cell substrate; and before inoculating undifferentiated stem cells, 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.

[0084] Aspect 9 relates to the method according to aspect 8, further comprising 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.

[0085] Aspect 10 relates to the method according to aspect 8 or aspect 9, further comprising washing the cell culture chamber with a washing solution during or after removing the coating solution.

[0086] Aspect 11 relates to the method according to aspects 8 to 10, wherein providing the coating solution comprises preparing a coating solution suitable for a specific cell culture application or a specific cell type.

[0087] Aspect 12 relates to the method according to aspects 8 to 11, wherein the coating solution comprises a material for enhancing cell attachment and / or growth on the cell substrate.

[0088] Aspect 13 relates to the method according to aspects 8 to 12, 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.

[0089] Aspect 14 relates to the method according to aspects 1 to 13, 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 the cell substrate and passing through the thickness of the cell substrate.

[0090] Aspect 15 relates to the method according to aspects 1 to 14, wherein the cell substrate comprises at least one of a molded polymer mesh sheet, a 3D printed mesh sheet, and a woven mesh sheet.

[0091] Aspect 16 relates to the method according to aspects 1 to 15, wherein the cell substrate comprises a polymeric material.

[0092] Aspect 17 relates to the method according to aspect 16, wherein the polymeric material is at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

[0093] Aspect 18 relates to the method according to aspects 1 to 17, wherein the bioreactor is configured to enable uniform fluid flow through the cell substrate and / or the cell culture chamber.

[0094] Aspect 19 relates to a system for culturing adherent cells in a bioreactor, the system comprising: a bioreactor vessel that includes a cell culture chamber and a cell substrate 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, the cell substrate being disposed in the cell culture chamber and configured to culture 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 differentiation medium container configured to supply a differentiation medium to the cell culture chamber to differentiate stem cells into a specific cell lineage, thereby converting the undifferentiated stem cells into differentiated cells.

[0095] Aspect 20 relates to the system according to aspect 19, wherein the differentiation medium container is fluidly connected to the cell culture chamber via the recirculation loop.

[0096] Aspect 21 relates to the system according to aspect 19 or aspect 20, further comprising a cell inoculum source for inputting undifferentiated cells into the cell culture chamber.

[0097] Aspect 22 relates to the system according to aspects 19 to 21, further comprising a coating solution container fluidly connected to the cell culture chamber and configured to hold a substrate coating solution.

[0098] Aspect 23 relates to the system according to aspects 19 to 22, further comprising one or more pumps for circulating at least one of a cell culture medium, a differentiation medium, a washing solution, a dissociation reagent, and a cell inoculum through the recirculation loop or into the cell culture chamber.

[0099] Aspect 24 relates to the system according to aspect 23, further comprising a controller for controlling the one or more pumps.

[0100] Aspect 25 relates to the system according to aspect 24, wherein controlling the one or more pumps includes controlling the flow rate or flow direction of the fluid in the bioreactor system.

[0101] Aspect 26 relates to the system according to aspect 24 or 25, wherein the controller includes a processor and a memory, the memory containing instructions and communicating with and readable by the processor.

[0102] Aspect 27 relates to the system according to aspect 26, wherein, when the processor executes the instructions, the controller receives signals for at least one of the following operations: injecting the undifferentiated cells into the cell culture chamber, injecting the differentiation medium into the cell culture chamber, injecting the washing solution into the cell culture chamber, injecting the coating solution into the cell culture chamber, injecting the cell culture medium into the cell culture chamber, and injecting the dissociation reagent into the cell culture chamber.

[0103] Definitions

[0104] "Fully synthetic" or "completely synthetic" refers to a cell culture article, such as the surface of a microcarrier or a culture vessel, which is composed entirely of synthetic source materials and does not contain any animal-derived or animal-sourced materials. The disclosed fully synthetic cell culture articles eliminate the risk of xenocontamination.

[0105] "Include", "includes" or similar terms mean to cover but not be limited to, i.e., inclusive rather than exclusive.

[0106] "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 a person who uses cells or cell products cultured and / or harvested according to the examples herein.

[0107] The use of "about" in describing embodiments of the present disclosure to modify, for example, amounts, concentrations, volumes, process temperatures, process 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, refers to, for example, variations in quantity that may occur due to the following reasons: typical measurement and processing procedures for preparing materials, compositions, complexes, concentrates, components, articles or 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 starting concentration or mixture.

[0108] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes the case where the event or circumstance occurs and the case where the event or circumstance does not occur.

[0109] 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.

[0110] 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).

[0111] The specific and preferred values and ranges thereof disclosed for components, ingredients, additives, dimensions, conditions and the like 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 described herein, or any combination of values, specific values, more specific values and preferred values, including intermediate values and ranges, whether explicit or implicit.

[0112] Unless otherwise expressly stated, no method set forth herein is intended to be construed as requiring that its steps be performed in a particular order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or where no particular order is otherwise specifically set forth in the claims or description, no particular order is intended to be inferred.

[0113] 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 those skilled in the art can conceive of modifications, combinations, sub - combinations and variations that are within the spirit and substance of the disclosed embodiments in combination, the disclosed embodiments should be construed to include all such within the scope of the appended claims and their equivalents.

Claims

1. A method for in-situ differentiating stem cells in a bioreactor using a cell culture bioreactor, the method comprising: Providing 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, which is disposed in the cell culture chamber and configured to culture cells thereon; Seeding undifferentiated stem cells onto the cell substrate in the cell culture chamber; and Perfusing a differentiation medium into the cell culture chamber, the differentiation medium being configured to promote the differentiation of the undifferentiated stem cells into a specific cell lineage, thereby causing the undifferentiated stem cells to become differentiated cells.

2. The method according to claim 1, further comprising, after perfusing the differentiation medium into the cell culture chamber, perfusing a dissociation reagent into the cell culture chamber, the dissociation reagent being configured to release the differentiated cells from the cell substrate.

3. The method according to claim 2, further comprising a harvesting step, the harvesting step comprising removing the released differentiated cells from the cell culture chamber.

4. The method according to claim 1, further comprising, after perfusing the differentiation medium into the bioreactor, washing the cell culture chamber with a washing solution.

5. The method according to claim 4, wherein the washing solution comprises a fluid medium or a phosphate buffered solution (PBS).

6. The method according to claim 2 or claim 3, further comprising, after washing the cell culture chamber with a washing solution, perfusing a dissociation reagent into the cell culture chamber, the dissociation reagent being configured to release the differentiated cells from the cell substrate.

7. The method according to claim 6, further comprising a harvesting step, the harvesting step comprising removing the released differentiated cells from the cell culture chamber.

8. The method according to any one of claims 1 to 7, further comprising: Providing a coating solution for coating the cell substrate; Before seeding the undifferentiated stem cells, 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.

9. The method according to claim 8, further comprising removing excess of the 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.

10. The method according to claim 8 or claim 9, further comprising, during or after removing the coating solution, washing the cell culture chamber with a washing solution.

11. The method according to any one of claims 8 to 10, wherein providing the coating solution comprises preparing a coating solution suitable for a specific cell culture application or a specific cell type.

12. The method according to any one of claims 8 to 11, wherein the coating solution comprises a material for enhancing cell attachment and / or growth on the cell substrate.

13. The method according to any one of claims 8 to 12, 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.

14. The method according to any one of claims 1 to 13, 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 the cell substrate and passing through the thickness of the cell substrate.

15. The method according to any one of claims 1 to 14, wherein the cell substrate comprises at least one of a molded polymer mesh sheet, a 3D printed mesh sheet, and a woven mesh sheet.

16. The method according to any one of claims 1 to 15, wherein the cell substrate comprises a polymeric material.

17. The method according to claim 16, wherein the polymeric material is at least one of polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinyl chloride, polyethylene oxide, polypyrrole, and polypropylene oxide.

18. The method according to any one of claims 1 to 17, wherein the bioreactor is configured to enable uniform fluid flow over the cell substrate and / or the cell culture chamber.

19. A system for culturing adherent cells in a bioreactor, the system comprising: A bioreactor vessel, which comprises: 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 to culture cells on the cell substrate; A recirculation loop, which is configured to supply fluid to the bioreactor vessel via the inlet and remove fluid from the bioreactor vessel via the outlet; and A differentiation medium container, which is configured to supply a differentiation medium to the cell culture chamber to differentiate stem cells into a specific cell lineage, thereby converting the undifferentiated stem cells into differentiated cells.

20. The system according to claim 19, wherein the differentiation medium container is fluidly connected to the cell culture chamber via the recirculation loop.

21. The system according to claim 19 or claim 20, further comprising a cell inoculum source for inputting undifferentiated cells into the cell culture chamber.

22. The system according to any one of claims 19 to 21, further comprising a coating solution container, the coating solution container being fluidly connected to the cell culture chamber and being configured to contain a substrate coating solution.

23. The system according to any one of claims 19 to 22, further comprising one or more pumps for circulating at least one of a cell culture medium, a differentiation medium, a washing solution, a dissociation reagent, and a cell inoculum through the recirculation loop or into the cell culture chamber.

24. The system according to claim 23, further comprising a controller for controlling the one or more pumps.

25. The system according to claim 24, wherein controlling the one or more pumps includes controlling the flow rate or flow direction of fluid in the bioreactor system.

26. The system according to claim 24 or claim 25, wherein the controller comprises a processor and a memory, the memory containing instructions and communicating with and readable by the processor.

27. The system according to claim 26, wherein, When the processor executes the instructions, the controller receives signals for at least one of the following operations: injecting the undifferentiated cells into the cell culture chamber, injecting the differentiation medium into the cell culture chamber, injecting the washing solution into the cell culture chamber, injecting the coating solution into the cell culture chamber, injecting the cell culture medium into the cell culture chamber, and injecting the dissociation reagent into the cell culture chamber.

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