Systems and methods for oxygenating media in perfusion bioreactors

Abstract

A cell culture system is provided comprising a cell culture vessel enclosing an interior configured to culture cells in a liquid cell culture medium and a culture medium conditioning system. The culture medium adjusting system comprises a culture medium adjusting container and an oxygenation tower, and the culture medium adjusting container is used for adjusting a liquid culture medium. The oxygenation tower includes a housing containing an interior space, an oxygen deficient medium inlet fluidly connected to the interior space, an excess gas vent fluidly connected to the interior space, and a lower opening fluidly connected to the interior space. The lower opening is disposed at a lower end of the oxygenation tower and in a fluid path between the interior space and the medium conditioning container. The oxygenation tower is designed to mix the oxygen deficient medium with a gas comprising oxygen in a countercurrent manner and thus achieve better dissolved oxygen saturation in the medium.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 428,975, filed on November 30, 2022, under 35 U.S.C.§119, the content of which is hereby incorporated by reference in its entirety and made a part hereof. Technical Field

[0003] The present disclosure generally relates to media conditioning in cell culture bioreactor systems, and more particularly, to media conditioning systems and methods for aerating cell culture media in perfusion bioreactors. Background Art

[0004] In the bioprocessing industry, large - scale cell culture is performed to produce hormones, enzymes, antibodies, vaccines, and cell therapies. The cell and gene therapy market is growing rapidly, and promising therapies are entering clinical trials and moving quickly towards commercialization. However, a single dose of cell therapy 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. The process efficiency of bioreactor cell culture should be high to achieve economically viable production. In the field of bioreactor design, the bioprocessing industry is moving towards high - density culture. For a given production capacity, high - density systems are more compact in size and more cost - effective.

[0005] However, there are limitations to increasing cell density and thus improving reactor performance. These limitations include operation, aeration, and media formulation. In a cell culture reactor, cells must grow under controlled conditions, which include being suspended or perfused in a cell culture medium, which is a liquid medium containing nutrients essential for cell life and growth. The inclusions of the cell culture medium must be controlled, including the pH and the content of dissolved gases (including, for example, dissolved oxygen), as well as the temperature of the medium and / or the cells, to optimize cell growth and bioreactor performance. Therefore, media conditioning systems are used in combination with or integrated into bioreactors to condition the media in the bioreactors. These media conditioning systems can, for example, control the temperature, pH, carbon dioxide content, dissolved oxygen content, and other aspects of the media. Depending on the cells being grown or the stage of the culture process, specific media conditioning requirements can vary. Generally, media conditioning can be performed in a hollow tank or vessel (e.g., a beaker or bottle) that has a complex probe system for controlling the composition of the media, one or more stirrers for mixing the media, and some type of temperature - control jacket around the vessel. The probes can enter the vessel through a lid on the vessel body, and sterility is always an issue, especially when the system is opened or opened during use.

[0006] The cell density in a reactor is typically determined by the oxygen delivered to the cells. The mass transfer rate from the gas to the culture medium should be balanced with the oxygen consumption of the cells. Currently, there are several aeration methods used in the bioprocessing industry. These aeration methods include surface aeration, membrane aeration, macro-sparging, and micro-sparging. Compared with other technologies, sparged cultures achieve higher oxygen transfer rates. However, the sparging rate is limited by cell lysis and foaming problems. One of the methods for further increasing the oxygenation rate is described in U.S. Patent No. 9,388,375, which proposes combining the gas blanket and gas sparging processes in the same reactor. U.S. Patent No. 9,512,392 also discloses a method for increasing the dissolved oxygen (DO) in the culture medium, which lays the foundation for designing and manufacturing an effective mammalian cell culture bioreactor. Historically, the bioprocessing industry was based on the design principles of traditional microbial fermenters, which relied heavily on stainless steel technology. Therefore, most of the sparging systems seen in stirred tank bioreactors are not suitable for mammalian cell culture. The systems rely on high-shear mixers to break the bubbles. However, mammalian cell culture requires gentle mixing and a lower gas shear rate, which requires different engineered sparging.

[0007] Expanding the capacity of biomanufacturing processes is crucial for process development and the production of biotherapeutics. The bioreactor process set points, acceptable ranges, and general operating parameters for large-scale use are typically based on the parameters developed at the lower-cost bench top or small scale. Scaling up bioreactor processes, especially oxygenation efficiency, is challenging. For example, it is difficult to maintain equivalent bioreactor characteristics, such as bubble size, distribution, residence time, and comparable bubble surface area. Therefore, mass transfer of the gas remains one of the key and difficult parameters for bioreactor control. Sufficient O2 or air delivery is required not only to support cell growth, metabolism, and biopharmaceutical production but also to control the accumulation of CO2 in the culture medium, which can adversely affect the performance endpoints.

[0008] Accordingly, there is a need for an improved culture medium regulation system that can meet the requirements of high-density cell culture in terms of gas-liquid mass transfer to the cell culture medium. SUMMARY OF THE INVENTION

[0009] According to an embodiment of the present disclosure, there is provided a cell culture system comprising a cell culture container and a culture medium regulating system. The cell culture container encloses an interior configured to culture cells in a liquid cell culture medium. The culture medium regulating system comprises a culture medium regulating container and an oxygenation tower. The culture medium regulating container is used to regulate the liquid culture medium. The oxygenation tower comprises a housing enclosing an interior space, a hypoxic culture medium inlet fluidly connected to the interior space, an excess gas outlet fluidly connected to the interior space, and a lower opening fluidly connected to the interior space. The lower opening is disposed at the lower end of the oxygenation tower and in a fluid path between the interior space and the culture medium regulating container. The oxygenation tower is designed to mix the hypoxic culture medium with a gas comprising oxygen in a countercurrent manner, and thus achieve a better dissolved oxygen saturation in the culture medium. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Shows a bioreactor system known in the art having a culture medium regulating container.

[0011] Figure 2 Schematic diagram showing a cell culture system having an oxygenation tower according to one or more embodiments of the present disclosure.

[0012] Figure 3 Enlarged schematic diagram showing an oxygenation tower according to one or more embodiments of the present disclosure.

[0013] Figure 4 Close-up schematic diagram showing the top section of an oxygenation tower according to an embodiment.

[0014] Figure 5A Shows a culture medium inlet tube from the top plate of an oxygenation tower according to one or more embodiments of the present disclosure.

[0015] Figure 5B Shows according to an embodiment Figure 5A Alternative view of the culture medium inlet tube.

[0016] Figure 6A Shows an example of multiple culture medium inlet tubes when filling the top plate with culture medium according to an embodiment.

[0017] Figure 6B Shows an example of multiple culture medium inlet tubes when filling the top plate with culture medium according to an embodiment. DETAILED DESCRIPTION

[0018] Various embodiments of the present disclosure will be described in detail with reference to the accompanying drawings, if any. References to various embodiments 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 and merely illustrate some of the many possible embodiments of the claimed invention.

[0019] Figure 1 A cell culture system 10 is shown, in which a conventional culture medium conditioning vessel 12 is attached to a bioreactor vessel 13. The culture medium 20 flows out of the culture medium conditioning vessel 12 via an outlet 14 to an inlet 15 of the bioreactor vessel 13. The culture medium 20 within the bioreactor vessel 13 is used to feed cells (not shown) within the bioreactor vessel 13 and can be perfused through it to an outlet 17. The culture medium 20 can then be recycled, for example, via an inlet 11 to the culture medium conditioning vessel 12. The culture medium conditioning vessel 12 can be a simple hollow tank or container (e.g., a glass or plastic beaker or bottle). A complex probe system 16 is used to control the composition of the culture medium 20, and one or more stirrers 18 are used to mix the culture medium 20. A temperature control jacket 19 (e.g., a water jacket) is shown surrounding the culture medium conditioning vessel 12, and the temperature control jacket can be used to control the temperature of the culture medium 20. The probe 16 can comprise a combination of sensors and feeding devices for various components (e.g., oxygen) and can access the culture medium conditioning vessel 12 through a lid on the vessel body. During operation of the cell culture system 10, as the culture medium 20 is perfused through the bioreactor vessel 13, the oxygen dissolved in the culture medium 20 is consumed by cells located in a fixed bed. The oxygen-depleted culture medium returns to the culture medium conditioning vessel 12, where the culture medium is re-oxygenated and excess CO2 is stripped from the culture medium by a gas transfer process on the surface of bubbles introduced through a gas sparger 17. The excess gas leaves the culture medium conditioning vessel through a sterile exhaust filter 21. In a high-intensity bioprocess, the oxygen consumption rate in the fixed bed reactor 13 exceeds the oxygenation rate in the culture medium conditioning vessel 12. As a result, the cells enter a state of oxygen starvation, thus reducing bioprocess efficiency and productivity. To overcome this limitation and enhance system performance, embodiments of the present disclosure provide an oxygenation system to significantly increase the culture medium oxygenation rate.

[0020] Embodiments of the present disclosure relate to a culture medium conditioning vessel and system, and a cell culture system incorporating a culture medium conditioning system. In particular, embodiments of the present disclosure provide systems and methods for improving gas-liquid mass transfer in a bioreactor. According to an embodiment, an oxygenation tower is provided, within which a cell culture medium is exposed to the surface of a packed bed material. The cell culture medium enters through the top of the oxygenation tower, and gas enters the tower via an inlet in the bottom portion of the oxygenation tower. The gas and liquid flow past each other within the oxygenation tower. The oxygenation tower can comprise a packed bed of material to increase the surface area of the liquid-gas interface and thus enhance gas-liquid mass transfer to re-oxygenate an oxygen-depleted culture medium and strip excess levels of CO2 from the culture medium.

[0021] Traditionally, one of the parameters that has been difficult to adjust in bioprocesses is medium aeration and CO2 stripping. These parameters depend on multiple primary bioreactor parameters such as mixing, gas flow rate, bubbling efficiency, bubble size, bubble residence time. According to the embodiments disclosed herein, the aeration module enables enhanced gas exchange and operates independently of the above parameters due to its ability to provide a constantly increasing liquid-gas interface surface area. The aeration tower allows the end user to significantly enhance the bioprocess in terms of the oxygen consumption of the cultured cells. This implementation results in a higher productivity compared to standard bioreactors that rely solely on gas bubbling.

[0022] In some embodiments, it is contemplated that the bioreactor is a fixed bed or packed bed bioreactor having a high-density scaffold for cell growth. The system also allows for temperature regulation of the medium and control of other aspects such as pH.

[0023] Figure 2 A schematic diagram of a cell culture system 100 according to some embodiments is shown, the cell culture system comprising a bioreactor vessel 103 for growing cells and / or viral vectors and a medium conditioning system 102 for conditioning the medium 200 supplied to the bioreactor vessel 103. As Figure 2 shown, the system 100 can be configured in a recirculation loop, where the medium 200 flows out of the medium conditioning system 102 via an outlet 104 and flows to the bioreactor vessel 103 via a pipe 140 or other fluid connector. Inside the bioreactor vessel 103, the medium supplies essential nutrients to the cells and maintains a healthy cell environment. The perfused medium 200 then returns to the medium conditioning vessel 102 via a return pipe 142 or other connector. The returned medium passes through the aeration tower 250 before or simultaneously with re-entering the medium conditioning vessel. Embodiments of the present disclosure include the entire cell culture system 100, as well as the individual medium conditioning system 102 and / or aeration tower 250.

[0024] Figure 3 An enlarged schematic diagram of an aeration tower 250 according to some embodiments is shown. The aeration tower 250 includes a housing 252 enclosing an internal space 254. Oxygen-depleted medium 256 enters the aeration tower 250 via a medium inlet 258, passes through the internal space 254, and exits the aeration tower 250 via a lower opening 260. Excess gas (e.g., air or oxygen) escaping from the medium conditioning vessel 102 enters the aeration tower 250 via the lower opening 260, flows upward through the aeration tower 250, and exits through a gas discharge port 262. Thus, countercurrent conditions exist within the internal space 254, where the returned medium flows downward through the aeration tower 250 while the gas flows upward through the aeration tower 250. According to equation (1), the operating principle of the aeration tower 250 is to increase the surface area of the liquid-gas interface to increase the gas transfer rate from the gas to the liquid (i.e., the medium):

[0025]

[0026] where C L is the oxygen concentration in the culture medium, C L * is the oxygen saturation concentration in the culture medium, t is time, k L is defined as the mass transfer coefficient, and a is the surface area of the culture medium - gas interface.

[0027] In aspects of the embodiments, the internal space 254 may contain materials for increasing the surface area of the culture medium - gas interface to improve gas transfer into the culture medium. For example, the oxygenation tower may be filled with a highly porous material 265. It is contemplated that this highly porous material may take various forms, and thus the embodiments are not limited to a particular porous material. The porous material may be a polymer, metal, ceramic, glass, or other suitable material compatible with bioprocessing applications. In some preferred embodiments, the porous material 265 comprises polyethylene terephthalate (PET). According to an example of an embodiment, the porous material may be in the form of PET sheets that are stacked (see Figure 3 ) or wound into rolls within the oxygenation tower 250. The PET sheets may be formed from a reticulated material having a defined ordered array of fibers and a plurality of openings between the ordered arrays. In an embodiment, the reticulate is a woven reticulate of one or more PET fibers, or is 3D printed, or is a punched or molded reticulated material.

[0028] The oxygenation tower may include a top plate 270, as Figure 3 shown. Figure 4 A magnified view showing an example of a top plate 370 according to some embodiments is shown. As Figure 4As shown, the culture medium inlet 358 feeds an oxygen-depleted culture medium into the aeration tower and to the top plate 370, while the gas discharge port 362 discharges excess gas from the top of the aeration tower. One or more gas outlets 364 inside the filled aeration tower allow excess gas from within the aeration tower to rise to the headspace 371 above the top plate 370. The culture medium 300 entering via the culture medium inlet 358 falls onto the top plate 370, where the culture medium 300 pools before rising to a level sufficient to enter one or more culture medium inlet openings 372. After rising to the level of the openings in the culture medium inlet openings 372, the culture medium 300 flows through the top plate 370 via the culture medium inlet openings 372 and into the interior of the packed bed of the aeration tower containing the porous material. A plurality of culture medium inlet openings 372 and gas outlets 364 may be provided to improve the uniformity of the culture medium entering the packed bed region and the gas leaving the packed bed region. The culture medium inlet openings 372 are configured to equalize the flow of the culture medium across the top plate 370 and distribute the flow of the culture medium evenly among all the culture medium inlet openings 372 in the top plate 370. For example, the culture medium inlet openings 372 may include culture medium inlet tubes 374, as Figure 5A and 5B shown, having inclined cutout flow openings in the walls of the culture medium inlet tubes 374. The flow openings become wider towards the top of the culture medium inlet tubes 374. Thus, the flow rate through each culture medium inlet tube 374 is proportional to the liquid level in the top plate, as Figure 6A and 6B shown. The horizontal placement of the top flow distribution plate will provide for self-regulating uniform flow distribution through the flow inlet tubes 374.

[0029] According to an embodiment, the aeration towers 250, 350 can be additional components that can be attached post-sale to the culture medium conditioning container. In other embodiments, the aeration tower can be integrated with the culture medium conditioning container or pre-installed on the culture medium conditioning container.

[0030] Optionally, the culture medium conditioning container 102 is temperature-controlled. That is, the temperature of the space inside the container 102 can be heated or cooled to control the temperature of the gas and / or culture medium entering or passing through the gas culture medium conditioning container 102 or the aeration tower 250. In some embodiments, the temperature control is used to heat or cool the gas, and then the gas heats or cools the culture medium inside the culture medium conditioning container 102 or the aeration tower. Optionally, this temperature control can be achieved by an integrated temperature control device 109 (such as a heater or cooler). Alternatively, the gas can be temperature-controlled before entering the enclosure.

[0031] The bioreactor system 100 may further include one or more sensors 106 for detecting the quality of the gas and / or culture medium. The sensors 106 can be disposed in the culture medium conditioning container 102, asFigure 2 shown. However, the sensor may also be supplied with the culture medium upstream or downstream of the culture medium inlet 101 and the culture medium outlet 104, or may be supplied in the bioreactor vessel itself, or may be supplied in the aeration towers 250, 350. Based on feedback from the sensor, the composition of the gas via the inlet and / or the temperature control device may be controlled to achieve the desired composition and temperature of the cell culture medium, thereby achieving optimal performance of the culture. For example, the gas composition may be adjusted to achieve the desired O2 and / or CO2 levels; and the pH may be adjusted.

[0032] The enclosure 109 of the culture medium conditioning container 102 may be a thermal enclosure. As used herein, "thermal enclosure" means that the enclosure is thermally insulated, and thus the temperature inside the thermal enclosure can be more easily controlled. As an alternative or supplement to the thermal enclosure, the enclosure 102 may include a heat source for controlling the temperature inside the enclosure or the temperature of the gas inside the enclosure, as discussed above. The heat source may be an integral structure and function of the enclosure 109, or the heat source may be a separate component that the enclosure 109 is configured to receive as needed. According to some embodiments, the heat source provides heat to the gas, the heat being sufficient to control the temperature of the culture medium in the culture medium exchange system within the desired range for cell culture.

[0033] In some embodiments, the culture medium conditioning system further includes one or more sensors for sensing the properties of the gas inside the enclosure, or the culture medium inside the aeration tower, or the culture medium before entering the bioreactor, after leaving the bioreactor, or inside the bioreactor. The one or more sensors may measure temperature, pH, oxygen (O2), CO2, or any one of a plurality of variables related to the cell culture operation being performed.

[0034] The culture medium conditioning systems and containers disclosed herein have many advantages. For example, the design allows the culture medium conditioning container to be a single-use or disposable container. The reusable or single-use components of the system can reduce operating costs while using the aeration tower to achieve gas delivery to the culture medium. The gas can also be temperature controlled in the system, which thus effectively controls the temperature of the culture medium. Additionally, due to the possible embodiments, the system can handle large volumes of culture medium and do so efficiently. Furthermore, the simplified design can avoid the use of adhesives or high particulate materials, thus avoiding potential complexities or unwanted components in the bioprocessing industry.

[0035] Upon consideration, a cell culture system can be used with a packed bed bioreactor having a fixed bed cell culture substrate. In conventional large-scale cell culture bioreactors, different types of packed bed bioreactors have been used. Generally, these packed beds contain a porous matrix to retain adherent or suspended cells and support growth and proliferation. The packed bed matrix provides a high surface area to volume ratio, so the cell density can be higher than that in other systems. However, the packed bed typically acts as a depth filter where cells are physically trapped or entangled in the fibers of the matrix. Thus, due to the linear flow of the cell inoculum through the packed bed, the cells experience non-uniform distribution inside 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 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.

[0036] 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 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 significant nutrient and metabolite gradients, thereby having a negative impact on overall cell culture and bioreactor performance. Cells located in low medium perfusion regions will starve and often die due to lack of nutrients or metabolite poisoning. Cell collection 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 inside the packed bed, and the cell recovery rate is extremely low. This significantly limits the utilization of such bioreactors in bioprocesses where live cells are the product. Thus, the non-uniformity causes regions to experience different flow rates and shear forces, effectively reducing the available cell culture area, causing uneven culturing, and interfering with transfection efficiency and cell release.

[0037] 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 can achieve efficient and high-yield cell culture of adherent-dependent cells and production of cell products (such as proteins, antibodies, virus particles). Embodiments include porous cell culture matrices made of an array of ordered and regular porous substrate materials, which can achieve uniform cell seeding and medium / nutrient perfusion, as well as efficient cell collection. Embodiments can also achieve scalable cell culture solutions, where the substrate and bioreactor can inoculate and grow cells and / or collect cell products from process development scale to full production scale without sacrificing the uniform performance of the embodiments. For example, in some embodiments, the bioreactor can be easily scaled up from process development scale to production scale, having a comparable number of viral genomes per unit substrate surface area (VG / cm 2 ) throughout the production scale. The collectability and scalability of the embodiments herein enable these embodiments to be used for efficient seed train to grow cell populations at multiple scales on the same cell substrate. Additionally, the embodiments herein provide a cell culture matrix with a high surface area, which, in combination with the other features described, can achieve a high-yield cell culture solution. In some embodiments, for example, the cell culture substrates and / or bioreactors described herein can produce 10 16 to 10 18 viral genomes (VG) per batch.

[0038] In one embodiment, a matrix 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 packed 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 culture matrices disclosed herein support the attachment and proliferation of adherent-dependent cells in a high volume density form. Uniform cell seeding of such matrices can be achieved, as well as efficient collection of cells or other products from the bioreactor. Additionally, the embodiments of the present disclosure support cell culture to provide a uniform cell distribution during the seeding step, and achieve monolayer or multilayer confluence of adherent cells on the disclosed matrices, and can avoid the formation of large and / or uncontrollable 3D cell aggregates where nutrient diffusion is limited and metabolite concentration increases. Thus, the matrix eliminates diffusion limitations during the operation of the bioreactor. Additionally, the matrix can simply and efficiently collect cells from the bioreactor. The structurally defined matrix of one or more embodiments can completely recover cells from the packed bed of the bioreactor and continuously collect cells.

[0039] According to some embodiments, a method of culturing cells using a bioreactor having a matrix is also provided for bioprocessing to produce therapeutic proteins, antibodies, viral vaccines, or viral vectors.

[0040] Compared with 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 a cell culture substrate 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 the packed bed. This configuration can improve cell seeding, nutrient delivery, cell growth, and cell collection. According to one or more specific embodiments, the matrix is formed from a substrate material having a flake-like structure having 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 side and the second side of the substrate. Additionally, a plurality of holes or openings are formed through the thickness of the substrate. The substrate material between the openings has a size and geometry that allows cells to attach 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 into a molded polymer sheet; a polymer sheet with openings punched through 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 matrix has a high surface area to volume ratio for culturing adherent-dependent cells. According to various embodiments, the matrix can be arranged or filled in the bioreactor in certain ways discussed herein for uniform cell seeding and growth, uniform medium perfusion, and efficient cell collection.

[0041] Embodiments of the present disclosure can achieve a viral vector platform of practical size, 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, or about 10 16 to about 10 19 viral genomes per batch, or about 1016 from 1 to 10 18 viral genomes, or about 10 17 to about 10 19 viral genomes, or about 10 18 to about 10 19 viral genomes, or about 10 18 or more viral genomes per batch.

[0042] 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 enable the collection of live cultured cells. The inability to collect live cells is a significant shortcoming of current platforms and makes it difficult to build and maintain a sufficient number of cells to achieve production capacity. According to aspects of the embodiments of the present disclosure, live cells can be collected 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 collected 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.

[0043] 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, and the plurality of openings can be defined by one or more widths or diameters. The size and shape of the openings can vary based on the weaving type (e.g., the number, shape and size of the filaments; the angle between the 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 a three-dimensional structure is formed due to the undulations of the cross fibers of 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 large surface area for culturing adherent cells, and the structural rigidity of the mesh can provide a consistent and predictable cell culture matrix structure, which enables uniform fluid flow.

[0044] In one or more embodiments, the fibers can have a diameter in the range of about 50 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μm to about 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400 μm; about 200 μm to about 300 μm; or about 150 μm to about 300 μm. At the microscale, due to the scale difference between the fibers and cells (e.g., fiber diameter is greater than that of cells), the surface of a single filament fiber presents as an approximately 2D surface for adherent cells to attach and proliferate. The fibers can be woven into a mesh having openings in the range of about 100 μm × 100 μm to about 1000 μm × 1000 μm. In some embodiments, the openings can have a diameter of about 50 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μm to about 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400 μm; or about 200 μm to about 300 μm. These ranges of filament diameter and 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 provide efficient and uniform fluid flow through the substrate when, for example, the cell culture substrate comprises multiple adjacent mesh layers (e.g., a stack or roll of individual layers of the mesh).

[0045] Factors such as fiber diameter, opening diameter, and weave type / pattern will determine the surface area available for cell attachment and growth. Additionally, when the cell culture substrate comprises other arrangements of stacked, rolled, or overlapping substrates, the packing density of the cell culture substrate will affect the surface area of the packed bed matrix. 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, then 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 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 may not overlap, e.g., when the lowest point of the upper layer is in direct contact with the highest point of the lower layer. For certain applications, it may be desirable to provide a cell culture substrate with a lower layer packing density (e.g., when higher permeability is prioritized) or a higher packing density (e.g., when maximizing the substrate surface area is prioritized). According to one or more embodiments, the packing thickness can be about 50 μm to about 1000 μm; about 100 μm to about 750 μm; about 125 μm to about 600 μm; about 150 μm to about 500 μm; about 200 μm to about 400 μm; about 200 μm to about 300 μm.

[0046] The above structural factors can determine the surface area of the cell culture substrate, whether it is a monolayer cell culture bottom or a cell culture substrate with a multi-layer bottom). For example, in a specific embodiment, a monolayer woven mesh bottom with a circular shape and a 6 cm diameter can have an effective surface area of approximately 68 cm 2 As used herein, "effective surface area" is the total surface area of the fibers in that portion of the substrate material that is available for cell attachment and growth. Unless otherwise stated, the "surface area" mentioned refers to this effective surface area. According to one or more embodiments, a single woven mesh base layer with a diameter of 6 cm can have an effective surface area of approximately 50 cm 2 to approximately 90 cm 2 ; approximately 53 cm 2 to approximately 81 cm 2 ; approximately 68 cm 2 ; approximately 75 cm 2 ; or approximately 81 cm 2 These effective surface area ranges are provided only as examples, and some embodiments may have different effective surface areas. The cell culture substrate can also be characterized by porosity, which will be discussed in the examples herein.

[0047] The base 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 base can have different patterns or weaves, including, for example, knitting, warp knitting, or weaving (such as plain weave, twill weave, Dutch weave, five-needle weave).

[0048] It may be necessary to modify the surface chemistry of the mesh filaments to provide the desired cell adhesion properties. Such modifications can be carried out by chemical treatment of the polymer material of the mesh or by grafting cell adhesion molecules to the filament surface. Alternatively, a thin layer of biocompatible hydrogel exhibiting cell adhesion properties, including, for example, collagen or Alternatively, through a treatment process using various types of plasmas, process gases, and / or chemicals known in the industry, the surface of the filament fibers of the mesh can be made to have cell adhesion properties. However, in one or more embodiments, the mesh is capable of providing an efficient cell growth surface without surface treatment.

[0049] As described herein, the same materials used for the cell culture bottom can also be used for the packed bed regions of the aeration towers 250, 350.

[0050] Figure 2System 100 includes a bioreactor 103 that houses a cell culture substrate incorporating one or more embodiments disclosed herein. As described above, bioreactor 103 can be fluidly connected to a culture medium conditioning vessel 102, and the system is capable of supplying the cell culture medium within conditioning vessel 102 to bioreactor 103. Culture medium conditioning vessel 102 can include sensors and control components that are present in typical bioreactors in the bioprocessing industry for suspension batch culture, fed-batch culture, or perfusion culture. These sensors and control components include, but are not limited to, a DO oxygen sensor, a pH sensor, an aerator / 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 gas flow controllers for N2, O2, and CO2 gases. Culture medium conditioning vessel 102 can also contain a pump or impeller for culture medium mixing. All culture medium parameters measured by the sensors listed above can be controlled by a culture medium conditioning control unit that communicates with culture medium conditioning vessel 102 and is capable of measuring the conditions of the cell culture medium and / or adjusting the conditions to a desired level. As Figure 2 shown, the provided culture medium conditioning vessel 102 is a vessel separated from bioreactor vessel 103. The advantage of this is that the conditioning medium can be separated from where the cells are cultured and then the conditioned medium can be supplied to the cell culture space. However, in some embodiments, the culture medium conditioning can be performed within bioreactor vessel 103.

[0051] The culture medium from culture medium conditioning vessel 102 is delivered to bioreactor 103 via a connector or conduit 140, and the bioreactor can also include an injection port for inoculating a cell inoculum and initiating cell culture. Bioreactor vessel 103 can also include one or more outlets to another connector or conduit 142 through which the cell culture medium exits the vessel 103. To analyze the effluent from bioreactor 103, one or more sensors can be provided in the line. In some embodiments, system 100 includes a flow control unit for controlling the flow into and / or out of bioreactor 103 and / or culture medium conditioning system 100. For example, the flow control unit can receive signals from one or more sensors (e.g., an O2 sensor) and, based on the signals, adjust the flow into bioreactor 103 by sending signals to a pump (e.g., a peristaltic pump) located upstream of the inlet of bioreactor 103. Thus, based on one factor or a combination of factors measured by the sensors, the pump can control the flow into bioreactor 103 to obtain desired cell culture conditions.

[0052] The perfusion rate of the culture medium is controlled by a signal processing unit that collects and compares sensor signals from the culture medium regulation system 100 and sensors located, for example, within the bioreactor 103 or at the outlet of the bioreactor 103. Due to the pack flow nature of the culture medium perfusion through the packed bed bioreactor, nutrient, pH, and oxygen gradients are formed along the packed bed. The perfusion flow rate of the bioreactor can be automatically controlled by a flow control unit operably connected to a peristaltic pump.

[0053] Exemplary embodiments

[0054] The following is a description of various aspects of the disclosed subject matter. Each aspect may include one or more of the various features, characteristics, or advantages of the disclosed subject matter. The embodiments are intended to illustrate several aspects of the disclosed subject matter and should not be regarded as a comprehensive or exhaustive description of all possible embodiments.

[0055] Aspect 1 relates to a cell culture system comprising: a cell culture vessel enclosing an interior configured to culture cells in a liquid cell culture medium, the cell culture vessel including a bioreactor inlet for supplying the cell culture medium to the interior and a bioreactor outlet for removing the cell culture medium from the interior; and a culture medium regulation system comprising: a culture medium regulation vessel configured to regulate a liquid culture medium; and an oxygenation tower, the oxygenation tower including a housing enclosing an interior space, an oxygen-depleted culture medium inlet fluidly connected to the interior space, an excess gas discharge port fluidly connected to the interior space, and a lower opening fluidly connected to the interior space, wherein the lower opening is disposed at the lower end of the oxygenation tower and in a fluid path between the interior space and the culture medium regulation vessel, and wherein the oxygenation tower is configured to mix the oxygen-depleted culture medium with a gas comprising oxygen in a countercurrent manner.

[0056] Aspect 2 relates to the cell culture system according to aspect 1, wherein the oxygen-depleted culture medium inlet is disposed at the upper end of the oxygenation tower and in the fluid path to receive the culture medium from the interior of the bioreactor vessel.

[0057] Aspect 3 relates to the cell culture system according to aspect 1 or 2, wherein the excess gas discharge port is disposed at the upper end of the oxygenation module.

[0058] Aspect 4 relates to the cell culture system according to aspects 1 to 3, wherein the lower opening is configured to supply oxygen-rich gas to the interior space and supply the culture medium from the oxygenation tower to the culture medium regulation vessel.

[0059] Aspect 5 relates to a cell culture system according to aspects 1 to 4, wherein the oxygenation tower includes a porous material in the internal space.

[0060] Aspect 6 relates to the cell culture system according to aspect 5, wherein the porous material is configured to increase the area of the gas-medium interface in the oxygenation tower.

[0061] Aspect 7 relates to the cell culture system according to aspect 5 or 6, wherein the porous material is a packed bed of the porous material.

[0062] Aspect 8 relates to the cell culture system according to any one of aspects 5 to 7, wherein the porous material is the same as the material used as the cell culture medium bottom disposed in the interior of the bioreactor vessel.

[0063] Aspect 9 relates to the cell culture system according to any one of aspects 1 to 8, wherein the porous material includes an ordered physical structure including a pore array.

[0064] Aspect 10 relates to the cell culture system according to aspect 9, wherein the porous material includes a woven mesh material.

[0065] Aspect 11 relates to the cell culture system according to aspect 9 or 10, wherein the porous material includes a plurality of sheets of the porous material stacked in the internal space.

[0066] Aspect 12 relates to the cell culture system according to any one of aspects 1 to 11, wherein the cell culture system is arranged in a perfusion circuit.

[0067] Aspect 13 relates to the cell culture system according to any one of aspects 1 to 12, wherein the oxygenation module is configured to passively regulate the flow rate of the hypoxic medium through the internal space based on the rate at which the hypoxic medium fills the top plate of the oxygenation tower.

[0068] Aspect 14 relates to the cell culture system according to aspect 13, wherein the top plate includes one or more openings configured to allow the hypoxic medium to flow through the one or more openings at a higher rate as the height of the hypoxic medium on the top plate increases.

[0069] Aspect 15 relates to the cell culture system according to aspect 13 or 14, wherein the one or more openings include a medium inlet tube that rises into the top plate and enters the headspace above the top plate.

[0070] Aspect 16 relates to the cell culture system according to aspect 15, wherein the culture medium inlet tube includes a culture medium inlet opening configured to allow an oxygen-depleted culture medium to flow from the headspace above the top plate to below the top plate.

[0071] Aspect 17 relates to the cell culture system according to aspect 16, wherein the culture medium inlet opening includes a variable width.

[0072] Aspect 18 relates to the cell culture system according to aspect 16 or 17, wherein the width of the culture medium inlet opening is narrower at the bottom of the culture medium inlet tube than at the top of the culture medium inlet tube.

[0073] Aspect 19 relates to the cell culture system according to any one of aspects 16 to 18, wherein the width of the culture medium inlet opening increases along the height of the culture medium inlet tube from the bottom of the culture medium inlet tube.

[0074] Definitions

[0075] "Fully synthetic" or "completely synthetic" refers to a cell culture product 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 products eliminate the risk of xenobiotic contamination.

[0076] "Include", "includes" or similar terms mean to cover but not be limited to, that is, inclusive rather than exclusive.

[0077] "User" refers to a person who uses the systems, methods, products or kits disclosed herein, and includes a person who cultures cells to collect cells or cell products, or a person who uses cells or cell products cultured and / or collected according to the examples herein.

[0078] When describing the embodiments of the present disclosure, using "about" to modify values such as the amount, concentration, volume, processing temperature, processing time, yield, flow rate, pressure, viscosity and similar values and their ranges of components in a composition, or the dimensions and similar values and their ranges of components, means that the change in the numerical amount may occur for the following reasons, for example: typical measurement and handling procedures for preparing materials, compositions, complexes, concentrates, components, products or using formulations; inadvertent errors in these procedures; differences in the manufacture, source or purity of the starting materials or components used to implement the method; and similar considerations. The term "about" also covers amounts that are different due to the aging of a composition or formulation having a specific initial concentration or mixture, and amounts that are different due to mixing or processing a composition or formulation having a specific initial concentration or mixture.

[0079] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur.

[0080] Unless otherwise specified, as used herein, the indefinite articles "a" or "an" and their corresponding definite article "the" mean at least one, or one or more.

[0081] Abbreviations well known to those of ordinary skill in the art may be used (e.g., "h" or "hrs" for one or more hours, "g" or "gm" for one gram or more grams, "mL" for milliliters, and "rt" for room temperature, "nm" for nanometers, and similar abbreviations).

[0082] 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 that are explicit or implicit.

[0083] 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.

[0084] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the spirit or scope of the disclosed embodiments. Since modifications, combinations, sub - combinations and variations of the disclosed embodiments that are within the spirit and substance of the combined embodiments can be envisioned by those skilled in the art, the disclosed embodiments should be construed to include all such within the scope of the appended claims and their equivalents.

Claims

1. A cell culture system, comprising: A cell culture container that encloses an interior configured to culture cells in a liquid cell culture medium, the cell culture container including a bioreactor inlet for supplying the cell culture medium to the interior and a bioreactor outlet for removing the cell culture medium from the interior; And A culture medium conditioning system, comprising: A culture medium conditioning container configured to condition a liquid culture medium; And An oxygenation tower, the oxygenation tower including a housing that houses an interior space, a hypoxic culture medium inlet fluidly connected to the interior space, an excess gas discharge port fluidly connected to the interior space, and a lower opening fluidly connected to the interior space, Wherein the lower opening is disposed at a lower end of the oxygenation tower and in a fluid path between the interior space and the culture medium conditioning container, Wherein the oxygenation tower is configured to mix the hypoxic culture medium with a gas including oxygen in a countercurrent manner.

2. The cell culture system according to claim 1, wherein the hypoxic culture medium inlet is disposed at an upper end of the oxygenation tower and in a fluid path to receive the culture medium from the interior of the bioreactor container.

3. The cell culture system according to claim 1 or claim 2, wherein the excess gas discharge port is disposed at an upper end of the oxygenation module.

4. The cell culture system according to any one of claims 1 to 3, wherein the lower opening is configured to supply oxygen-rich gas to the interior space and supply the culture medium from the oxygenation tower to the culture medium conditioning container.

5. The cell culture system according to any one of claims 1 to 4, wherein the oxygenation tower includes a porous material in the interior space.

6. The cell culture system according to claim 5, wherein the porous material is configured to increase the area of the gas-medium interface in the oxygenation tower.

7. The cell culture system according to claim 5 or claim 6, wherein the porous material is a packed bed of the porous material.

8. The cell culture system according to any one of claims 5 to 7, wherein the porous material is the same as the material used as the bottom of the cell culture medium disposed in the interior of the bioreactor container.

9. The cell culture system according to any one of claims 1 to 8, wherein the porous material includes an ordered physical structure including a pore array.

10. The cell culture system according to claim 9, wherein the porous material includes a woven mesh material.

11. The cell culture system according to claim 9 or claim 10, wherein the porous material includes a plurality of sheets of the porous material stacked in the interior space.

12. The cell culture system according to any one of claims 1 to 11, wherein the cell culture system is arranged in a perfusion loop.

13. The cell culture system according to any one of claims 1 to 12, wherein the oxygenation module is configured to passively regulate the flow rate of the hypoxic culture medium through the interior space based on the rate at which the hypoxic culture medium fills the top plate of the oxygenation tower.

14. The cell culture system according to claim 13, wherein the top plate includes one or more openings configured to allow the hypoxic culture medium to flow through the one or more openings at a higher rate as the height of the hypoxic culture medium on the top plate increases.

15. The cell culture system according to claim 13 or claim 14, wherein the one or more openings include a culture medium inlet tube that rises into the top plate and into the headspace above the top plate.

16. The cell culture system according to claim 15, wherein the culture medium inlet tube includes a culture medium inlet opening configured to allow the hypoxic culture medium to flow from the headspace above the top plate to below the top plate.

17. The cell culture system according to claim 16, wherein the culture medium inlet opening includes a variable width.

18. The cell culture system according to claim 16 or claim 17, wherein the width of the culture medium inlet opening is narrower at the bottom of the culture medium inlet tube than at the top of the culture medium inlet tube.

19. The cell culture system according to any one of claims 16 to 18, wherein the width of the culture medium inlet opening increases along the height of the culture medium inlet tube from the bottom of the culture medium inlet tube.

20. An oxygenation tower for oxygenating a cell culture medium, the oxygenation tower comprising: a housing that encloses an internal space; a hypoxic culture medium inlet fluidly connected to the internal space; an excess gas outlet fluidly connected to the internal space; and a lower opening fluidly connected to the internal space, wherein the lower opening is disposed at the lower end of the oxygenation tower and in a fluid path between the internal space and the exterior of the oxygenation tower, and wherein the oxygenation tower is configured to mix the hypoxic culture medium with a gas comprising oxygen in a countercurrent manner.

21. The oxygenation tower according to claim 20, wherein the hypoxic culture medium inlet is disposed at the upper end of the oxygenation tower and in a fluid path to receive the culture medium from the interior of a bioreactor vessel.

22. The oxygenation tower according to claim 20 or 21, wherein the excess gas outlet is disposed at the upper end of the oxygenation module.

23. The oxygenation tower according to any one of claims 20 to 22, wherein the lower opening is configured to supply oxygenated gas to the internal space and to supply the culture medium from the oxygenation tower to a culture medium conditioning vessel.

24. The oxygenation tower according to any one of claims 20 to 23, wherein the oxygenation tower includes a porous material in the internal space.

25. The oxygenation tower according to claim 24, wherein the porous material is configured to increase the area of the gas-medium interface in the oxygenation tower.

26. The oxygenation tower according to claim 25, wherein the porous material is a packed bed of the porous material.

27. The oxygenation tower according to any one of claims 20 to 26, wherein the porous material includes an ordered physical structure comprising an array of pores.

28. The oxygenation tower according to claim 27, wherein the porous material comprises a woven mesh material.

29. The oxygenation tower according to claim 27 or 28, wherein the porous material comprises a plurality of sheets of the porous material arranged in a stacked manner within the internal space.

30. The oxygenation tower according to any one of claims 20 to 29, wherein the oxygenation module is configured to passively adjust the flow rate of the oxygen-depleted culture medium through the internal space based on the rate at which the oxygen-depleted culture medium fills the top plate of the oxygenation tower.

31. The oxygenation tower according to claim 30, wherein the top plate comprises one or more openings configured to allow the oxygen-depleted culture medium to flow through the one or more openings at a higher rate as the height of the oxygen-depleted culture medium on the top plate increases.

32. The oxygenation tower according to claim 30 or 31, wherein the one or more openings comprise a culture medium inlet tube that rises into the top plate and into the headspace above the top plate.

33. The oxygenation tower according to claim 32, wherein the culture medium inlet tube comprises a culture medium inlet opening configured to allow the oxygen-depleted culture medium to flow from the headspace above the top plate to below the top plate.

34. The oxygenation tower according to claim 33, wherein the culture medium inlet opening comprises a variable width.

35. The oxygenation tower according to claim 33 or 34, wherein the width of the culture medium inlet opening is narrower at the bottom of the culture medium inlet tube than at the top of the culture medium inlet tube.

36. The oxygenation tower according to any one of claims 33 to 35, wherein the width of the culture medium inlet opening increases along the height of the culture medium inlet tube from the bottom of the culture medium inlet tube.

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