Systems and methods for oxygenating culture medium in perfusion bioreactors - Patents.com

Abstract

A cell culture system is provided that includes a cell culture vessel having an interior configured to culture cells in a liquid cell culture medium and a medium conditioning system. The medium conditioning system includes a medium conditioning vessel for conditioning the liquid medium and an oxygenation column. The oxygenation column includes a housing containing an interior space, an oxygen-depleted medium inlet fluidly connected to the interior space, an excess gas vent fluidly connected to the interior space, and a bottom opening fluidly connected to the interior space. The bottom opening is disposed at a lower end of the oxygenation column and within a fluid pathway between the interior space and the medium conditioning vessel. The oxygenation column is designed to mix the oxygen-depleted medium with an oxygen-containing gas in a countercurrent manner, thereby achieving better dissolved oxygen saturation in the medium.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 428,975, filed November 30, 2022, the contents of which are herein relied upon and incorporated by reference in their entirety. [Technical Field]

[0002] The present disclosure relates generally to medium conditioning in cell culture bioreactor systems, and more particularly to medium conditioning systems and methods for oxygenating cell culture medium in perfusion bioreactors. [Background technology]

[0003] The bioprocessing industry conducts large-scale cultivation of cells for the production of hormones, enzymes, antibodies, vaccines, and cell therapies. The cell and gene therapy market is growing rapidly, with promising therapies advancing into clinical trials and rapidly toward commercialization. However, a single cell therapy dose can require billions of cells or trillions of viruses. Therefore, being able to provide large amounts of cell product in a short period of time is critical for clinical success. The process efficiency of bioreactor cell culture should be high for economically viable production. In the field of bioreactor design, the bioprocessing industry is moving toward the development of high-density cultures. For a given production capacity, high-density systems are more compact in size and more cost-effective.

[0004] However, there are limitations to increasing cell density and therefore reactor performance. These include operation, aeration, and media formulation. In cell culture reactors, cells must be grown under controlled conditions, including being suspended or perfused in a cell culture medium, a liquid medium containing nutrients necessary for cell survival and growth. The contents of the cell culture medium, including pH and content of dissolved gases (including, for example, dissolved oxygen), as well as the temperature of the medium and / or cells, must be controlled to optimize cell growth and bioreactor performance. Therefore, medium conditioning systems are used in conjunction with or integrated into bioreactors to condition the medium therein. These medium conditioning systems can control, for example, temperature, pH, carbon dioxide content, dissolved oxygen content, and other aspects of the medium. Depending on the cells being grown or the stage of the culture process, specific medium conditioning needs may vary. Typically, medium conditioning is performed in a hollow container or vessel (e.g., a beaker or bottle) with a complex system of probes to control the medium's composition, one or more agitators to mix the medium, and some type of temperature-controlled jacket around the vessel. The probe can enter the container through a cap on the container body, and sterility is always a concern, especially if the system is open or will be open during use.

[0005] The cell density in a reactor is often determined by the oxygen supply to the cells. The mass transfer rate from the gas to the medium must balance the oxygen consumption rate by the cells. Currently, several aeration methods are used in the bioprocessing industry. These include surface aeration, membrane aeration, macrosparging, and microsparging. Sparged cultures achieve higher oxygen transfer rates compared to other techniques. However, sparging rates are limited by cell disruption and foaming problems. One approach to further increasing oxygenation rates is described in U.S. Pat. No. 9,388,375, which proposes combining gas overlay and gas sparging processes within the same reactor. U.S. Pat. No. 9,512,392 also discloses a method for increasing dissolved oxygen (DO) in the culture medium, which forms the basis for designing and fabricating effective mammalian cell culture bioreactors. Historically, the bioprocessing industry has been based on traditional microbial fermentor design principles, which rely heavily on stainless steel technology. For this reason, most sparging systems found in stirred-tank bioreactors are not suitable for mammalian cell culture. They relied on high shear mixers to break up the gas bubbles, however mammalian cell culture requires gentle mixing and lower gas shear rates, which requires a different sparge design.

[0006] The ability to scale up biomanufacturing processes is essential for process development and biopharmaceutical production. Bioreactor process setpoints, acceptable ranges, and general operating parameters used at large scales are typically based on those developed at benchtop or small scales, which are less costly. Scaling up bioreactor processes, particularly oxygenation efficiency, is challenging. Simultaneously maintaining comparable bioreactor characteristics, such as bubble size, distribution, residence time, and comparable bubble surface area, is difficult. Therefore, gas mass transfer remains one of the critical yet challenging parameters used to control bioreactors. Sufficient O2 or air delivery is necessary not only to support cell growth, metabolism, and biopharmaceutical production, but also to control CO2 accumulation in the medium, which can adversely affect performance endpoints.

[0007] Therefore, there is a need for an improved medium conditioning system that can meet the demands of high density cell culture in terms of gas-liquid mass transfer to the cell culture medium. Summary of the Invention

[0008] According to one embodiment of the present disclosure, a cell culture system is provided, including a cell culture vessel having an interior configured to culture cells in a liquid cell culture medium, and a medium conditioning system. The medium conditioning system includes a medium conditioning vessel for conditioning the liquid medium, and an oxygenation column. The oxygenation column includes a housing containing an interior space, an oxygen-depleted medium inlet fluidly connected to the interior space, an excess gas vent fluidly connected to the interior space, and a bottom opening fluidly connected to the interior space. The bottom opening is disposed at a lower end of the oxygenation column and within a fluid pathway between the interior space and the medium conditioning vessel. The oxygenation column is designed to mix the oxygen-depleted medium with an oxygen-containing gas in a countercurrent manner, thereby achieving better dissolved oxygen saturation in the medium. [Brief explanation of the drawings]

[0009] [Figure 1]1 shows a bioreactor system with a medium conditioning vessel as known in the art. [Figure 2] FIG. 1 shows a schematic diagram of a cell culture system having an oxygenation column in accordance with one or more embodiments of the present disclosure. [Figure 3] FIG. 1 shows an expanded schematic view of an oxygenation column according to one or more embodiments of the present disclosure. [Figure 4] FIG. 1 shows an enlarged schematic view of an upper section of an oxygenation column, according to an embodiment. [Figure 5A] 1 shows a medium inlet tube from the top plate of the oxygenation column, according to one or more embodiments of the present disclosure. [Figure 5B] 5B shows an alternative view of the medium inlet tube of FIG. 5A, according to an embodiment. [Figure 6A] 10 shows an example of multiple medium inlet tubes as medium fills the top plate, according to an embodiment. [Figure 6B] 10 shows an example of multiple medium inlet tubes as medium fills the top plate, according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0010] Various embodiments of the present disclosure will now be described in detail with reference to the drawings, where available. References to various embodiments do not limit the scope of the invention, which is limited only by the claims appended hereto. Additionally, any examples set forth herein are not limiting and merely describe some of the many possible embodiments of the claimed invention.

[0011] FIG. 1 shows a cell culture system 10 having a conventional medium conditioning vessel 12 attached to a bioreactor vessel 13. Medium 20 flows from the medium conditioning vessel 12 to the inlet 15 of the bioreactor vessel 13 via outlet 14. The medium 20 in the bioreactor vessel 13 can be used to feed cells (not shown) in the bioreactor vessel 13, through which it can be perfused to outlet 17. The medium 20 can then be recirculated, for example, via inlet 11 back to the medium conditioning vessel 12. The medium conditioning vessel 12 can be a simple hollow container or vessel (e.g., a glass or plastic beaker or bottle). A complex system of probes 16 is used to control the composition of the medium 20, and one or more agitators 18 are used to mix the medium 20. A temperature control jacket 19 (e.g., a water jacket) is shown around the medium conditioning vessel 12, which can be used to control the temperature of the medium 20. Probe 16, which may include a combination of sensors and supplies for various components (e.g., oxygen), can enter medium conditioning vessel 12 through the cap of the vessel body. During operation of cell culture system 10, as medium 20 is perfused through bioreactor vessel 13, oxygen dissolved in medium 20 is consumed by cells positioned within the fixed bed. The oxygen-depleted medium is returned to medium conditioning vessel 12 where it is re-oxygenated, and excess CO2 is removed from the medium by a gas transfer process on the surface of gas bubbles introduced through gas sparge 17. Excess gas exits the medium conditioning vessel through sterile vent filter 21. In high-intensity bioprocesses, the oxygen consumption rate within fixed-bed reactor 13 exceeds the oxygenation rate within medium conditioning vessel 12. As a result, cells become oxygen-starved, thereby reducing the efficiency and productivity of the bioprocess. To overcome this limitation and enhance system performance, embodiments of the present disclosure provide an oxygenation system for significantly improving medium oxygenation rates.

[0012] Embodiments of the present disclosure are directed to medium conditioning vessels and systems, as well as cell culture systems incorporating medium conditioning systems. In particular, embodiments of the present disclosure provide systems and methods for improving gas-liquid mass transfer within a bioreactor. According to embodiments, an oxygenation column is provided in which cell culture medium is exposed to the surface of a packed bed material. The cell culture medium enters the oxygenation column through an upper portion, and gas enters the oxygenation column via an inlet in the lower portion of the column. The gas and liquid flow past each other within the oxygenation column. The oxygenation column includes a packed bed of material to increase the liquid-gas interfacial surface area, thereby enhancing gas-liquid mass transfer to reoxygenate oxygen-depleted medium and remove excess levels of CO2 from the medium.

[0013] Traditionally, one of the parameters difficult to scale in bioprocesses is medium oxygenation and CO2 removal. These parameters depend on multiple primary bioreactor parameters, such as mixing, gas flow rate, sparging efficiency, bubble size, and bubble residence time. According to embodiments disclosed herein, the oxygenation module allows for enhanced gas exchange and operates independently of the above parameters due to its ability to provide a constant, increased surface area of ​​the liquid-gas interface. The oxygenation column allows end users to significantly enhance their bioprocess in the area of ​​oxygen consumption by cultured cells. This allows for higher productivity compared to standard bioreactors that rely solely on gas sparging.

[0014] In some embodiments, the bioreactor is envisioned to be a fixed-bed or packed-bed bioreactor with a high density scaffold for cell growth. This system also allows for control of other aspects of the culture medium, such as temperature regulation and pH.

[0015] FIG. 2 shows a schematic diagram of a cell culture system 100 including both 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, according to some embodiments. As shown in FIG. 2, the system 100 can be configured in a recirculation loop, with the medium 200 flowing from the medium conditioning system 102 via outlet 104 and through tubing 140 or other fluid connectors to the bioreactor vessel 103. Within the bioreactor vessel 103, the medium provides the cells with necessary nutrients and maintains a healthy cellular environment. The perfusion medium 200 is then returned to the medium conditioning vessel 102 via return tubing 142 or other connector. The returned medium passes through an oxygenation column 250 before entering the medium conditioning vessel or during re-entry. Embodiments of the present disclosure include the entire cell culture system 100 as well as the individual medium conditioning system 102 and / or oxygenation column 250.

[0016] 3 shows an enlarged schematic diagram of an oxygenation column 250, according to some embodiments. The oxygenation column 250 includes a housing 252 that encloses an interior space 254. Oxygen-depleted medium 256 enters the oxygenation column 250 through a medium inlet 258, passes through the interior space 254, and exits the oxygenation column 250 through a bottom opening 260. Excess gas (e.g., air or oxygen) leaking from the medium conditioning vessel 102 enters the oxygenation column 250 through the bottom opening 260, passes through the oxygenation column 250, and exits through a gas vent 262. Therefore, a counterflow condition exists within the interior space 254, where gas flows upward through the oxygenation column 250 while returning medium flows downward through the oxygenation column 250. The operating principle of the oxygenation column 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., medium), according to equation (1):

number

[0017] In aspects of embodiments, the interior space 254 can include a material to increase the surface area of ​​the medium-gas interface for improved gas transfer to the medium. For example, the oxygenation column can be filled with a highly porous material 265. This highly porous material can take a variety of forms, and therefore, it is contemplated that embodiments are not limited to a particular porous material. The porous material can 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). The porous material can be in the form of a PET sheet that is stacked into a roll (see FIG. 3) or wound within the oxygenation column 250, according to example embodiments. The PET sheet can be formed from a mesh material having fibers defined in an ordered array and having a plurality of openings therebetween in the ordered array. In embodiments, the mesh is a woven mesh of one or more PET fibers, or a 3D printed, stamped, or molded mesh material.

[0018] The oxygenation column can include a top plate 270, as shown in FIG. 3. FIG. 4 shows an enlarged view of an example top plate 370, according to some embodiments. As shown in FIG. 4, a medium inlet 358 supplies oxygen-depleted medium to the oxygenation column and top plate 370, while a gas vent 362 exhausts excess gas from the top of the oxygenation column. One or more gas outlets 364 inside the packed oxygenation column allow excess gas from within the oxygenation column to rise to a head space 371 above the top plate 370. Medium 300 flowing in through the medium inlet 358 falls onto the top plate 370, where it pools before rising to a sufficient level to flow into one or more medium inlet openings 372. After rising to the level of the openings in the medium inlet openings 372, the medium 300 flows through the top plate 370 via the medium inlet openings 372 and into the interior of the oxygenation column, which contains a packed bed of porous material. Multiple medium inlet openings 372 and gas outlets 364 may be provided to improve the uniformity of medium flow entering and gas exiting the packed bed zone. The medium inlet openings 372 are configured to equalize medium flow across the upper plate 370 and distribute the medium flow evenly among all of the medium inlet openings 372 within the upper plate 370. For example, the medium inlet openings 372 may include medium inlet tubes 374 with sloped, notched flow openings in their walls, as shown in FIGS. 5A and 5B. The flow openings widen toward the top of the medium inlet tubes 374. As a result, the flow through each medium inlet tube 374 is proportional to the liquid level within the upper plate, as shown in FIGS. 6A and 6B. The horizontal orientation of the upper flow distribution plate will provide conditions for self-regulating, uniform flow distribution through the medium inlet tubes 374.

[0019] According to embodiments, the oxygenation column 250, 350 may be an add-on component that can be installed on the medium conditioning vessel after the fact. In other embodiments, the oxygenation column may be integrated with or pre-installed on the medium conditioning vessel.

[0020] Optionally, the medium conditioning vessel 102 is temperature controlled. That is, the temperature of the space within the vessel 102 can be heated or cooled to control the temperature of the gas and / or medium entering or passing through the gas medium conditioning vessel 102 or oxygenation column 250. In some embodiments, temperature control is used to heat or cool the gas, which in turn heats or cools the medium in the medium conditioning vessel 102 or oxygenation column. Optionally, this temperature control can be achieved by an integrated temperature control device 109, such as, for example, a heater or cooler. Alternatively, the gas can be temperature controlled before entering the enclosure.

[0021] The bioreactor system 100 can also include one or more sensors 106 for detecting gas and / or medium quality. The sensors 106 can be provided in the medium conditioning vessel 102, as shown in FIG. 2. However, sensors can also be provided along the medium, either upstream or downstream of the medium inlet 101 and medium outlet 104, or within the bioreactor vessel itself, or within the oxygenation column 250, 350. Based on feedback from the sensors, the composition of the gases via the inlets and / or temperature control devices can be controlled to achieve the desired composition and temperature of the cell culture medium for optimal performance of the culture. For example, the gas composition can be adjusted to achieve the desired O and / or CO levels, and the pH can be adjusted.

[0022] The housing 109 for the medium conditioning vessel 102 can be a thermal housing. As used herein, "thermal housing" means that the housing is insulated so that the temperature inside the thermal housing can be more easily controlled. As an alternative to, or in addition to, a thermal housing, the housing 102 can include a heat source for controlling the temperature inside the housing, or the temperature of the gas inside the housing, as discussed above. The heat source can be an integral structure and function of the housing 109, or can be a separate component that the housing 109 is configured to receive, if desired. According to some embodiments, the heat source provides sufficient heat to the gas to control the temperature of the medium in the medium exchange system within a desired range for cell culture.

[0023] In some embodiments, the medium conditioning system further includes one or more sensors for sensing the properties of the gas within the enclosure, or the properties of the medium within the oxygenation column, or the properties of the medium before entering the bioreactor, after exiting the bioreactor, or while within the bioreactor. The one or more sensors may measure temperature, pH, oxygen (O), CO, or any of a number of variables related to the cell culture operation being performed.

[0024] The medium conditioning system and vessel disclosed herein have many advantages. For example, the design allows the medium conditioning vessel to be a disposable or single-use vessel. The reusable or single-use components of the system can reduce operating costs while still achieving gas delivery to the medium using an oxygenation column. The gas can also be temperature controlled within the system, thus effectively controlling the temperature of the medium. Additionally, due to possible embodiments, the system can process large volumes of medium and do so efficiently. Furthermore, the simplified design can avoid the use of adhesives or high-particulate materials, thus avoiding potential complications or undesirable components in the bioprocessing industry.

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

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

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

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

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

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

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

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

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

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

[0035] Factors such as fiber diameter, opening diameter, and weave type / pattern determine the surface area available for cell attachment and growth. Additionally, when a cell culture substrate includes a stack, roll, or other arrangement of overlapping substrates, the packing density of the cell culture substrate affects the surface area of ​​the packed-bed substrate. Packing density can vary depending on the packing thickness of the substrate material (e.g., the space required for a layer of substrate). For example, if a stack of cell culture substrates has a certain height, each layer of the stack can be said to have a packing thickness determined by dividing the total height of the stack by the number of layers in the stack. Packing thickness varies based on fiber diameter and weave, but can also vary based on the alignment of adjacent layers within the stack. For example, due to the three-dimensional nature of woven layers, there is a degree of interlocking or overlap that allows adjacent layers to accommodate each other based on their alignment. In one alignment, adjacent layers can fit closely together, while in a second alignment, adjacent layers may have zero overlap, such as when the bottommost point of the top layer is in direct contact with the topmost point of the bottom layer. In certain applications, it may be desirable to provide a cell culture substrate with a lower packing density of layers (e.g., when higher permeability is a priority) or a higher packing density (e.g., when maximizing substrate surface area is a priority). According to one or more embodiments, the packing thickness can be from about 50 μm to about 1000 μm, from about 100 μm to about 750 μm, from about 125 μm to about 600 μm, from about 150 μm to about 500 μm, from about 200 μm to about 400 μm, or from about 200 μm to about 300 μm.

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

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

[0038] The surface chemistry of the mesh filaments may need to be modified to provide the desired cell adhesion properties. Such modification can be achieved through chemical treatment of the mesh's polymeric material or by grafting cell adhesion molecules onto the filament surface. Alternatively, the mesh can be coated with a thin layer of a biocompatible hydrogel that exhibits cell adhesion properties, including, for example, collagen or Matrigel®. Alternatively, the surfaces of the mesh's filament fibers can be rendered cell adhesive through various types of plasma, process gas, and / or chemical treatment processes known in the art. However, in one or more embodiments, the mesh can provide an efficient cell growth surface without surface treatment.

[0039] As described herein, the same materials used for the cell culture substrate can also be used in the packed bed zone of the oxygenation columns 250, 350.

[0040] The system 100 of FIG. 2 includes a bioreactor 103 containing a cell culture substrate according to one or more embodiments disclosed herein. The bioreactor 103 can be fluidly connected to a medium conditioning vessel 102, and as described above, the system can supply cell culture medium in the conditioning vessel 102 to the bioreactor 103. The medium conditioning vessel 102 can include sensor and control components found in typical bioreactors used in the bioprocessing industry for suspension batch, fed-batch, or perfusion culture. These include, but are not limited to, a DO oxygen sensor, a pH sensor, an oxygen generator / gas sparging unit, a temperature probe, and nutrient and base addition ports. The gas mixture supplied to the sparging unit can be controlled by gas flow controllers for N, O, and CO gases. The medium conditioning vessel 102 also contains a pump or impeller for medium mixing. All medium parameters measured by the sensors listed above can be controlled by a medium conditioning control unit, which communicates with the medium conditioning vessel 102 and can measure and / or adjust the cell culture medium conditions to desired levels. 2, the medium conditioning vessel 102 is provided as a separate vessel from the bioreactor vessel 103. This can have advantages in that the medium can be conditioned separately from where the cells are cultured and then the conditioned medium can be supplied to the cell culture space. However, in some embodiments, the medium conditioning can be performed within the bioreactor vessel 103.

[0041] Culture medium from the medium conditioning vessel 102 is pumped into the bioreactor 103 via connector or tubing 140, which may also include an injection port for a cell inoculum to initiate cell seeding and cultivation. The bioreactor vessel 103 may also include one or more outlets to another connector or tubing 142 through which the cell culture medium exits the vessel 103. One or more sensors may be provided in the line to analyze the contents of the outflow from the bioreactor 103. In some embodiments, the system 100 includes a flow control unit for controlling flow to and / or from the bioreactor 103 and / or medium conditioning system 100. For example, the flow control unit may receive a signal from one or more sensors (e.g., an O sensor) and, based on the signal, adjust the flow into the bioreactor 103 by sending a signal to a pump (e.g., a peristaltic pump) upstream of the inlet to the bioreactor 103. Therefore, based on one or a combination of the factors measured by the sensors, the pump can control the flow into the bioreactor 103 to obtain the desired cell culture conditions.

[0042] The medium perfusion rate is controlled by the medium conditioning system 100 and a signal processing unit that collects and compares sensor signals from sensors located, for example, in or at the outlet of the bioreactor 103. Due to the packed flow nature of the medium perfusion through the packed-bed bioreactor, nutrient, pH, and oxygen gradients develop 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.

[0043] Exemplary Implementations Below are descriptions of various aspects of implementations of the disclosed subject matter. Each aspect may include one or more of various features, characteristics, or advantages of the disclosed subject matter. The embodiments are intended to illustrate some aspects of the disclosed subject matter and should not be considered a comprehensive or exhaustive description of all possible implementations.

[0044] Aspect 1 relates to a cell culture system, the 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 comprising 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 medium conditioning system, the medium conditioning system comprising: a medium conditioning vessel configured to condition a liquid medium; and an oxygenation column, the oxygenation column comprising: a housing containing an interior space; an oxygen-depleted medium inlet fluidly connected to the interior space; an excess gas vent fluidly connected to the interior space; and a bottom opening fluidly connected to the interior space, the bottom opening being disposed in a fluid pathway between the interior space and the medium conditioning vessel at a lower end of the oxygenation column; and the oxygenation column configured to mix the oxygen-depleted medium with an oxygen-containing gas in a countercurrent manner.

[0045] Aspect 2 relates to the cell culture system of aspect 1, wherein the oxygen-depleted medium inlet is disposed at an upper end of the oxygenation column and in a fluid path for receiving medium from the interior of the bioreactor vessel.

[0046] Aspect 3 relates to the cell culture system of aspect 1 or 2, wherein the excess gas vent is disposed at an upper end of the oxygenation module.

[0047] A fourth aspect relates to the cell culture system according to any one of the first to third aspects, wherein the lower opening is configured to supply oxygen-rich gas to the interior space and supply culture medium from the oxygenation column to the culture medium conditioning vessel.

[0048] A fifth aspect relates to the cell culture system according to any one of the first to fourth aspects, wherein the oxygenation column comprises a porous material in the interior space.

[0049] A sixth aspect relates to the cell culture system of the fifth aspect, wherein the porous material is configured to increase the area of ​​the gas-medium interface within the oxygenation column.

[0050] Aspect 7 relates to the cell culture system of aspect 5 or 6, wherein the porous material is a packed bed of porous material.

[0051] Aspect 8 relates to the cell culture system according to any one of Aspects 5 to 7, wherein the porous material is the same material as that used as the cell culture substrate disposed inside the bioreactor vessel.

[0052] A ninth embodiment relates to the cell culture system of any one of the first to eighth embodiments, wherein the porous material comprises an ordered physical structure comprising an array of pores.

[0053] Example 10 relates to the cell culture system of example 9, wherein the porous material comprises a woven mesh material.

[0054] Example 11 relates to the cell culture system of example 9 or 10, wherein the porous material comprises a plurality of sheets of porous material in a stacked arrangement within the interior space.

[0055] A twelfth aspect relates to the cell culture system according to any one of the first to eleventh aspects, wherein the cell culture system is arranged in a perfusion loop.

[0056] Example 13 relates to the cell culture system of any one of Examples 1 to 12, wherein the oxygenation module is configured to passively regulate the flow of the oxygen-depleted medium through the interior space based on a rate at which the oxygen-depleted medium fills the upper plate of the oxygenation column.

[0057] Example 14 relates to the cell culture system of Example 13, wherein the top plate comprises one or more openings, the one or more openings configured to allow a higher rate of flow of the oxygen-depleted medium through the one or more openings as the height of the oxygen-depleted medium on the top plate increases.

[0058] Example 15 relates to the cell culture system of example 13 or example 14, wherein the one or more openings comprise a medium inlet tube that ascends in the upper plate to a headspace above the upper plate.

[0059] Example 16 relates to the cell culture system of Example 15, wherein the medium inlet conduit comprises a medium inlet opening configured to allow flow of oxygen-depleted medium from a head space above the top plate to below the top plate.

[0060] Example 17 relates to the cell culture system of example 16, wherein the medium inlet opening comprises a variable width.

[0061] Example 18 relates to the cell culture system of example 16 or example 17, wherein the width of the medium inlet opening is narrower at the bottom of the medium inlet tube than at the top of the medium inlet tube.

[0062] Aspect 19 relates to the cell culture system of any one of aspects 16 to 18, wherein the width of the medium inlet opening increases along the height of the medium inlet tube from the bottom of the medium inlet tube. definition

[0063] "Wholely synthetic" or "fully synthetic" refers to a cell culture article, such as a microcarrier or culture vessel surface, that is composed entirely of synthetic-source materials and does not contain any animal-derived or animal-origin materials. The disclosed fully synthetic cell culture articles eliminate the risk of xenocontamination.

[0064] The words "include," "includes," or similar terms are meant to be non-exclusive, i.e., inclusive and not exclusive.

[0065] "User" refers to anyone who uses a system, method, article, or kit disclosed herein, including anyone who is culturing cells to harvest cells or cell products, or who is using cells or cell products that have been cultured and / or harvested according to embodiments herein.

[0066] When describing embodiments of the present disclosure, the term "about" as used to modify, for example, the amount, concentration, volume, process temperature, process time, yield, flow rate, pressure, viscosity, and similar values ​​and ranges of components in a composition, or the dimensions of components and similar values ​​and ranges, refers to variations in the numerical amount that may occur, for example, through typical measuring and handling procedures used to prepare a material, composition, composite, concentrate, component, article of manufacture, or formulation for use; through inadvertent errors in these procedures; through differences in the manufacture, source, or purity of starting materials or components used to carry out the method; and through similar considerations. The term "about" also encompasses different amounts due to aging of a composition or formulation having a particular initial concentration or mixture, and different amounts due to mixing or processing a composition or formulation having a particular initial concentration or mixture.

[0067] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not occur.

[0068] As used herein, the indefinite article "a" or "an" and its corresponding definite article "the" mean at least one, or one or more, unless otherwise specified.

[0069] Abbreviations known to those skilled in the art may be used (e.g., "h" or "hrs" for hour(s), "g" or "gm" for gram(s), "mL" for milliliter, and "rt" for room temperature, "nm" for nanometer, and similar abbreviations).

[0070] Specific preferred values ​​disclosed for components, ingredients, additives, dimensions, conditions, and similar aspects, as well as ranges thereof, are for illustrative purposes only and do not exclude other defined or other values ​​within the defined ranges. The systems, kits, and methods of the present disclosure can include any value or any combination of the values, specific values, more specific values, and preferred values ​​set forth herein, including any stated or implied intermediate values ​​and ranges.

[0071] Unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps are to be followed, or the claim or description specifically states that the steps are to be limited to a particular order, no particular order is intended to be inferred.

[0072] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope or spirit of the disclosed embodiments. Since modifications, combinations, subcombinations, and variations of the disclosed embodiments incorporating the spirit and content of the embodiments may occur to those skilled in the art, the disclosed embodiments should be construed as including all within the scope of the appended claims and their equivalents.

Claims

1. 1. A 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 comprising a bioreactor inlet for supply of cell culture medium to the interior and a bioreactor outlet for removal of cell culture medium from the interior; a medium conditioning system, a medium conditioning vessel configured to condition a liquid medium; an oxygenation column, the oxygenation column comprising: a housing containing an interior space; an oxygen-depleted 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 column and within a fluid path between the interior space and the medium conditioning vessel; The cell culture system, wherein the oxygenation column is configured to mix the oxygen-depleted medium with an oxygen-containing gas in a countercurrent manner.

2. 10. The cell culture system of claim 1, wherein the oxygen-depleted medium inlet is disposed at an upper end of the oxygenation column and in a fluid path for receiving medium from the interior of a bioreactor vessel.

3. 3. The cell culture system of claim 1, wherein the excess gas vent is disposed at the upper end of the oxygenation module.

4. 4. The cell culture system according to claim 1, wherein the lower opening is configured to supply an oxygen-rich gas to the interior space and to supply culture medium from the oxygenation column to the culture medium conditioning container.

5. The cell culture system according to any one of claims 1 to 4, wherein the oxygenation column comprises a porous material within the interior space.

6. 6. The cell culture system of claim 5, wherein the porous material is configured to increase the gas-medium interfacial area within the oxygenation column.

7. 7. The cell culture system of claim 5 or 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 material as that used as a cell culture substrate disposed in the interior of the bioreactor vessel.

9. The cell culture system of any one of claims 1 to 8, wherein the porous material comprises an ordered physical structure comprising an array of pores.

10. The cell culture system of claim 9 , wherein the porous material comprises a woven mesh material.

11. 11. The cell culture system of claim 9 or 10, wherein the porous material comprises a plurality of sheets of the porous material in a stacked arrangement within 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. 13. The cell culture system of claim 1, wherein the oxygenation module is configured to passively regulate the flow of oxygen-depleted medium through the interior space based on a rate at which the oxygen-depleted medium fills the upper plate of the oxygenation column.

14. 14. The cell culture system of claim 13, wherein the top plate comprises one or more openings configured to allow a higher rate of flow of the oxygen-depleted medium through the one or more openings as the height of the oxygen-depleted medium above the top plate increases.

15. 15. The cell culture system of claim 13 or 14, wherein the one or more openings comprise a medium inlet tube that rises in the upper plate to a head space above the upper plate.

16. 16. The cell culture system of claim 15, wherein the medium inlet tube comprises a medium inlet opening configured to allow flow of the oxygen-depleted medium from the head space above the top plate to below the top plate.

17. 17. The cell culture system of claim 16, wherein the medium inlet opening comprises a variable width.

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

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

20. 1. An oxygenation column for oxygenating a cell culture medium, comprising: a housing that contains an internal space; an oxygen-depleted medium inlet fluidly connected to the interior space; an excess gas vent fluidly connected to the interior space; a lower opening fluidly connected to the interior space; the lower opening is disposed at a lower end of the oxygenation column and within a fluid path between the interior space and an exterior of the oxygenation column; The oxygenation column is configured to mix oxygen-depleted medium with an oxygen-containing gas in a countercurrent manner.

21. 21. The oxygenation column of claim 20, wherein the oxygen-depleted medium inlet is disposed at an upper end of the oxygenation column and in a fluid path for receiving medium from an interior of a bioreactor vessel.

22. 22. The oxygenation column of claim 20 or 21, wherein the excess gas vent is disposed at the upper end of the oxygenation module.

23. 23. The oxygenation column of any one of claims 20 to 22, wherein the lower opening is configured to supply oxygen-rich gas to the interior space and supply culture medium from the oxygenation column to a culture medium conditioning vessel.

24. 24. The oxygenation column of any one of claims 20 to 23, wherein the oxygenation column comprises a porous material within the interior space.

25. 25. The oxygenation column of claim 24, wherein the porous material is configured to increase gas-medium interfacial area within the oxygenation column.

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

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

28. 28. The oxygenation column of claim 27, wherein the porous material comprises a woven mesh material.

29. 29. The oxygenation column of claim 27 or 28, wherein the porous material comprises a plurality of sheets of the porous material in a stacked arrangement within the interior space.

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

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

32. 32. The oxygenation column of claim 30 or 31, wherein the one or more openings comprise a medium inlet tube that rises in the top plate to a head space above the top plate.

33. 33. The oxygenation column of claim 32, wherein the medium inlet tube comprises a medium inlet opening configured to allow flow of the oxygen-depleted medium from the head space above the top plate to below the top plate.

34. 34. The oxygenation column of claim 33, wherein the medium inlet opening comprises a variable width.

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

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

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