Bioreactor System

Abstract

A cell culture system is provided that includes at least one multi-layer container for culturing cells and a cabinet having an internal cavity surrounded by one or more sidewalls. The cabinet is configured to accommodate the multi-layer container within the internal cavity. The multi-layer container includes a cell culture space within the multi-layer container. The cabinet is capable of changing the orientation of the multi-layer container from an upright orientation to an inclined orientation.

Description

Related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 072,517, filed on 31 August 2020 under Section 119 of the U.S. Patent Act, and relies upon and is incorporated herein by reference in its entirety. [Technical Field]

[0002] This disclosure relates to systems and methods for culturing cells, and more particularly to systems and methods for combining multiple cell culture vessels to enable high yields of cell culture products while minimizing the required vessel space and manual operation. [Background technology]

[0003] Various cell culture products are configured to provide stacked or stackable units for culturing cells. For example, T-flasks are typically manufactured with flat top and bottom surfaces to allow for stacking of T-flasks, resulting in space savings. Some modified T-flasks have multiple parallel culture surfaces within the flask to reduce the time and effort associated with filling and unfilling. Another type of incubator is a multi-component assembly with multiple parallel or stacked culture surfaces. In most such stacked culture assemblies, each culture layer is separated to reduce hydrostatic pressure on the culture layer below. As the number of stacked layers increases, the potential effect of hydrostatic pressure increases.

[0004] One exemplary cell culture is Corning's HYPERStack® system. The HYPERStack® system comprises multiple modules formed from individual stack layers that can be interconnected by flexible tubing connected to tubing connectors. The modules are interconnected for filling and removing the HYPERStack® system. Valves and other devices can be used to control the inflow of fluid into and outflow of the HYPERStack® system. The HYPERStack® 36-layer vessel has lowered the barrier to entry for users seeking to conduct Phase I and Phase II clinical trials. However, the current protocols for using HYPERStack® can be laborious and require manual operation. For example, the current filling and removal process for the HYPERStack® system involves tilting the HYPERStack® system at various stages to obtain better results. This tilting not only requires user attention and manual operation, but can also lead to inconsistent results if the protocol is not applied consistently. When using multiple HYPERStack® containers for larger cell culture needs, this manual operation requires increased user workload during critical moments in cell or virus production, such as during seeding, transfection, refeeding, and harvesting. As a result, each individual handling the containers may introduce variations in the virus production process, potentially reducing efficiency. Damage to the containers can also occur during these operational steps.

[0005] In addition, if customers require larger-scale cell cultures (e.g., entering Phase III clinical trials or beyond), the HYPERStack® system can be adjusted to require a larger footprint, and the required space may become unmanageable for some users. Using manual operation of the HYPERStack® system during filling and unfilling can exacerbate space issues, as manual operation often results in inefficient use of space between multiple HYPERStack® units. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] What is needed are systems and methods for cell culture and virus production that are more controlled, require less manual labor, and are space-efficient, enabling scaling up production. [Means for solving the problem]

[0007] Embodiments of the present disclosure provide a cell culture system comprising at least one multilayer container for culturing cells, the multilayer container containing a cell culture space, and a cabinet having an internal cavity surrounded by one or more side walls, the cabinet capable of housing the multilayer container within the internal cavity. The cabinet can change the orientation of the multilayer container from an upright orientation to a tilted orientation.

[0008] In one embodiment of several configurations, the system further includes at least one sensor for detecting characteristics within the cell culture space. The sensor may include at least one of a collection monitor and an analyte monitor. The sensor may be integrated into a multilayer vessel. In another configuration, the sensor is mounted in a cabinet and is configured to detect characteristics within the cell culture space when the multilayer vessel is placed inside the cabinet.

[0009] In one of several embodiments, the multilayer vessel includes at least one sensor window, the sensor configured to detect characteristics within the cell culture space through the sensor window.

[0010] In some embodiments, the cabinet includes a support surface configured to support at least one multilayer container.

[0011] According to some embodiments, at least one multilayer vessel includes a plurality of multilayer cell culture modules. At least some of the plurality of multilayer cell culture modules can be connected to one another.

[0012] A multilayer vessel may include an inlet and an outlet, the inlet being configured to supply liquid culture medium to the cell culture space, and the outlet being configured to allow liquid or gas to flow into or out of the cell culture space. In one aspect of several embodiments, the inlet is located at the bottom of the multilayer vessel. The outlet may be located at the top of the multilayer vessel. [Brief explanation of the drawing]

[0013] [Figure 1] This is a perspective view of a cell culture vessel according to one or more embodiments illustrated and described herein. [Figure 2] This is a schematic diagram of multiple stack layers for use with the cell culture vessel shown in Figure 1, according to one or more embodiments illustrated and described herein. [Figure 3] This is a side view of a multi-position support that supports the cell culture vessel shown in Figure 1 in an upright configuration, according to one or more embodiments illustrated and described herein. [Figure 4] Figure 3 is a perspective view of a multi-position support according to one or more embodiments illustrated and described herein. [Figure 5] This is a plan view of the multi-position support shown in Figure 4, according to one or more embodiments illustrated and described herein. [Figure 6]A side view showing the multi-position support shown in FIG. 3 in an inclined configuration according to one or more embodiments illustrated and described herein. [Figure 7] An end view of the multi-position support shown in FIG. 6 in an inclined configuration according to one or more embodiments illustrated and described herein. [Figure 8] A cross-sectional view of a 2D cell culture module according to one or more embodiments illustrated and described herein. [Figure 9] A cross-sectional view of a 3D cell culture module according to one or more embodiments illustrated and described herein. [Figure 10] A cell culture vessel having a plurality of cell culture modules according to one or more embodiments illustrated and described herein. [Figure 11A] A side view showing the middle of the filling operation of the cell culture vessel shown in FIG. 10 according to one or more embodiments illustrated and described herein. [Figure 11B] A side view showing the middle of the filling operation of the cell culture vessel shown in FIG. 11A according to one or more embodiments illustrated and described herein. [Figure 11C] A side view showing the completion of the filling operation of the cell culture vessel shown in FIGS. 11A and 11B according to one or more embodiments illustrated and described herein. [Figure 12] A side view of a cell culture system including a cabinet for accommodating a plurality of cell culture vessels according to one or more embodiments illustrated and described herein. [Figure 13] A side view of a cell culture system including a cabinet and a gas-impermeable enclosure according to one or more embodiments illustrated and described herein. [Figure 14A] A schematic side view showing a cell culture system in an upright configuration on a cart according to one or more embodiments illustrated and described herein. [Figure 14B] A schematic side view showing a cell culture system in an inclined configuration on a cart according to one or more embodiments illustrated and described herein.

[0014] Drawings are not necessarily to scale. Similar symbols used in drawings refer to similar components, steps, etc. However, it should be understood that using a symbol to refer to a component in a given drawing is not intended to limit the component to another drawing bearing the same symbol. Furthermore, using different symbols to refer to a component is not intended to indicate that components with different symbols cannot be identical or similar. [Modes for carrying out the invention]

[0015] The following detailed description refers to the accompanying drawings, which form part of this specification and illustrate several specific embodiments of the apparatus, systems, and methods. It should be understood that other embodiments are conceivable and may be implemented without departing from the scope or spirit of this disclosure. Therefore, the following detailed description should not be construed as restrictive.

[0016] All scientific and technical terms used herein have their common meanings in the art unless otherwise specified. The definitions provided herein are intended to facilitate the understanding of certain terms that are frequently used herein and are not intended to limit the scope of this disclosure.

[0017] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include embodiments having multiple references unless otherwise specified. As used herein and in the appended claims, the term “or” is generally used to include “and / or” unless otherwise specified.

[0018] In this specification, "have," "having," "include," "comprise," and "comprising" are used without restriction on their meaning and generally mean "to include, but not limited to."

[0019] When describing embodiments of this disclosure, the term "about" used to modify values ​​such as the amount, concentration, structural dimensions, volume, process temperature, process time, yield, flow rate, pressure, viscosity, and their ranges, refers to variations in numerical quantities that may occur, for example: due to typical measurement and operation procedures used to prepare materials, compositions, composites, concentrates, or formulations used; due to accidental errors in these procedures; due to differences in the manufacture, source, or purity of the starting materials or components used to carry out the method; and for similar reasons. The term "about" also includes different amounts based on aging of a composition or formulation having a particular initial concentration or mixture, and different amounts based on mixing or processing of a composition or formulation having a particular initial concentration or mixture.

[0020] In vitro cell culture provides the materials necessary for pharmacological, physiological, and toxicological research. Recent advances in drug screening techniques have enabled pharmaceutical companies to rapidly screen vast libraries of compounds against therapeutic targets. These large-scale screening techniques require a large number of cells to be grown and maintained in vitro. Maintaining these large numbers of cells requires large quantities of cell growth media and reagents, as well as a large and diverse range of laboratory cell culture vessels and equipment. This task can be labor-intensive.

[0021] Cell culture vessels have been developed to increase the surface area for cell proliferation while also providing the necessary gas exchange means. These systems utilize conventional cell culture vessels, including common flasks, roller bottles, and cell culture dishes, as well as multilayer cell proliferation vessels, which may include multilayer flasks, multilayer cell culture dishes, bioreactors, cell culture bags, and similar articles that may include special surfaces designed to improve cell culture parameters, including proliferation density and differentiation factors. Examples of closed-system cell culture articles specifically developed for high-yield cell proliferation include HYPERFlask® and HYPERStack® products (available from Corning, Inc.) which have a gas-permeable film that provides a cell proliferation surface and allows gas exchange with the surrounding environment.

[0022] This disclosure, in particular, describes systems and methods for the production of cells and cell-derived products in a more controlled and / or compact manner than conventional multilayer vessels. This specification describes cell culture systems and methods that can provide a closed, automated or semi-automated system for culturing anchorage-dependent or adherent cells, or three-dimensional ("3D") cell cultures, using one or more cell culture vessels. These systems and methods enable a denser cell culture footprint, saving space and increasing yield in cell culture production facilities.

[0023] According to embodiments of this disclosure, a cell culture vessel may include one or more cell culture surfaces and at least one port for allowing material to flow into and out of the cell culture vessel. In several embodiments, the system is configured to automatically fill one or more cell culture vessels with cell culture medium, detach cells cultured in one or more cell culture vessels from one or more cell culture surfaces, and remove (e.g., harvest) cells cultured in one or more cell culture vessels. In several embodiments, the system is a closed system. As used herein, a “closed” system means that the cell culture vessel can be operated without being exposed to the external environment during the culture process. Several embodiments provide high-yield cell culture systems and methods that minimize the required space (e.g., small footprint) and allow for greater control over the operation of the cell culture vessel to reduce variability in cell culture conditions.

[0024] Embodiments of the present disclosure include cell culture systems and methods that include or use one or more cell culture vessels (e.g., at least one cell culture vessel, more than one cell culture vessel, two or more cell culture vessels, etc.) that can be configured for culturing multiple scaffold-dependent or adherent cells or for 3D cell culture. In some embodiments, the cell culture vessel includes multiple parallel cell culture surfaces within multiple stacked or multilayer units, compartments, or modules (e.g., multiple cell culture surfaces are parallel to each other). Nevertheless, according to some embodiments, substantially all cell culture vessels can be adapted for use with the systems described herein. For example, any cell culture vessel having multiple stacked layers, or that can be stacked to form multiple layers, can be adapted for use with the systems described herein. Examples of such cell culture vessels include T flasks, TRIPLE-FLASK cell culture vessels (Nunc, Intl.), HYPERFLASK cell culture vessels (Corning, Inc.), CELLSTACK culture chambers (Corning, Inc.), CELLCUBE modules (Corning, Inc.), HYPERSTACK cell culture vessels (Corning, Inc.), CELLFACTORY incubators (Nunc, Intl.), and cell culture products / vessels described in International Publication No. 2007 / 015770, entitled "MULTILAYERED CELL CULTURE APPARATUS," published on February 8, 2007, whose entire contents are incorporated herein by reference to the extent that they do not conflict with this disclosure. Of course, cell culture vessels that do not have stacked layers, or are generally not stackable, may be used in some embodiments.

[0025] A multilayer cell culture vessel may include multiple cell culture modules, each containing multiple growth or culture surfaces within its cell culture chambers, and connected to one another via a manifold to form a cell culture vessel. The cell culture vessel can further be connected to additional cell culture vessels via a manifold to form a stacked or horizontally connected cell culture apparatus. In some embodiments, the manifold may include an integrated column structure formed as a single component of the manifold. The column structure has an inlet port and provides at least a portion of a fluid channel from the inlet port, which is fluidly connected to individual cell culture chambers within the cell culture modules. The manifold and associated column structure can provide a closed system in which the column structure can be connected to a flexible tube to isolate the cell culture chambers from the environment during use of the cell culture vessel.

[0026] A cell culture vessel may include multiple cell culture surfaces connected via a manifold. These culture surfaces may be stacked in a multi-layer configuration. The manifold may include multiple fluid connection ports, which are used to separate individual cell culture chambers or groups of cell culture chambers. Generally, cell or growth culture surfaces are positioned parallel to the ground during the cell culture process. To disperse materials such as cell culture medium within the cell culture vessel, the vessel may be positioned or moved so that the multiple cell culture surfaces are not positioned parallel to the ground, thereby allowing the material to be evenly distributed within all chambers / units and across all multiple cell culture surfaces.

[0027] A cell culture vessel may contain multiple cell culture modules, each of which has a cell culture space and / or includes a multilayer cell proliferation surface. In another embodiment, cell culture vessels can be combined with each other, thereby enabling large-scale cell proliferation and, consequently, large-scale production of viruses, extracellular vesicles, cells, and other cell-derived products.

[0028] Cell culture vessels or parts thereof as described herein may be formed from any suitable material. Preferably, a material intended to come into contact with cells or culture media is suitable for cells and culture media. Typically, cell culture units are formed from polymer materials. Examples of suitable polymer materials include polystyrene, polymethyl methacrylate, polyvinyl chloride, polycarbonate, polysulfone, polystyrene copolymer, fluoropolymer, polyester, polyamide, polystyrene-butadiene copolymer, fully hydrogenated styrene polymer, polycarbonate-PDMS copolymer, and polyolefins such as polyethylene, polypropylene, polymethylpentene, polypropylene copolymer, and cyclic olefin copolymer.

[0029] In some embodiments, the culture vessel (and / or units / compartments within the culture vessel) has a gas-permeable, liquid-impermeable film, thereby allowing gas to move between the cell culture chamber and the outside of the cell culture assembly. Such a culture vessel may have spacers or spacer layers positioned adjacent to the film outside the chamber to allow airflow between the stacked units. One commercially available example of a cell culture vessel including such stacked gas-permeable culture units is Corning's HYPERFLASK cell culture vessel. Such cell culture units may be manufactured in any suitable manner, for example, U.S. Patent Application No. 61 / 130421, filed May 30, 2008, entitled "Assembly of Cell Culture Vessels," which is incorporated herein by reference in its entirety, insofar as it does not conflict with this disclosure. Examples of suitable gas-permeable polymer materials useful for forming films include polystyrene, polyethylene, polycarbonate, polyolefin, ethylene vinyl acetate, polypropylene, polymethylpentene, polysulfone, polytetrafluoroethylene (PTFE), or compatible fluoropolymers, silicone rubber or copolymers, poly(styrene-butadiene-styrene), or combinations of these materials. A variety of polymer materials may be used, as long as their suitability for manufacturing and cell growth permits. Preferably, the film is of a thickness that allows for efficient gas transfer across the film. For example, a polystyrene film may be about 0.003 inches (about 75 μm) thick, but cell growth is possible at various thicknesses. Thus, the membrane may be any, preferably about 25–250 μm, or about 25–125 μm thick. The membrane can take any size or shape, allowing for free gas exchange between the assembly chamber and the external environment. In some embodiments, the gas-permeable film eliminates the need for an oxygen supply, as oxygen can be transferred from the surrounding environment through the gas-permeable substrate. Preferably, the membrane is durable for the manufacture, handling, and operation of the equipment.

[0030] Multiple embodiments of the present disclosure include a single housing or cabinet for accommodating one or more cell culture vessels, each having an internal cabinet cavity, thereby forming an optimized production system with a compact footprint and minimizing manual handling and manipulation of the vessels. The cabinet is sized to accommodate multiple cell culture vessels at once. In some embodiments, multiple cell culture means can be pre-configured or combined to form a single unit, and the cabinet is sized to accommodate one or more of these combined units. In some embodiments, the cabinet is portable. For example, the cabinet may be provided with wheels to facilitate handling and positioning. Being portable allows the cabinet to be placed in a convenient location with a laboratory or production facility, and multiple cabinets can be arranged in a compact array when it is convenient to do so (e.g., when manual user intervention is not required).

[0031] To minimize manual handling of cell culture vessels (or combined units), the cabinet is designed to perform operations on the cell culture vessels required during the culture process (e.g., repositioning or tilting). These operations may be semi-automated, fully automated, or involve manual operation by the user. For example, the cabinet may include mechanical or electromechanical means (as described herein) for tilting or repositioning the cell culture vessels while they are housed within the internal cavity. In some embodiments, the cabinet includes levers that can be manually operated by the user to tilt the entire cabinet or at least a portion of the cabinet containing the cell culture vessels. This tilting operation may be performed, for example, during filling and unfilling, thereby facilitating these operations when required for the multilayer cell culture vessels described herein.

[0032] For example, to disperse materials such as cell culture media, buffers, and proteolytic enzymes within a cell culture vessel, the cell culture vessel can be configured to tilt or change orientation during operation. Orienting the vessel in one or more different positions can facilitate the culture process of scaffold-dependent or adherent cells within the vessel. According to various embodiments, the vessel may be provided with a mechanism for changing the orientation of the vessel or tilting the vessel while housed in the cabinet described herein. In some embodiments, the cabinet is configured to change the orientation of the vessel or tilt the vessel. For example, the cabinet may be configured to provide mechanical lifting means such as an extension piston, a robotic arm, a lever, a mooring means, or an inclined shelf within the internal cavity of the cabinet. In some embodiments, a significant portion, most or all of the cabinet itself can change orientation or tilt, and consequently, one or more vessels stored within the cabinet can also change orientation or tilt. That is, according to various embodiments, tilting the vessel can result in a changing orientation of the vessel relative to the cabinet, or, if the orientation of the cabinet itself is changed, it can maintain the relative orientation of the vessel to the cabinet.

[0033] The possible orientations of the container may include one or more filling positions, one or more removal positions, one or more culture positions, etc. One or more filling positions may be defined as positions that can operate to fill the cell culture container (e.g., effectively fill it), and similarly, one or more removal positions may be defined as positions that can operate to empty the cell culture container (e.g., effectively empty it). Furthermore, there may be one or more filling positions, since there may be different optimal filling positions for each stage of the filling cycle. For example, to effectively fill the cell culture container, the container may be tilted at one or more specific or selected angles during the early stages of the filling cycle, and then tilted at one or more specific or selected angles different from the angles in the early stages during the later stages of the filling cycle. Furthermore, there may also be one or more removal positions, since there may be different optimal removal positions for each stage of the removal cycle. For example, to effectively empty the cell culture container, the container may be tilted at one or more specific or selected angles during the early parts of the removal cycle, and then tilted at one or more specific or selected angles different from the angles in the early stages during the later parts of the removal cycle. One or more culture locations may generally include a location where the cell culture or growth surface is parallel to the ground (for example, to promote effective cell proliferation). Furthermore, although several different locations have been described herein, the filling location, removal location, and incubation location / conditions may also be specific to the particular cell culture vessel used, and therefore the systems described herein may operate differently to suit the specific cell culture vessel used. In other words, the filling location, removal location, and incubation location / conditions described herein are not the only locations possible for the systems described herein, and furthermore, the systems described herein may be configured to suit multiple locations used for any particular cell culture vessel.

[0034] In some embodiments, the cell culture vessel and / or cabinet is configured to move the cell culture vessel about a first axis and a second axis, the first and second axes being perpendicular to each other and parallel to the ground (on which the cabinet is located). In some embodiments, the cabinet may be configured to move the cell culture vessel vertically along the vertical axis, thereby assisting, for example, the loading and unloading of the cell culture vessel into or on various other devices of the system for use. In some embodiments, only a simple tilt of the cell culture vessel about a single axis is used.

[0035] A cell culture vessel may include at least one port, which may be fluidly connected to a fluid source that can be supplied into the cell culture space of the vessel via a gravity feed or pressure feeder. The cell culture vessel may further include a manifold that fluidly connects each cell culture module or unit of the cell culture vessel to at least one port, thereby allowing material to be pressurized into and discharged from the cell culture vessel using at least one port.

[0036] The pumping device may be fluidly connected to each cell culture vessel. In at least one embodiment, the pumping device may include at least one pump for each cell culture vessel, for example, to maintain a closed system or to prevent cross-contamination when one pump is used for multiple cell culture vessels. In other words, the pumping device may include multiple pumps. Furthermore, the pumping device may include multiple valves, which may be used to selectively connect or fluidly connect one or more reservoirs to the pumping device, thereby allowing material placed in a reservoir to be pumped into a cell culture vessel and / or material placed in a cell culture vessel to be pumped into a reservoir. Each reservoir may be defined as a fluid-tight container or vessel configured to hold material. As used herein, “material” pumped, for example, into and from a cell culture vessel may be defined as any flowable material (e.g., liquid) that can be used in a cell culture process. For example, the materials may include cell culture medium (containing, for example, cells to be cultured), waste culture medium, proteolytic enzymes, quenching solutions, chelates, buffers, transfection agents, etc.

[0037] The pumping device and reservoir can be coupled to the operating device and / or any other part of the cell culture system, thereby integrating or incorporating the pumping device and reservoir into the cell culture system.

[0038] The cell culture systems described herein may further include a temperature control system. In several embodiments, the temperature control system includes an incubation apparatus. An incubation apparatus can generally be described as any apparatus capable of incubating a cell culture vessel to facilitate the incubation of cells within the cell culture vessel. For example, an incubation apparatus can apply heat to the cell culture vessel in the range of 30°C to about 40°C. In at least one embodiment, the incubation apparatus may completely enclose a cabinet. In at least one other embodiment, the incubation apparatus may be separate from the cabinet so that the cabinet can be placed inside the incubation apparatus for incubation together with the internal cell culture vessel and / or moved out of the incubation apparatus after incubation. In some embodiments, the incubation apparatus is incorporated into the cabinet, so that the cabinet controls the temperature within the internal cavity via an integrated temperature control device or incubation apparatus.

[0039] The thermally controlled environment may include controlled operation of ambient temperature (i.e., the temperature of the environment surrounding the system), or intermediate values ​​and ranges, such as approximately 15 to approximately 50°C, 15 to approximately 45°C, 27 to approximately 45°C, 30 to approximately 40°C, and 35 to approximately 38°C.

[0040] In embodiments where the cell culture system includes an incubator in which a cabinet and cell culture vessels are arranged, the incubator may include one or more ports that allow the passage of tubes and / or wires (or other signal carriers for power and / or sensors). Thus, cell culture operations (e.g., filling and removing) of the cell culture vessels can be performed while the cabinet remains inside the incubator. In some embodiments, one or more ports allow the passage of connectors from vessel sensors to detectors located outside the incubator, thereby eliminating the need to manufacture these detectors to withstand incubation temperatures and / or humidity.

[0041] As described above, embodiments of this disclosure include cell culture vessels having a gas-permeable film that provides a cell growth surface. Generally, passive gas exchange can establish appropriate soluble gas concentrations in the cell growth medium to meet the metabolic needs of cells in culture. The cell medium may rely on a carbon dioxide / bicarbonate buffer system to adjust the pH of the cell medium by interacting with dissolved carbon dioxide. This approach is sufficient for vessels placed in incubators where the carbon dioxide gas environment can be controlled. However, for vessels placed in thermally controlled environments such as incubators that do not have a controlled gas environment, laboratories, factories, and similar facilities, a good way to control pH is to change the composition of the growth medium, and the composition of the growth medium is not adjusted by carbon dioxide. Since many cell culturers are unwilling to change the composition of the growth medium or buffer, there may be a prejudice against using gas-permeable film vessels in thermally controlled environments.

[0042] Gas-permeable film containers (e.g., HYPERStack®, HYPERFlask®) are intended to provide users with a simple, passive gas diffusion system for supplying cells with the oxygen necessary for metabolism. However, when cells grow in a culture medium containing a carbon dioxide / bicarbonate-based buffer system, they do not function adequately in an environment without 5% carbon dioxide gas. Thermally controlled environments, such as high-temperature chambers, are large spaces maintained at a suitable incubation temperature of 370°C, but lack the humidity and gas control of typical incubators. Thermally controlled environments are typically used in relatively large containers, such as gas-permeable HYPERStack-36·120 stacked containers or gas-impermeable CellSTACK-10·40 stacked containers. Conventional gas-impermeable stacked cell culture vessels, such as CellSTACK-40 or Cell Factory-40, have headspace that allows for the addition of carbon dioxide gas during or before incubation, so that culture media containing carbon dioxide / bicarbonate-based buffer systems can still be used in a thermally controlled room environment after gas treatment.

[0043] Conventional gas-permeable film containers do not have an internal "headspace" like conventional containers. Therefore, by enclosing the entire gas-permeable film container in a gas-impermeable enclosure, a 5% carbon dioxide environment can be provided to all the gas-permeable film inside the container. This enclosure can be made of any suitable material, as long as gas impermeability is maintained. The enclosure may be, for example, flexible, such as a polybag, or inflexible, such as a rigid side enclosure. The gas-impermeable enclosure may be, for example, a flexible sheet, a semi-rigid sheet with sealable ends, a rigid sheet with sealable ends, and similar structures, or a combination thereof.

[0044] Those skilled in the art will understand the types of materials suitable for forming gas-impermeable enclosures. For example, conventional materials such as polyethylene terephthalate for flexible bag-shaped enclosures may be relatively thin (e.g., 4 mils (approximately 0.1016 mm)) but gas-impermeable. However, polypropylene materials may need to be thicker (e.g., 8 mils (approximately 0.2032 mm)) to reduce the gas permeability of polypropylene. Laminated materials may be used to provide certain properties, such as puncture resistance, strength in heat sealing, and excellent gas impermeability. Flexible bag-shaped enclosures do not need to be optically transparent, although this may be a desirable feature for the operator or user.

[0045] According to some embodiments, the gas-impermeable enclosure is sized to include at least one cell culture vessel having a gas-permeable film, as described herein. A gas inlet port penetrating the gas-impermeable enclosure may be provided to provide a gas communication between an external gas source and the cell culture vessel inside the enclosure. In some embodiments, the system may also be provided with an outlet port, or a passage between the cell culture vessel and an external outlet. The gas-impermeable enclosure may have ports for at least one sensor located inside or on the gas-impermeable enclosure to monitor the operation of the enclosed cell culture vessel. The ports may be hermetically sealed, for example, around a penetrating conduit, i.e., the conduit may be, for example, a tube for carrying gas, a cable for carrying optical elements, a wire for carrying signals, and similar functional structures. The inlet port may be fixed to the wall of the gas-impermeable enclosure bag, thereby allowing connection of tubes for actively exchanging gas inside and outside the enclosure, or any connection for locating fiber optic cables to, for example, various optical sensors.

[0046] The bag enclosure may be provided with an opening or vent (e.g., a manifold connection) to allow the container port to protrude for handling air and liquid. The opening may be elastic to form an airtight seal around the port, or it may be sealed by a tube or O-ring that can be added to the port after the bag enclosure is positioned to cover the container. The enclosure bag can be placed over the container and can be fastened, for example at the bottom, using, for example, a drawstring, cable tie, or other similar fastening means or method, thereby securing the bag enclosure around the container. Alternatively, the bag enclosure may have, for example, a zipper, a hook-and-loop structure (e.g., Velcro®) or a magnetic closure to allow the bag enclosure to cover and secure the outside of the container. Relatively less flexible gas-impermeable enclosures are also possible and may consist of, for example, pre-molded parts (e.g., molded material, thermoformed material, extruded material, etc.) that are appropriately sized to enclose the container to be sealed and can secure assembled parts. For example, a tubular structure including an airtight body, a base plate, a removable lid or cap, and the aforementioned communication sections, connection ports, and openings. A pre-formed enclosure assembly or enclosure lid or cap may include the aforementioned gas detection ports and connections required by the flexible enclosure.

[0047] In some embodiments, the system includes a control device, and at least one of the gas supply source, exhaust gas passage, and at least one of the at least one sensor communicates with the control device to control the gas characteristics (e.g., gas composition and gas concentration) within the gas-impermeable enclosure. The control device may be located outside the gas-impermeable enclosure enclosing the cell culture vessels and / or outside the cabinet, or it may be incorporated into one or more gas-impermeable enclosures or cabinets.

[0048] The gas source may be, for example, at least one of carbon dioxide (CO2), carbon dioxide equilibrium air, oxygen (O2), water vapor (humidity), and similar substances, or a combination thereof. At least one sensor may be, for example, at least one sensor for carbon dioxide, oxygen, pH, humidity, or a combination thereof. The relative percentages of carbon dioxide, oxygen, humidity, or a combination thereof within the gas-impermeable enclosure may be, for example, about 1 to 35% carbon dioxide, about 1 to 50% oxygen, and about 1 to 95% humidity. The given range of values ​​may include, for example, one or more of the following: about 1 to about 10% carbon dioxide; about 1 to about 30% oxygen; about 10 to about 95% relative humidity; and about 4 to about 9 acidity (pH).

[0049] Embodiments of the present disclosure include methods of using the cell culture system described herein, which includes a cell culture system having a gas-impermeable enclosure. Such methods may include, for example, monitoring the concentration or activity of at least one of the gases, gas mixtures, humidity, acidity (pH), or combinations thereof supplied to a gas-permeable film container having a cell culture contained in the cell culture system, and adjusting at least one of the gases, gas mixtures, humidity, acidity (pH), or combinations thereof. If the monitoring indicates a deviation from a predetermined range of values, a control device adjusts at least one of the gases, gas mixtures, humidity, acidity (pH), or combinations thereof to bring the system back to the predetermined range of values.

[0050] According to embodiments of the present disclosure, the spatial and operational requirements of high-yield cell culture vessels are reduced by providing a compact, low-floor high-yield culture system whose operation can be automated or semi-automated. The systems and methods of the present disclosure may include cell culture apparatus, enclosures (e.g., cabinets and / or gas-impermeable barriers or enclosures), sensors, fluid sources, connections and passages (e.g., tubing, fittings, manifolds, gas / medium sources, exhaust outlets, etc.), thermal control systems or temperature control systems, pumping systems and control systems.

[0051] The cell culture apparatus includes at least one cell culture vessel, a cabinet apparatus, a pumping apparatus, a monitoring apparatus, and a control apparatus. The at least one cell culture vessel is configured to culture cells using a plurality of parallel cell culture surfaces, and the at least one cell culture vessel includes at least one port configured to allow material to flow into and out of the at least one cell culture vessel. The cabinet is configured to hold the at least one cell culture vessel within its internal cavity. The cabinet is further configured to rotate the at least one cell culture vessel. Depending on the design of the cell culture vessel and the culture stage in which the vessel is rotated, the form and degree of rotation can be varied. In some embodiments, the rotation is a simple rotation about a single axis, while in other embodiments, it may be a complex rotation about a first rotation axis and a second rotation axis (for example, in this case the first rotation axis is perpendicular to the second rotation axis, and the first and second rotation axes are each parallel to the ground). The pumping device is fluidly connected to at least one port of at least one cell culture vessel and is configured to pump material into at least one cell culture vessel via at least one port and discharge it therefrom. The monitoring device is configured to monitor one or more parameters of at least one cell culture vessel, cabinet, and pumping device. The control device is operablely connected to the cabinet, pumping device, and monitoring device and is configured to adjust the movement of at least one cell culture vessel using the rotational movement of the cabinet, which involves pumping material into at least one culture vessel using the pumping device and discharging it therefrom.

[0052] In various embodiments, the control device is further configured to monitor one or more parameters of at least one cell culture vessel, cabinet, and pumping device using a monitoring device, and to adjust one or more parameters of at least one cell culture vessel, cabinet, and pumping device based on the one or more monitored parameters.

[0053] The cell culture systems described herein may include monitoring devices. Generally, monitoring devices may be configured to monitor any one or more parameters related to the cell culture system. For example, a monitoring device may be configured to monitor one or more of the following: cell culture vessels, cabinets, pumping devices, reservoirs, cell detachment devices, incubation devices, etc. Furthermore, monitoring devices may include position sensors, temperature sensors, pressure sensors, light sensors, fill position sensors, oxygen sensors, carbon dioxide sensors, pH sensors, gas concentration sensors, fluorescence imaging-based sensors, optical sensors, glucose sensors, lactate sensors, ammonium sensors, load cells (for example, for weighting cell culture vessels), electrical impedance sensors, ultrasonic impedance sensors, vision systems, and / or any other sensors that may be used in the cell culture system. Monitoring devices may be used by a control device of the cell culture system to monitor the cell culture system and provide feedback for adjusting one or more parameters related to the cell culture system. The cell culture vessels and modules of this disclosure may incorporate sensors for detecting cell aggregation and monitoring metabolites. In some embodiments, Raman probes may be used, and sensors for the Raman probes may be located outside the cabinet.

[0054] The control device for a cell culture system may include one or more computing devices capable of processing data. The control device may include, for example, a microprocessor, a programmable logic array, data storage (e.g., volatile or non-volatile memory and / or memory elements), input devices, output devices, etc. The control device may be programmed to carry out the methods or parts of the methods described herein and may be operably connected to each element of the cell culture system, for example, to monitor or adjust one or more parameters relating to each element of the cell culture system. For example, the control device may be operably connected to a cell culture vessel, an operating device, a pumping device, a reservoir, a cell detachment device, an incubation device, or a monitoring device.

[0055] Where the term "operably connected" is used herein, it may be defined as being connected (e.g., by wire or wireless) in such a way that information (e.g., image data, commands, etc.) can be transmitted between each object.

[0056] In several embodiments, the position sensor of the monitoring device may be configured to monitor the position of the cell culture vessel and / or the position of the cabinet or support surface within the cabinet, for example, the rotation of the cell culture vessel around a first axis parallel to the ground, the rotation of the cell culture vessel around a second axis parallel to the ground, the distance of the cell culture vessel from the ground, etc. Such position data may be used, for example, by a control device to confirm the movement made to the cell culture vessel during culture. In at least one other embodiment, the temperature sensor of the monitoring device may be configured to monitor the temperature inside or outside the cell culture vessel and / or cabinet, and / or the temperature inside the incubator device. Such temperature data may be used for monitoring and / or adjusting the incubator device.

[0057] In several embodiments, the monitoring device's pressure sensor may be configured to measure the pressure in each cell culture vessel or module, each reservoir, and / or incubation apparatus. In at least one embodiment, the monitoring device's fill level sensor or position sensor may be configured to monitor the amount of material (e.g., fill level) in the cell culture vessel or reservoir. Such fill level data may be used to determine whether or not the cell culture vessel is filled. In at least one embodiment, the monitoring device's oxygen sensor may be configured to monitor the oxygen concentration in the cell culture vessel, gas-impermeable enclosure, cabinet, reservoir, or incubation apparatus, and the monitoring device's carbon dioxide sensor may be configured to monitor the carbon dioxide concentration in the cell culture vessel, gas-impermeable enclosure, cabinet, reservoir, or incubation apparatus. In several embodiments, the control device may be configured to change the rate at which material is pumped into and discharged from each culture vessel using a pumping device based on one or more monitoring parameters of the culture vessel.

[0058] In several embodiments, the optical sensor of the monitoring device may be configured to image the material in the cell culture vessel (e.g., the cell culture medium), and the control device may be configured to provide the image to the user. Furthermore, the user may be located away from the system; for example, the user can view images of the cell culture without being positioned locally or nearby to the system. In other words, the cell culture system can provide a means of remote visualization of the cell culture (e.g., it can provide a rapid evaluation of cell aggregation). Moreover, such a remote visualization means can also be used to inspect the cells after they have been detached from the cell culture surface. In practice, the optical sensor of the monitoring device may also provide a remote microscope for observing the cell culture.

[0059] The cell culture systems described herein can provide semi-automated or fully automated solutions for achieving one or more processes used in seeding, growing, and harvesting adhesive cells from stacked cell culture vessels. Furthermore, the systems described herein can combine multiple separate components and integrate them into a single central computer-controlled machine, which may use one or more detection devices to provide feedback to the cell culturer or user. Furthermore, one or more cell culture systems described herein may include a human-machine interface (HMI) that allows a user to input numerical process variables specific to the needs of cell culture; a full computer or programmable logic control (PLC) for controlling one or more cell culture parameters, such as the filling rate, filling pressure and filling volume of each cell culture vessel; semi-automatic or fully automatic positioning means for vessels that coordinate with pump speed adjustments during filling / removal, equilibration and cell removal stages; manually controlled or semi-automatic or fully automatic valves for controlling the flow of medium into and out of the vessels; specific positioning means for vent filters to avoid wetting; automatic pressure testing means for ensuring vessel integrity; built-in safety features; and process monitoring means for time, temperature, pH, gas concentration and metabolites.

[0060] Embodiments of cell culture systems described herein may include monitoring devices, such as one or more sensors, that can be configured to detect flow rate, filling volume, temperature, pressure, etc., to the cell culture vessel. In at least one embodiment, the cell culture system may be configured to apply pressure to the cell culture vessel and monitor it to ensure vessel integrity. Furthermore, in at least one embodiment, the cell culture system may incorporate or include means for controlling the temperature and gas concentration inside and / or around the cell culture vessel.

[0061] Furthermore, in various embodiments, the cell culture system described herein may include one or more human-machine interfaces that can be configured to enable a human operator to monitor and adjust automated processes, as well as to monitor cell culture conditions such as pH, gas concentration, metabolites, and temperature. Moreover, such human-machine interfaces may be located remotely, thereby eliminating the need for, for example, the physical presence of a local human operator to the system.

[0062] The cell culture system may further include a cell detachment device. Generally, the cell detachment device may be operable to detach cells that have adhered, bound to, or fixed to the cell culture surface or growth surface of a cell culture vessel, for example, after the cells have been cultured. In at least one embodiment, the cell detachment device may include a shaker configured to shake the cell culture vessel at frequencies such as about 0.1 kHz, about 0.5 kHz, about 1 kHz or higher and / or about 5 kHz, about 10 kHz, about 15 kHz, about 20 kHz or lower, to detach at least a portion of the multiple cells adhering to the cell culture surface of the cell culture vessel. In at least one embodiment, the cell detachment device may include a shaker configured to shake the cell culture vessel with an amplitude of about 12 mm to about 26 mm. Furthermore, the shaking path may be oriented at a wide range of angles with respect to the cell culture surface of the cell culture vessel. For example, the shaker may be configured to move the cell culture vessel 12 in a circular path, vertically, parallel to the cell culture surface, and in a linear reciprocating motion. The shaking device may be such as that described in U.S. Provisional Patent Application No. 61 / 527,164, filed on August 25, 2011, entitled “METHODS OF RELEASING CELLS ADHERED TO A CELL CULTURE SURFACE,” which is incorporated herein by reference in its entirety, insofar as it does not conflict with the disclosures presented herein.

[0063] The shaking device may be integrated with or separate from the cabinet. For example, the shaking device may be coupled to the cabinet and configured to shake at least a portion of the cabinet, thereby shaking the cell culture vessels held by the cabinet. In some embodiments, the shaking device may be housed within the cabinet and configured to shake the cell culture vessels within the cabinet without shaking the entire cabinet. For example, support surfaces or shelves may be provided within the internal cavity of the cabinet, and the shaking device may include those shelves or equipment provided on the shelves. Furthermore, for example, the shaking device may be located away from the cabinet, at a distance, or separately. In this example, the cabinet or the shaking device can be moved relative to each other to position the shaking device and the cell culture vessels in contact with each other, thereby allowing the shaking device to shake the cell culture vessels and detach at least a portion of the cells adhering to the cell culture surface of the cell culture vessels. In several embodiments, the shaking device may be configured to contact or come very close to at least a portion of the cell culture vessel and to slide across the cell culture vessel or relative to the cell culture vessel, thereby supplying shaking energy to the portion of the cell culture vessel when the transducer slides relative to the vessel.

[0064] In several embodiments, the shaking device may include a platform on which cell culture vessels can be placed inside a cabinet. After the cell culture vessels are placed on the platform, the platform shakes, thereby shaking the cell culture vessels and causing at least some of the cells adhering to the cell culture surface of the vessels to detach.

[0065] In several embodiments, the cell detachment apparatus may include an ultrasonic transducer device configured to supply ultrasonic energy to a cell culture vessel at frequencies such as approximately 1 kHz, approximately 10 kHz, approximately 15 kHz or higher, and approximately 20 kHz, approximately 30 kHz, approximately 40 kHz or lower. Furthermore, the ultrasonic transducer device may be configured to supply ultrasonic energy to each cell culture vessel once or more times for approximately 5 to 30 seconds. For example, the ultrasonic transducer device may be such as that described in U.S. Patent Application Publication No. 2009 / 0298153, filed on 19 May 2009 and published on 3 December 2009, titled "METHOD FOR ULTRASONIC CELL REMOVAL," which is incorporated herein by reference in its entirety, insofar as it does not conflict with the disclosures presented herein. Furthermore, the ultrasonic transducer device may be movable relative to the cell culture vessel and / or cabinet, thereby being configured to supply ultrasonic energy to at least one of one or more chambers, units, modules, or compartments of the cell culture vessel. For example, an ultrasonic transducer device may be configured to contact or come very close to at least a portion of a cell culture vessel and to slide across or relative to the cell culture vessel, thereby supplying ultrasonic energy to the portion of the cell culture vessel when the transducer slides relative to the vessel. Furthermore, for example, the ultrasonic transducer device may be directional, thereby being configured to guide or spread ultrasonic energy across the cell culture vessel, for example using a horn.

[0066] Embodiments of the present disclosure include methods using the cell culture system described herein. The method may include the steps of manipulating one or more cell culture vessels and moving materials into or out of one or more cell culture vessels either during or after the manipulation. For example, the cell culture vessels can be manipulated to a filled position, and after being manipulated to the filled position, the method can begin transferring materials, such as cell culture media, from one or more reservoirs to the cell culture vessels. Furthermore, for example, the cell culture vessels can be manipulated to a removal position, and after being manipulated to the removal position, the method can begin transferring materials, such as waste culture medium, collected cells, etc., from the cell culture vessels to one or more reservoirs.

[0067] A filling and unfilling method for use with the cell culture system described herein may include multiple different positions to facilitate the filling and unfilling method. Thus, the method manipulates the cell culture vessel (e.g., to one or more positions) continuously, periodically, or as needed, while transferring material to and from the cell culture vessel (as indicated by arrows returning in a loop from process to process). In at least one embodiment, the manipulation of the cell culture vessel and the transfer of material can be performed simultaneously. In several embodiments, one or more sensors can detect the filling level of the culture vessel, thereby providing feedback to a control unit for the purpose of manipulating the vessel to the appropriate position during the filling and unfilling process. Any suitable sensor may be used to detect the filling level of the vessel during the filling or unfilling process. In several embodiments, a load sensor or another mass sensor may be used to measure the mass of each of at least one cell culture vessel for the purpose of detecting the filling level (e.g., data from the load sensor or another mass sensor may be used to coordinate the movement of at least one cell culture vessel and / or cabinet with the pumping of material into and out of at least one culture vessel using a pumping device). In several embodiments, one or more optical sensors, infrared sensors, etc., may be appropriately positioned along the culture vessel to detect the filling level.

[0068] This disclosure describes a cell culture system that enables semi-automated or fully automated filling and / or removal of liquid culture medium in cell culture vessels. Embodiments of the system may include different combinations of the various components described herein, including cell culture vessels; storage cabinets; filling sensors for detecting the filling level of the cell culture vessels; actuators for repositioning the cell culture vessels and / or cabinets during filling; control devices; pressure sensors; and one or more of various connecting means, fittings, tubing, and manifolds. Embodiments described herein use filling sensors to monitor the liquid culture medium level during filling or removal of the vessels and reposition the cell culture vessels or adjust the filling rate according to the filling level. The systems and methods disclosed herein can enable semi-automated or fully automated filling and / or removal of cell culture vessels. As a result, cell culture systems and methods are provided that reduce the risk of leaks, contamination, and other stresses in the cell culture system, thereby reducing the degree of user monitoring and attention required during the filling or removal procedure.

[0069] In embodiments of the present disclosure, one or more sensors may be used to measure the fill level in a cell culture vessel or manifold. Since the fill rate may vary slightly across each cell culture device, if a user attempts to fill multiple vessels at once, sensors in each cell culture device can determine the appropriate time to change the orientation of each particular vessel or to alter the fluid flow.

[0070] Referring to Figure 1, the cell culture vessel 10 has three cell culture modules 12, 14, and 16, each having multiple layers of cell culture chambers 18, one stacked on top of the other to form a multilayer cell culture vessel 10. Each cell culture module 12, 14, and 16 uses two manifolds 20 and 22. Through the first manifold 20, liquid can flow into and out of the cell culture modules 12, 14, and 16. Therefore, the first manifold 20 may also be called a fluid manifold. Through the second manifold 22, air can flow into and out of the cell culture modules 12, 14, and 16. Therefore, the second manifold 22 may also be called an air manifold.

[0071] Each of the cell culture modules 12, 14, and 16 may include multiple stack layers 24, which, when stacked together, form multiple cell culture chambers 18 with conduit spaces (air spaces) 25 between them, as shown in Figure 2. Figure 2 is a schematic diagram of multiple stack layers 24 stacked together to form stacked cell culture chambers 18 and cell culture surfaces 26 having a gas-permeable, liquid-impermeable film 28, for example, the stack layers 24 include conduit spaces 25 that allow gas to move between the cell culture chambers 18 and the outside of the cell culture vessel 10. Referring again to Figure 1, the cell culture modules 12, 14, and 16 may be isolated from each other by spacers 31, 33, and 35. The spacers 31, 33, and 35 can provide structural support for the individual cell culture modules 12, 14, and 16. In some embodiments, to increase the total number of cell culture chambers 18, the spacers 31 and / or 33 may be replaced with additional stack layers 24. Furthermore, a riser volume may be provided above the cell culture module 12 to capture residual air rather than the air present in the cell culture chamber 18.

[0072] In some embodiments, the culture module has a gas-permeable, liquid-impermeable film 28, which allows gas to move between the cell culture chamber 18 and ultimately outside the cell culture vessel. Such a culture module may have spacers or spacer layers positioned adjacent to the film outside the chamber to allow airflow between the stacked units. One commercially available example of a cell incubator including such stacked gas-permeable culture units is Corning's HYPERStack® cell incubator.

[0073] As described above, the cell culture modules 12, 14, and 16 can be connected to each other using manifolds 20 and 22. The manifold 20 has a sidewall base structure 30 and a column structure 32 formed as an integral part of the sidewall base structure 30, which provides an integrated manifold 20. The column structure 32 has a projection structure 34, which provides at least a portion of the fluid channels from the projection structure 34 that are fluidly connected to the individual cell culture chambers 18 in the cell culture modules 12, 14, and 16. The manifold 20 can be configured to allow filling and removing of the cell culture chambers 18.

[0074] The manifold 22 also has a side wall base structure 30' and a column structure 32' formed as an integral part of the side wall base structure 30' to provide an integrated manifold 22. The column structure 32' has a projection structure 34' which provides at least a portion of the fluid flow path from the individual cell culture chambers 18 in the cell culture modules 12, 14 and 16 to the projection structure 34'. The manifold 22 may be configured to allow filling and removing of the cell culture chambers 18 by enabling the inflow and outflow of air in the cell culture vessel 10. In some embodiments, the column structure 32' may be offset from the illustrated position to control the inflow of the medium into the column structure 32'.

[0075] Regarding a typical filling procedure, the cell incubator 10 may be positioned with its left side facing down, towards the support surface or tray. In this orientation, the front of the cell incubator 10, equipped with manifolds 20 and 22, is tilted downward to become the first filling orientation at the start of filling (see side view in Figure 3). The flow of liquid medium into the cell culture vessel is then initiated. For example, the medium may be pumped into the lower column structure 32 via the projection structure 34 using a peristaltic pump, or the vessel may be filled by a flow generated by gravity. Once the liquid medium in the cell incubator 10 has risen to the first filling level in a predetermined position, the cell incubator 10 (and the filling tray, if used) is reoriented to a second filling position. In this second filling orientation, filling can continue until the liquid medium reaches the final filling level in the cell incubator 10. Once the final filling level is reached, the flow of medium is stopped, and the inlets and outlets from the manifolds 20 and 22 may be blocked or closed to shut off the system. At this time, the cell culture device 10 is ready for use in cell culture.

[0076] Embodiments of the present disclosure include, as described above, a type of packing tray or multi-position support. This multi-position support may be incorporated into a support surface within the internal cavity of a cabinet. For example, the multi-position support may be positioned on the support surface, or the support surface may take the form of a multi-position support. Embodiments are not limited to using the illustrated multi-position support. However, to illustrate the tilting operation of cell culture vessels in some embodiments, the multi-position support shown in the accompanying drawings is described below. Further details of the multi-position support can be found in U.S. Provisional Patent Application No. 63 / 056,913, filed July 27, 2020, which is incorporated herein by reference.

[0077] Referring to Figure 3, the cell culture vessel 10 can be filled and removed using the multi-position support 50 by tilting it with its side surface 40 facing downwards, as shown in Figure 3. The cell culture vessel 10 can be reliably positioned at its side surface 40 at a predetermined inclination angle θ1 (e.g., about 10 to 12 degrees) relative to the support member 42 or the horizontal plane using the multi-position support 50. The side surface 40 closest to the fluid manifold 20 is positioned on the multi-position support 50 such that the fluid manifold 20 is lower than the air manifold 22. As will be described in more detail below, the multi-position support 50 may be tilted between an upright configuration (as shown in Figure 3) and an inclined configuration for positioning the cell culture vessel 10 at different angles relative to the horizontal plane.

[0078] Referring to Figures 4 and 5, the multi-position support 50 is shown alone and is formed as a single bent plate having a bottom 52, an upper 54, opposing ends 56 and 58, and opposing sides 60 and 62. At the sides 62, the multi-position support 50 has positioning tabs 64 and 66, which help to engage the lower edge 68 (Figure 3) of the cell culture vessel 10 with the multi-position support 50 in an upright position, thereby holding the cell culture vessel 10 in a predetermined position on the multi-position support 50. In some embodiments, the lower edge 68 of the cell culture vessel 10 may be provided with recesses 71 and 73 that are sized and positioned to receive the positioning tabs 64 and 66. The positioning tabs 64 and 66 may have bends 75 that can be used to grip the lower edge 68 and prevent the cell culture vessel 10 from moving laterally away from the multi-position support 50.

[0079] The multi-position support 50 has a main base 70, which in the upright configuration of the multi-position support 50 as shown in the figure is supported by a support member (e.g., a table or laboratory bench). A main support surface 72 is provided, which is vertically offset from the main base 70 in the upright configuration and supports the cell culture vessel 10. The multi-position support 50 further has an intermediate surface 74 extending between the main base 70 and the main support surface 72. The intermediate surface 74 is in contact with the main base 70 at a connecting portion 76 formed as a bent portion extending at an angle with respect to the sides 60 and 62 of the multi-position support 50. The intermediate surface 74 is also in contact with the main support surface 72 at a connecting portion 77 formed as a bent portion extending at an angle with respect to the sides 60 and 62. In some embodiments, the angles of the connecting portions 76 and 77 may be approximately the same (e.g., within 5 degrees) as with respect to the sides 60 and 62, or they may be different.

[0080] The multi-position support 50 further has a sub-base 79 supported by a support member in the upright configuration. In the upright configuration, a sub-support surface 78 is provided, which is vertically offset from the sub-base 79 and supports the cell culture vessel 10. The sub-support surface 78 and the main support surface 72 are located on the same plane, forming a predetermined angle with respect to the horizontal plane and also inclined with respect to the main base 70 and the sub-base 79. The multi-position support 50 further has another intermediate surface 80 extending between the sub-base 79 and the sub-support surface 78. The intermediate surface 80 is in contact with the sub-base 79 at a connecting portion 82 formed as a bent portion extending perpendicularly to the sides 60 and 62 of the multi-position support 50. Yet another intermediate surface 84 extends between the main base 70 and the sub-support surface 78. The intermediate surface 84 is in contact with the main base 70 at a connecting portion 86 which is formed as a bent portion that also extends perpendicularly to the side portions 60 and 62. A gripping portion 88 is provided at the end portion 56. The gripping portion 88 may have a support flange 90 that is vertically offset from the sub-base 79 in the upright configuration and supports the cell culture vessel 10. The end portion 58 is provided with a support flange 94 that extends vertically outward from the main support surface 72 and is used to hold the cell culture vessel 10 on the main support surface 72.

[0081] The multi-position support 50 shown in Figure 3 supports the cell culture vessel 10 in an upright configuration. In the upright configuration, the cell culture vessel 10 has a rear section 100 that is higher than the front section 102 at an angle θ1 (10 to 12 degrees) with respect to the horizontal plane. However, the vertical angle is parallel (zero degrees) to the horizontal plane. This upright configuration allows the cell culture vessel 10 to be positioned at the initial filling position when filling begins, in which case the front section 102 is lower than the rear section 100, which provides a gentler filling angle, reduces foaming in the fluid, and facilitates air discharge through the air manifold and the filter connected thereto.

[0082] When the cell culture vessel 10 is filled using the upright multi-position support 50, the fluid level inside the cell culture vessel 10 rises toward the air manifold 22 and toward the filter connected to the air manifold. If the filter gets wet, it may reduce the airflow rate from the cell culture vessel 10, which can pressurize the inside and create an undesirable environment inside the cell culture vessel 10. To reduce the possibility of fluid reaching the filter, the multi-position support 50 is provided with a tilt configuration that allows the multi-position support 50 to rotate together with the cell culture vessel 10 without lifting the multi-position support 50 or the cell culture vessel 10. By simply applying a force F to the rear corner 110 of the cell culture vessel 10, the multi-position support 50 can be manually tilted together with the cell culture vessel 10, thereby rotating the multi-position support 50 and the cell culture vessel 10 around the connection 76. Since the connection portion 76 extends obliquely to the sides 60 and 62 of the multi-position support 50, this inclination changes both the angle from front to rear and the angle from top to bottom, thereby increasing the rise of the top of the air manifold to which the filter is connected. According to the embodiments described below, this tilting operation may be performed by an automated cell culture system without the application of a manual force F. However, the same multi-position support 50 described in the figures may be used for both manual and automated tilting.

[0083] Referring to Figure 6, the multi-position support 50 and the cell culture vessel 10 are shown in an inclined configuration, in which the front portion 102 is higher than the rear portion 100, resulting in an angle θ2 (11 to 13 degrees) with respect to the horizontal plane. As can be seen, in the inclined configuration, the corner portion 112 between the side portion 40 and the rear portion 100 of the cell culture vessel 10 is supported by a support member. Referring to Figure 7, the upper portion 116 is higher than the lower portion 114 at an angle θ3 (7 to 9 degrees) with respect to the horizontal plane. Thus, the inclined configuration provides the multi-position support 50 and the cell culture vessel 10 with a composite angle of both θ2 (front to rear) and θ3 (upper to lower), which can be called the final filled position. When the cell culture vessel 10 is filled, the side portion 60 of the multi-position support 50 closest to the upper portion 116 of the cell culture vessel 10 may be rotated upward until the cell culture vessel 10 is in an upright position. Therefore, the cell culture vessel 10 can be operated using only the multi-position support 50 throughout the entire filling process without the need to lift the cell culture vessel 10 from the multi-position support 50. Removal from the cell culture vessel 10 may be performed in the reverse order.

[0084] The multi-position support described above can be used to operate cell incubators without the need to handle the cell incubator separately from the multi-position support during filling or removing operations. This allows the multi-position support to increase processing efficiency and save user time based on higher filling and removing speeds and simple, quick angle change procedures. The multi-position support can further provide a clear and concise control protocol that can reduce errors, decrease the possibility of product failure and / or damage, and reduce angle changes caused by the support method and fixed tilt angle using the multi-position support. By providing a multi-position support with a composite tilt angle, infiltration changes of filters attached to air manifolds are reduced. In some embodiments, the multi-position device can be formed from stainless steel, which can provide improved durability and meet Good Manufacturing Practices (GMP). The multi-position device can be formed from sheet material in metal brakes to reduce manufacturing costs. Changes can be made without incurring significant costs for tool remanufacturing.

[0085] The orientation of the multi-position support can be performed manually in some embodiments. In another embodiment, the tilting of the multi-position support is automated and controlled by the control system of the cell culture system described above.

[0086] Figure 8 is a cross-sectional view of a cell culture vessel 200 according to another embodiment. Similar to the embodiments shown in Figures 1 and 2, the vessel 200 is a multilayer cell culture vessel. In Figure 8, the vessel 200 includes a cell culture space 201 illustrated with 10 cell culture layers 202. However, it is understood that multiple embodiments may include vessels with more or fewer layers. Each layer includes a polymer support surface (layer 202) for growing scaffold-dependent or adhesive cells and a gas-permeable film 204. The vessel 200 is shown filled with liquid medium 206 used during the cell culture process. The vessel 200 may have a planar surface on the layers 202 for 2D cell culture. When used for static cell culture (as opposed to perfusion cell culture), the vessel 200 may be provided with a vent 208, which can, for example, release exhaust gases from the culture space within the vessel 200.

[0087] Figure 9 shows a variation of the embodiment shown in Figure 8 adapted for 3D cell culture. Specifically, Figure 9 shows a cell culture vessel 200' having a structure similar to vessel 200, but with a cell proliferation surface formed by a gas-permeable film 204'. The gas-permeable film 204' has a 3D surface, on which wells or microcavities for 3D cell culture are formed.

[0088] In some embodiments, multiple containers 200 and 200' shown in Figures 8 and 9 can be stacked or joined together to form one larger cell culture vessel. In such cases, containers 200 and 200' each function as individual modules of the larger vessel. Figure 10 shows a front view of an example of a container 210 containing multiple such cell culture modules 212a-212e (similar to 200 or 200'). Although five modules 212a-212e are shown in Figure 10, several embodiments may have more or fewer modules 212 within the container 210. The lower module 212a is provided with an inlet 214, which is fluidly connected to the cell culture space within the lower module 212a. Each module 212a-212e is connected to an adjacent module so that the cell culture spaces within all modules 212a-212e are fluidly connected to each other. Thus, culture medium can be filled into the entire cell culture space of the container 210 via the inlet 214. The outlet 215 of module 212e is also provided with a vent 216, which allows gas to be discharged from the cell culture space of the container 210. The vent 216 may be equipped with a filter and is used to allow exhaust gas to pass through during static cell culture. For example, the vent 216 allows air to escape from the container 210 if the air is moved by the fluid filling the container 210, and also allows air to flow into the container 210 if the fluid is discharged from the container 210 through the inlet 214 during static culture. In perfusion culture, the outlet 215 may be connected to a tube for carrying the liquid out of the cell culture space of the container 210. The inlet 214 and outlet 215 are located at opposite corners on the diagonal of the container 210. That is, the inlet 214 is located at the lower right corner of the front of the container 210 (as seen in Figure 10), and the outlet 215 is located at the upper left corner of the back of the container 210 (as seen in Figure 10). The relative positioning of the inlet 214 and outlet 215 may affect the operations required for the filling and removal process.The arrangement on opposite sides along the diagonal shown in FIG. 10 is one preferred embodiment, but other positions can be considered as well.

[0089] Compared with the container shown in FIG. 1 having an inlet (32) and an outlet or vent (32') on the same side of the container 10, the inlets 214 and outlets 215 on opposite sides along the diagonal greatly simplify the operation of the container 210 during filling and removal, and provide the advantage that there is no need to expand the container and operate it individually. This enables perfusion flow as well, if desired by the user.

[0090] Optionally, as described herein, one or more of the modules 212a - 212e may be provided with a sensor 218 for measuring parameters of cell culture. The sensor 218 can be used, for example, to detect cell aggregates or monitor metabolites.

[0091] In some embodiments, the container 210 shown in FIG. 10 can provide a cell culture area of more than 7,000 cm 2 sup, more than 14,000 cm 2 sup, more than 18,0,00 cm 2 sup, more than 36,000 cm 2 sup, or more than 50,000 cm 2 sup. For example, in the embodiment shown in FIG. 10, the surface area is about 50,000 cm 2 , which is larger than the 18,000 cm 2 surface area provided by a commercially available HYPERStack® unit. Viewed another way, an embodiment occupying an installation area of 50,000 cm 2 is smaller than the installation area occupied by several HYPERStack® units required to fit or exceed a surface area of 50,000 cm 2 . Further, in some embodiments, the hydrostatic head pressure required for the operation of the container 210 shown in FIG. 10 may be only about 0.5 psi (3447.38 Pa).

[0092] Figures 11A–11C show three stages of the filling process for container 210. Container 210 is shown positioned on a horizontal or flat surface 220 (i.e., surface 220 is parallel to the ground). While filling the medium 222 through the inlet 214, container 210 is tilted to an angle θ defined by the angle between the bottom surface 224 of container 210 and the support surface 220. The filling angle θ allows the container 210 to be filled to a predetermined filling pressure while minimizing bubble formation by the container 210 as the level of the culture medium 222 rises and air is expelled through the outlet 215, as shown in Figures 11A–11C. Based on the horizontal positioning of the inlet 214 and outlet 215 on opposite sides (see Figure 10), a tilt angle θ of simply 5° may be used, or angles between 1° and 10° may be used, or angles between 5° and 20° may be used, or angles between 10° and 45° may be used.

[0093] Figure 12 shows one embodiment of a cell culture system 300 using a cabinet 302 for housing multiple cell culture vessels 210. The form and structure of the vessels 210 correspond to those shown in Figures 10 and 11, but the cabinet 302 can also be similarly adapted for use with other types of vessels. The cabinet 302 includes an internal cavity 304 and may include one or more support surfaces 306 arranged to support one or more cell culture vessels 210. As shown, multiple cell culture vessels 210 may be provided on each support surface 306, and multiple support surfaces 306 enable a high-density cell culture system with a small footprint. The inlets 214 of the vessels 210 are connected to each other by tubes 308 connecting the various inlets 214 in this example. Thus, culture medium can be supplied to all vessels 210 via a main input line 310 connected to the vessels 210 via ports 312 of the cabinet 302.

[0094] During operation, the container 210 is loaded by the user into the internal cavity 304 of the cart 302. The user can then connect different containers 210 on a single support surface 306, for example via tubes 308. These containers 210 connected on each support surface 306 can be connected to containers connected on another support surface 306 via additional tubes 308. The main input line 310 is connected to a new culture medium or, in the case of resupply, to a waste container (not shown). The flow can be controlled using valves or clamps, thereby limiting individual filling to each shelf and avoiding an increase in hydrostatic pressure. The cart 302 also has a tilting capability, for example, used during the filling or removal of containers 210. The cart 302 may further include an electrical plug for supplying power to the system in the cabinet 302. The cabinet 302 may include, for example, an electromechanical valve, an electromechanical tilting mechanism, or an incubation system for maintaining a heated environment for cell culture, as described herein.

[0095] In some embodiments, sensors are positioned in front of the vent filter 216 and wirelessly connected to valves at each container inlet 214, thereby regulating the flow of culture medium to reach the appropriate filling level and preventing overfilling. A human / machine interface (HMI) provided on (or communicating with) the cart 302 can enable programming of the sensors and valves, thereby allowing filling and removal without manual operation of clamps.

[0096] Figure 13 shows a cell culture system 350 of another embodiment according to the present disclosure. Similar to Figure 12, the system 350 is provided with a cabinet 302 for housing a plurality of cell culture vessels 210. The components and structure of the cabinet 302 and vessels 210 corresponding to those described above will not be repeated with reference to Figure 13. As shown, the cabinet 302 is surrounded by a gas-impermeable enclosure 352, which extends vertically to receive tubing through ports 356 for controlling gases within the internal cavity of the cabinet 302. The gas-impermeable enclosure 352 may have flexible or rigid sides and may be formed as part of a cart or as a separate entity. The ports 356 or additional ports through the gas-impermeable enclosure 352 are available for sensor connections or any other monitoring or control systems. The gas-impermeable enclosure may act to retain heat generated by the cart and disperse gases that may be humidified or heated.

[0097] According to some embodiments, multiple containers may be joined to one another via structural members or rails, thereby forming a combined container unit. These structural members may include rails along the sides and corners or edges of the containers. While the rails may have the effect of separating the individual containers, structural support means and connecting means may also be provided between the containers. These rails can also protect the containers from collisions with each other during transport or operation, while also allowing for a denser footprint.

[0098] Incubation may be required during cell culture. In some embodiments, a temperature control system that enables incubation can be incorporated into the cabinet 302. In other embodiments, the cabinet 302, which may be portable, may be moved into an incubator that houses the entire cabinet 302.

[0099] Figures 14A and 14B illustrate another embodiment of the cell culture system. As described herein, the cell culture system allows the cell culture vessels to be tilted while they are in the system's cabinet. In some embodiments, this is achieved by tilting the entire cabinet, as shown in Figure 14B. For example, the cabinet 400 is mounted on a support or cart 402 in an upright configuration, as shown in Figure 14A. The cart 402 can also be tilted into an inclined configuration, as shown in Figure 14B, if desired. For example, for the system disclosed herein, a tilt of just 5° may suffice. This tilting may be the only operation required during the culture process, and therefore the cart 402 and cabinet can be simple structures for achieving this tilting operation. The cart 402 also allows the vessels to be maintained in a tight, high-density format, which can be done inside the incubator without the need to move the vessels from the incubator over a table or cart for fluid exchange, as required with existing cell culture vessels. Alternatively, the incubator may include a mechanism that causes tilting instead of carts. Optionally, the cart handles may be removed to save space once the HYPERBioreactor is ready for incubation.

[0100] Embodiments of cell culture systems and related methods have been disclosed. Those skilled in the art will recognize that the cell culture systems and methods described herein may be carried out in embodiments other than those disclosed. The disclosed embodiments are presented for illustrative purposes only, and not for limiting purposes.

[0101] Exemplary Embodiments The following describes various aspects of the embodiments of the disclosed subject matter. Each aspect may include one or more of the various mechanisms, features, 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 embodiments.

[0102] Embodiment 1 relates to a cell culture system comprising at least one multilayer container configured for culturing cells, the multilayer container containing a cell culture space, and a cabinet having an internal cavity surrounded by one or more side walls, the cabinet configured to house the multilayer container within the internal cavity, the cabinet being configured to change the orientation of the multilayer container from an upright orientation to a tilted orientation.

[0103] Embodiment 2 relates to the cell culture system according to Embodiment 1, further comprising at least one sensor configured to detect characteristics within a cell culture space.

[0104] Embodiment 3 relates to the cell culture system according to Embodiment 2, wherein the sensor includes at least one of a collection monitor and an analyte monitor.

[0105] Embodiment 4 relates to the cell culture system according to Embodiment 2 or 3, wherein the sensor is incorporated into a multilayer container.

[0106] Embodiment 5 relates to the cell culture system according to Embodiment 2 or 3, wherein a sensor is mounted on a cabinet and is arranged to detect characteristics within the cell culture space when a multilayer container is placed inside the cabinet.

[0107] Embodiment 6 relates to a cell culture system according to any one of Embodiments 2 to 5, wherein the multilayer container has at least one sensor window, and the sensor is configured to detect characteristics within the cell culture space through the sensor window.

[0108] Embodiment 7 relates to a cell culture system according to any one of Embodiments 1 to 6, wherein the cabinet has a plurality of support surfaces configured to support at least one multilayer container, each of which is configured to support at least one multilayer container.

[0109] Embodiment 8 relates to a cell culture system according to any one of Embodiments 1 to 7, wherein at least one multilayer container has a plurality of multilayer cell culture modules.

[0110] Embodiment 9 relates to the cell culture system according to Embodiment 8, wherein at least some of a plurality of multilayer cell culture modules are connected to one another.

[0111] Embodiment 10 relates to a cell culture system according to any one of Embodiments 1 to 9, wherein the multilayer container has an inlet and an outlet, the inlet is configured to supply a liquid culture medium to a cell culture space, and the outlet is configured to allow a liquid or gas to flow into or out of the cell culture space.

[0112] Embodiment 11 relates to the cell culture system according to Embodiment 10, wherein the inlet is located at the bottom of the multilayer container.

[0113] Embodiment 12 relates to the cell culture system according to Embodiment 10 or 11, wherein the outlet is located at the top of the multilayer container.

[0114] Embodiment 13 relates to the cell culture system according to Embodiment 12, wherein the outlet is located on the opposite side of the multilayer container diagonally from the inlet.

[0115] Embodiment 14 relates to a cell culture system according to any one of Embodiments 10 to 13, wherein the outlet has a vent port configured to allow gas to escape from or into the cell culture space.

[0116] Embodiment 15 relates to a cell culture system according to any one of Embodiments 10 to 14, wherein the outlet has a filter.

[0117] Embodiment 16 is a multilayer container with a minimum capacity of 18,000 cm². 2 The present invention relates to a cell culture system according to any one of embodiments 1 to 15, having a cell culture surface area.

[0118] Appearance 17 is a cell culture surface area of ​​approximately 50,000 cm². 2 This relates to the cell culture system described in embodiment 16.

[0119] Embodiment 18 relates to a cell culture system according to any one of Embodiments 1 to 17, wherein, in an inclined orientation, the bottom of the multilayer container forms an angle of approximately 5° with respect to the horizontal plane.

[0120] Embodiment 19 relates to a cell culture system according to any one of Embodiments 1 to 18, wherein the tilted orientation is rotated by approximately 5° relative to the upright orientation.

[0121] Embodiment 20 relates to a cell culture system according to any one of Embodiments 7 to 19, wherein a plurality of multilayer containers are arranged on each of a plurality of support surfaces.

[0122] Embodiment 21 relates to the cell culture system according to Embodiment 20, wherein the inlets of multiple multilayer containers, which are located on one of a plurality of support surfaces, are connected to one another.

[0123] Embodiment 22 relates to the cell culture system according to Embodiment 20 or 21, wherein the inlets of a plurality of multilayer containers arranged on a plurality of support surfaces are connected to one another.

[0124] Embodiment 23 relates to a cell culture system according to any one of Embodiments 1 to 22, wherein the cabinet has a main inlet configured to be fluidly connected to the cell culture space of at least one multilayer vessel and to supply a liquid culture medium to the cell culture space.

[0125] Embodiment 24 relates to the cell culture system according to Embodiment 23, wherein the main inlet is fluidly connected to a plurality of multilayer containers.

[0126] Embodiment 25 relates to the cell culture system according to Embodiments 1 to 24, wherein the cabinet has a gas port configured to supply gas to an internal cavity.

[0127] Embodiment 26 relates to the cell culture system according to Embodiment 25, further comprising a gas supply unit fluidly connected to a gas port.

[0128] Embodiment 27 relates to the cell culture system according to embodiments 1 to 26, further comprising a temperature control system configured to control the temperature of an internal cavity.

[0129] Embodiment 28 relates to the cell culture system according to Embodiment 27, wherein the temperature control system includes at least one of a heat source and a cooling system.

[0130] Embodiment 29 relates to the cell culture system according to Embodiments 1 to 28, wherein the cabinet is configured to change the orientation of the multilayer container by changing the orientation of the cabinet.

[0131] Embodiment 30 relates to the cell culture system according to Embodiment 29, wherein the orientation of the multilayer container is fixed relative to the cabinet.

[0132] Embodiment 31 relates to a cell culture system according to embodiments 1 to 30, wherein one or more side walls have an opening to an internal cavity, the opening being sized to allow insertion or removal of a multilayer container.

[0133] Embodiment 32 relates to the cell culture system according to Embodiment 31, wherein the cabinet has a door that covers an opening, and the door is configured to seal the internal cavity when a multilayer container is placed inside the internal cavity.

[0134] Embodiment 33 relates to the cell culture system according to embodiments 1 to 32, wherein the cabinet has a gas-impermeable enclosure within an internal cavity, and the gas-impermeable enclosure is configured to surround at least one multilayer vessel.

[0135] Embodiment 34 relates to the cell culture system according to embodiments 25 to 33, wherein the gas port is connected to the opening of a gas-impermeable enclosure.

[0136] Embodiment 35 relates to the cell culture system according to embodiments 1 to 34, further comprising an incubation enclosure configured to house a cabinet.

[0137] Embodiment 36 relates to the cell culture system according to Embodiment 35, wherein the incubation enclosure has one or more ports configured for at least one of supplying liquid culture medium to a multilayer container and transmitting signals from sensors of the cell culture system to the outside of the incubation enclosure.

[0138] Embodiment 37 relates to a cell culture system according to any one of Embodiments 1 to 28, wherein the orientation of the multilayer container is variable with respect to the orientation of the cabinet.

[0139] Embodiment 38 relates to a cell culture system according to any one of Embodiments 1 to 37, wherein the multilayer container has a gas-permeable substrate that isolates the cell culture space from the internal cavity.

[0140] Embodiment 39 relates to a cell culture system according to any one of Embodiments 1 to 36, wherein the multilayer container has at least one of a 2D adhesive cell culture film and a 3D microcavity film.

[0141] Preferred embodiments of the present invention are described below in separate sections.

[0142] Embodiment 1 A cell culture system, A multilayer container configured for culturing cells, the multilayer container comprising a cell culture space within the multilayer container, A cabinet having an internal cavity surrounded by one or more side walls, wherein the cabinet is configured to house the multilayer container within the internal cavity. Includes, The cabinet is configured to change the orientation of the multilayer container from an upright orientation to an inclined orientation. Cell culture system.

[0143] Embodiment 2 The cell culture system according to Embodiment 1, further comprising at least one sensor configured to detect characteristics within the cell culture space.

[0144] Embodiment 3 The cell culture system according to Embodiment 2, wherein the sensor includes at least one of a collection monitor and an analyte monitor.

[0145] Embodiment 4 The cell culture system according to embodiment 2 or 3, wherein the sensor is incorporated into the multilayer container.

[0146] Embodiment 5 The cell culture system according to Embodiment 2 or 3, wherein the sensor is mounted on the cabinet and is configured to detect the characteristics in the cell culture space when the multilayer container is placed inside the cabinet.

[0147] Embodiment 6 A cell culture system according to any one of embodiments 2 to 5, wherein the multilayer container has at least one sensor window, and the sensor is configured to detect the characteristics in the cell culture space through the sensor window.

[0148] Embodiment 7 A cell culture system according to any one of embodiments 1 to 6, wherein the cabinet has a plurality of support surfaces configured to support at least one of the multilayer containers.

[0149] Embodiment 8 A cell culture system according to any one of embodiments 1 to 7, wherein at least one of the multilayer containers has a plurality of multilayer cell culture modules.

[0150] Embodiment 9 A cell culture system according to Embodiment 8, wherein at least some of the multiple multilayer cell culture modules are connected to one another.

[0151] Embodiment 10 A cell culture system according to any one of Embodiments 1 to 9, wherein the multilayer container has an inlet and an outlet, the inlet is configured to supply a liquid culture medium to the cell culture space, and the outlet is configured to allow liquid or gas to flow into or out of the cell culture space.

[0152] Embodiment 11 The cell culture system according to Embodiment 10, wherein the inlet is located at the bottom of the multilayer container.

[0153] Embodiment 12 The cell culture system according to embodiment 10 or 11, wherein the outlet is located at the top of the multilayer container.

[0154] Embodiment 13 The cell culture system according to Embodiment 12, wherein the outlet is located on the opposite side of the multilayer container diagonally from the inlet.

[0155] Embodiment 14 A cell culture system according to any one of embodiments 10 to 13, wherein the outlet has a vent port configured to allow gas to escape from or into the cell culture space.

[0156] Embodiment 15 A cell culture system according to any one of embodiments 10 to 14, wherein the outlet has a filter.

[0157] Embodiment 16 The aforementioned multilayer container has a capacity of at least 18,000 cm³. 2A cell culture system according to any one of Embodiments 1 to 15, having a cell culture surface area.

[0158] Embodiment 17 The aforementioned cell culture surface area is approximately 50,000 cm². 2 The cell culture system according to Embodiment 16.

[0159] Embodiment 18 A cell culture system according to any one of Embodiments 1 to 17, wherein, in the tilted orientation, the bottom of the multilayer container is at an angle of approximately 5° with respect to the horizontal plane.

[0160] Embodiment 19 A cell culture system according to any one of Embodiments 1 to 18, wherein the tilted orientation is rotated by approximately 5° relative to the upright orientation.

[0161] Embodiment 20 A cell culture system according to any one of embodiments 7 to 19, wherein multiple multilayer containers are arranged on each of the multiple support surfaces.

[0162] Embodiment 21 A cell culture system according to Embodiment 20, wherein the inlets of a plurality of multilayer containers, which are located on one of the plurality of support surfaces, are connected to one another.

[0163] Embodiment 22 A cell culture system according to embodiment 20 or 21, wherein the inlets of a plurality of multilayer containers arranged on a plurality of support surfaces are connected to one another.

[0164] Embodiment 23 A cell culture system according to any one of embodiments 1 to 22, wherein the cabinet has a main inlet configured to be fluidly connected to the cell culture space of at least one of the multilayer containers and to supply a liquid culture medium to the cell culture space.

[0165] Embodiment 24 The cell culture system according to embodiment 23, wherein the main inlet is fluidly connected to a plurality of multilayer containers.

[0166] Embodiment 25 A cell culture system according to any one of embodiments 1 to 24, wherein the cabinet has a gas port configured to supply gas to the internal cavity.

[0167] Embodiment 26 The cell culture system according to Embodiment 25, further comprising a gas supply unit fluidly connected to the gas port.

[0168] Embodiment 27 A cell culture system according to any one of embodiments 1 to 26, further comprising a temperature control system configured to control the temperature of the internal cavity.

[0169] Embodiment 28 The cell culture system according to Embodiment 27, wherein the temperature control system includes at least one of a heat source and a cooling system.

[0170] Embodiment 29 A cell culture system according to any one of Embodiments 1 to 28, wherein the cabinet is configured to change the orientation of the multilayer container by changing the orientation of the cabinet.

[0171] Embodiment 30 The cell culture system according to Embodiment 29, wherein the orientation of the multilayer container is fixed with respect to the cabinet.

[0172] Embodiment 31 A cell culture system according to any one of embodiments 1 to 30, wherein one or more of the side walls have an opening to the internal cavity, the opening being sized to allow insertion or removal of the multilayer container.

[0173] Embodiment 32 The cell culture system according to Embodiment 31, wherein the cabinet has a door that covers the opening, and the door is configured to seal the internal cavity when the multilayer container is placed inside the internal cavity.

[0174] Embodiment 33 A cell culture system according to any one of embodiments 1 to 32, wherein the cabinet has a gas-impermeable enclosure within the internal cavity, and the gas-impermeable enclosure is configured to surround at least one of the multilayer containers.

[0175] Embodiment 34 A cell culture system according to any one of embodiments 25 to 33, wherein the gas port is connected to the opening of the gas-impermeable enclosure.

[0176] Embodiment 35 A cell culture system according to any one of embodiments 1 to 34, further comprising an incubation enclosure configured to house the cabinet.

[0177] Embodiment 36 The cell culture system according to Embodiment 35, wherein the incubation enclosure has one or more ports configured for at least one of supplying liquid culture medium to the multilayer container and transmitting signals from sensors of the cell culture system to the outside of the incubation enclosure.

[0178] Embodiment 37 A cell culture system according to any one of Embodiments 1 to 28, wherein the orientation of the multilayer container is variable with respect to the orientation of the cabinet.

[0179] Embodiment 38 A cell culture system according to any one of embodiments 1 to 37, wherein the multilayer container has a gas-permeable substrate that isolates the cell culture space from the internal cavity.

[0180] Embodiment 39 The cell culture system according to any one of embodiments 1 to 36, wherein the multilayer container has at least one of a 2D adhesive cell culture film and a 3D microcavity film.

Claims

1. A cell culture system, A plurality of multilayer containers configured for culturing cells, each of which contains a cell culture space, A cabinet having an internal cavity surrounded by one or more side walls, wherein the cabinet is configured to house the multilayer container within the internal cavity, A multi-position support within the cabinet, comprising a plurality of multi-position supports configured to support the plurality of multilayer containers, Includes, The multi-position support is, The main base and the sub-base, which are both horizontal when the multi-position support is in an upright configuration, A main support surface is offset vertically from the main base and configured to support the multilayer container, A sub-support surface is offset vertically from the sub-base and configured to support the multilayer container, A first intermediate surface extending between the main base and the main support surface, A second intermediate surface extending between the sub-base and the sub-support surface, Includes, The first intermediate surface extends at an angle with respect to the main base and also extends at an angle with respect to the main support surface, The second intermediate surface extends at an angle with respect to the sub-base and also extends at an angle with respect to the sub-support surface, The main support surface and the secondary support surface are located on the same plane, but form an angle with respect to the main base and the secondary base. The multi-position support is configured to have an upright configuration in which it is oriented vertically and an inclined configuration in which it is oriented horizontally. Cell culture system.

2. The cell culture system according to claim 1, further comprising at least one sensor configured to detect characteristics within the cell culture space, wherein the sensor comprises at least one of a confluence monitor and an analyte monitor.

3. The cell culture system according to claim 2, wherein at least one of the sensors is incorporated into the multilayer container.

4. The cell culture system according to claim 2, wherein at least one of the sensors is mounted on the cabinet and is configured to detect the characteristics in the cell culture space when the multilayer container is placed inside the cabinet.

5. The cell culture system according to any one of claims 1 to 4, wherein the multilayer container comprises a plurality of multilayer cell culture modules having the cell culture space, and at least some of the plurality of multilayer cell culture modules are connected to one another.

6. The cell culture system according to any one of claims 1 to 5, wherein each of the plurality of multilayer containers has an inlet and an outlet, the inlet being located at the bottom of the multilayer container and configured to supply liquid culture medium to the cell culture space, and the outlet being located at the top of the multilayer container and configured to allow liquid or gas to flow into or out of the cell culture space.

7. The cell culture system according to claim 6, wherein the outlet is located on the opposite side of the multilayer container diagonally from the inlet.

8. The cell culture system according to claim 6 or 7, wherein the outlet has a vent port configured to have a filter and to allow gas to escape from or into the cell culture space.

9. The plurality of multilayer containers total at least 18,000 cm³ ２ A cell culture system according to any one of claims 1 to 8, having a cell culture surface area.

10. The cell culture system according to any one of claims 1 to 9, wherein the tilted orientation is rotated by approximately 5° relative to the upright orientation.

11. A cell culture system according to any one of claims 6 to 8, wherein a plurality of the multilayer containers are arranged in each of the plurality of the multiposition supports, and the inlets are connected to one another.

12. The cell culture system according to any one of claims 1 to 11, wherein the cabinet has a main inlet configured to be fluidly connected to the cell culture space of at least one of the multilayer containers and to supply a liquid culture medium to the cell culture space, and the cabinet has a gas port configured to supply gas to the internal cavity.

13. The cell culture system according to any one of claims 1 to 12, further comprising a temperature control system configured to control the temperature of the internal cavity.

14. The cell culture system according to any one of claims 1 to 13, wherein the cabinet has a gas-impermeable enclosure within the internal cavity, and the gas-impermeable enclosure is configured to surround at least one of the multilayer containers.

15. The cell culture system according to any one of claims 1 to 14, wherein the orientation of the plurality of multilayer containers is variable with respect to the orientation of the cabinet.

Images