Culture Device

Abstract

A culture device is provided having a base having a first surface and a second surface fixedly disposed relative to the first surface, and a flexible sidewall extending from the base to define an interior volume of the culture device, wherein the first surface and the second surface are non-coplanar.

Description

[Technical Field]

[0001] The present invention relates to a device, particularly a culture device for culturing cells, a method for culturing cells, and a system for culturing cells. [Background technology]

[0002] In the field of cell culture, it is commonly known that cell growth progresses from an initial "lag phase," during which cells adapt to the culture environment and grow at a slower rate, to a "log phase," during which cells exponentially grow and consume nutrients in the growth medium. As cells consume nutrients in the growth medium, it becomes necessary to remove the spent medium and introduce new growth medium so that the cells can continue to grow. Furthermore, as cells grow, the density of the cell culture increases, necessitating the transfer of the cell culture from one culture device to another, larger culture device containing fresh growth medium. This process is called cell culture passaging. This is also necessary for adherent cell cultures, in which cells adhere (attach) to a suitable surface. In adherent cell cultures, adherent cells proliferate and eventually occupy the entire available area within the culture device. Therefore, transferring adherent cell cultures from one surface to another, larger surface, is necessary to maintain a desired growth rate.

[0003] Generally, the passaging process requires transferring cells from one culture vessel to another, often a larger vessel. This process is more complicated for adherent cells, as cells that adhere to a surface must be detached from that surface before being transferred to another culture vessel. Many dissociation procedures are known in the art, including shaking or agitating the culture vessel, vigorous pipetting, scraping the cells from the surface, or enzymatic dissociation using reagents such as trypsin, dispase, TrypLE® (ThermoFisher Scientific), collagenase, ethylenediaminetetraacetic acid (EDTA), Accutase® (Innovative Cell Technologies), etc.

[0004] Current processes for transferring cells between various culture vessels have many drawbacks. First, the use of interconnecting tubing, additional vessels, or other equipment that may not be adequately sterilized before use risks introducing or passing cells through a non-sterile environment. Second, the cell transfer process requires manual intervention, especially in adherent cell cultures, which can lead to operator error and compromise sterility during use. Third, such transfer processes often negatively impact cell yield because it is difficult to ensure that most or all of the cells have been removed from the first vessel and transferred to the second vessel. In particular, cells are often lost from the pipette tip or tubing during the transfer process. Fourth, with currently known culture vessels, removing the final cell product from the vessel at the end of the process is often cumbersome. Fifth, the use of multiple vessels is an inefficient use of space, affecting the scalability of the process. Cells are often removed by pipetting or pumping mechanisms, which adds additional complexity to the system and carries the risk of compromising sterility and operator error. Furthermore, such pipetting requires the highest level of personal protective equipment and a laminar flow hood, which requires a particulate-free environment. Thus, typical pipetting is a costly, time-consuming, and labor-intensive process that requires highly skilled operators.

[0005] Therefore, it is an object of the present invention to solve or at least mitigate the above-mentioned drawbacks. In particular, it is an object of the present invention to provide a culture device and method that does not require the transfer (i.e., "passaging") of cells between two or more culture devices, e.g., two, three, four, or more culture devices. More specifically, it is an object of the present invention to provide a culture device and method that allows cells to be passaged or transferred within a single culture device, thereby ensuring a sterile environment and higher cell yield. Such a culture device and method may also be more amenable to automation. It is an object of the present invention to provide a culture device that allows materials, such as cells, to be more easily removed. Furthermore, it is an object of the present invention to provide a culture device that allows materials, such as cells, to be mixed, resuspended in medium, or otherwise agitated. Summary of the Invention

[0006] According to one aspect of the present invention, there is provided a device comprising a base including a first surface and a second surface fixedly disposed relative to the first surface, and a sidewall extending from the base to define an interior volume of the device, wherein the first surface and the second surface are non-coplanar.

[0007] That is, the present invention provides, for example, a culture device, or a device suitable for culturing materials, such as cellular materials. The device includes a base and a sidewall extending from the base. The base includes one or more surfaces, i.e., a plurality of faces. At least one of these surfaces forms an angle with a horizontal plane, i.e., a transverse plane, extending perpendicular to a central longitudinal axis of the device, at a predetermined, preset, or fixed angle. That is, the device includes a plurality of faces, each of which is fixedly disposed (i.e., immovable) relative to one another. The transverse plane may be at least partially defined by the base, such as by a particular point of the base disposed within the transverse plane or by a side of the base extending within the transverse plane, as described further below.

[0008] In other words, without reference to a particular plane, e.g., a horizontal plane or a transverse plane, the base is non-coplanar. That is, the base includes multiple surfaces, at least one of which is non-coplanar with respect to the other surface(s). That is, at least one surface (e.g., a first surface) may be at an angle of 1 to 179 degrees relative to the other surface (e.g., a second surface). The angle may be positive (+) or negative (-) relative to the transverse plane. That is, one culture surface may be at an angle of +1 to +179 degrees, or -1 to -179 degrees, relative to the other culture surface. As a specific example, one culture surface may be at an angle of 90 to 179 degrees relative to the other culture surface.

[0009] In other words, the base includes multiple surfaces, e.g., a first surface and a second surface, each of which extends in a plane. The planes of the first surface and the second surface are not coplanar (non-coplanar). That is, the plane of the first surface and the plane of the second surface form an angle. This angle may be one of the angles described above or may be an angle described in more detail below.

[0010] In some examples, a culture device is provided that includes a base and a sidewall extending axially from the base along a central longitudinal axis, the sidewall defining an interior volume of the device, the base including a plurality of surfaces, at least one of the surfaces being non-coplanar with respect to the other surfaces.

[0011] Preferably, the sidewall is a flexible sidewall. Optionally, the flexible sidewall is a compressible sidewall. Alternatively, the sidewall is a rigid sidewall.

[0012] This provides the advantage that each surface can be independently positioned horizontally, i.e., in a horizontal or transverse plane. In this way, by rotating the device from one horizontal surface to the other, materials such as cell cultures can be transferred, or "passaged," between the surfaces without leaving the device. This eliminates the need for multiple devices. This ensures a continuous sterile environment, reduces operator workload and associated errors, and improves suitability for automated processes. Furthermore, this provides the advantage that low-volume cell cultures, such as suspension cell cultures, can be mixed, agitated, or resuspended before adding additional cell culture medium to the vessel. For example, settled suspension cells can be resuspended in a volume of approximately 10 mL to approximately 200 mL of medium before adding more medium to the vessel, which is often referred to as "volume expansion."

[0013] It should be noted that any suitable material may be cultured in the culture devices described herein, including, but not limited to, biological material, bacteria, algae, synthetic meat, human or animal tissue, or human or animal cells. In particular, the culture devices described herein are suitable for culturing adherent-type cells and / or suspension-type cells.

[0014] Each of the multiple surfaces can be fixedly positioned relative to one another, i.e., each of the multiple surfaces is provided in a fixed state relative to one another. In this respect, the surfaces are statically positioned, i.e., cannot move relative to one another. This can be achieved by a rigid surface and / or a rigid base.

[0015] Optionally, in the case of a substantially circular base, each face is formed as an angled or tilted sector (i.e., each face may be formed as an inclined wedge). Optionally, in the case of a substantially circular base, each face is formed as an angled or tilted cylindrical sector. Optionally, each sector may be tilted downward toward a point or axis of the base, such as a central origin or axis of the base.

[0016] Optionally, the plurality of surfaces includes a first surface and a second surface, optionally the second surface being separate from the first surface, and the first and second surfaces may be in any suitable arrangement, for example, juxtaposed, adjacent, or concentric.

[0017] Optionally, the first surface, or the plane on which the first culture surface is disposed or located, forms a first angle with respect to a horizontal plane, i.e., a transverse plane extending substantially perpendicular to a central longitudinal axis of the device.

[0018] Optionally, the first angle is between about 1 degree and about 179 degrees.

[0019] Optionally, the first angle is between about 1 degree and about 90 degrees.

[0020] Optionally, the first angle is between about 1 degree and about 45 degrees.

[0021] Optionally, the first angle is between about 1 degree and about 30 degrees.

[0022] Optionally, the first angle is between about 5 degrees and about 30 degrees.

[0023] Optionally, the first angle is between about 5 degrees and about 25 degrees.

[0024] Optionally, the second surface, or the plane in which the second surface is disposed or located, forms a second angle with respect to a horizontal plane, i.e., a transverse plane extending substantially perpendicular to a central longitudinal axis of the device.

[0025] Optionally, the second angle is between about 1 degree and about 179 degrees.

[0026] Optionally, the second angle is between about 1 degree and about 90 degrees.

[0027] Optionally, the second angle is between about 1 degree and about 45 degrees.

[0028] Optionally, the second angle is between about 1 degree and about 30 degrees.

[0029] Optionally, the second angle is between about 5 degrees and about 30 degrees.

[0030] Optionally, the second angle is between about 5 degrees and about 25 degrees.

[0031] Optionally, the first angle and the second angle are different or unequal.

[0032] Optionally, each of the first angles is between about 1 degree and about 45 degrees, optionally between about 5 degrees and about 25 degrees, and each of the first and second angles is different or unequal. Any of the above contemplated ranges may be used in combination.

[0033] Optionally, the first area and the second area are equal, or the first area and the second area are unequal.

[0034] Optionally, the ratio between the area of ​​the first surface and the area of ​​the second surface is from about 1:9 to about 1:1.

[0035] Optionally, the ratio is about 1:9, or about 1:8, or about 1:7, or about 1:6, or about 1:5, or about 1:4, or about 1:3, or about 1:2, or about 1:1.

[0036] It will be understood that such a ratio may represent the ratio of the area of ​​the first surface to the area of ​​the second surface, or the ratio of the area of ​​the second surface to the area of ​​the first surface.

[0037] Optionally, the device and / or base (i.e., the device, the base, or the device and the base) can be movable about an axis, such that each of the first and second surfaces can be positioned in a horizontal plane. This can also be the case when the first, second, or both surfaces are angled relative to the horizontal plane. The axis can be coaxial with the adjacent region between the first and second surfaces. In some examples, the axis can be non-coaxial with the adjacent region between the first and second surfaces.

[0038] Optionally, the base further comprises a third surface fixedly disposed relative to each of the first and second surfaces and non-coplanar with the first and second surfaces, i.e., the third surface may be fixedly disposed relative to the first and second surfaces, i.e., the third surface may be non-coplanar with the first and second surfaces.

[0039] Optionally, the device and / or base (i.e., the device, the base, or the device and the base) can move about two axes, thereby causing each of the first, second, and third surfaces to lie in a horizontal plane. That is, the base can move about a first axis, thereby causing one or more surfaces to lie in a horizontal plane. That is, the base can move about a second axis, thereby causing one or more surfaces to lie in a horizontal plane. In some examples, the axes can be coaxial with adjacent regions between the surfaces. In some examples, the axes can be perpendicular to each other. Optionally, the base can move about two or more axes, thereby causing each of the first, second, and third surfaces to lie in a horizontal plane. Any number of axes is contemplated.

[0040] Optionally, the third surface is separated from the first and second surfaces. The first, second, and third surfaces may be arranged in any suitable configuration, such as, for example, a side-by-side, adjacent, concentric, or triangular, i.e., circular or cylindrical sector, configuration.

[0041] Optionally, the third surface, or the plane in which the third surface is disposed or located, forms a third angle with respect to a horizontal plane, i.e., a transverse plane extending substantially perpendicular to the central longitudinal axis of the device.

[0042] Optionally, the third angle is between about 1 degree and about 179 degrees.

[0043] Optionally, the third angle is between about 1 degree and about 90 degrees.

[0044] Optionally, the third angle is between about 1 degree and about 45 degrees.

[0045] Optionally, the third angle is between about 1 degree and about 30 degrees.

[0046] Optionally, the third angle is between about 5 degrees and about 30 degrees.

[0047] Optionally, the third angle is between about 5 degrees and about 25 degrees.

[0048] Optionally, the first angle, the second angle, and the third angle are different or unequal.

[0049] Optionally, each of the first angle, second angle, and third angle is selected from the range of about 1 degree to about 45 degrees, optionally about 1 degree to about 30 degrees, optionally about 5 degrees to about 25 degrees, and each of the first angle, second angle, and third angle is different or unequal. Any of the above contemplated ranges may be utilized in combination.

[0050] Optionally, the first angle is about 20 degrees, the second angle is about 30 degrees, and the third angle is about 45 degrees.

[0051] Although three surfaces are described above, it will be readily understood that other surfaces, such as a fourth, fifth, sixth, etc., may be formed as part of the device. Optionally, each surface is formed at a different angle relative to a horizontal plane, i.e., a transverse plane extending substantially perpendicular to the central longitudinal axis of the device. Optionally, if there are multiple surfaces, the device and / or base (i.e., the device, the base, or the device and the base) may be movable about two (or more) axes, thereby allowing each surface to lie in a horizontal plane. That is, the base may be movable about a first axis, thereby allowing one or more surfaces to lie in a horizontal plane. That is, the base may be movable about a second axis, thereby allowing one or more surfaces to lie in a horizontal plane. In some examples, these axes may be coaxial with adjacent regions between the surfaces. That is, the number of axes may be equal to the number of adjacent regions between the surfaces. In some examples, the axes may be formed perpendicular to each other. Optionally, the base may be movable about more than one axis, thereby allowing each surface to lie in a horizontal plane. Any number of axes may be envisioned.

[0052] Optionally, the area of ​​the first surface, the area of ​​the second surface, and the area of ​​the third surface are equal, or the area of ​​the first surface, the area of ​​the second surface, and the area of ​​the third surface are unequal.

[0053] Optionally, the ratio of the areas of the first surface, the second surface, and the third surface is approximately 1:2:3.

[0054] Optionally, the ratio of the areas of the first surface, the second surface, and the third surface is about 1:3:6.

[0055] Optionally, the area of ​​the first face is 25cm 2 and the area of ​​the second surface is 75 cm 2 and the area of ​​the third face is 150 cm 2 is.

[0056] This provides the advantage that the cells can be transplanted over a larger area as they grow.

[0057] Optionally, the base further comprises at least one port. Optionally, the base further comprises at least one outlet.

[0058] This provides the advantage that material such as cells can be removed from the device.

[0059] Optionally, one or each face includes a port or outlet. A port or outlet may be provided in the first face, the second face, and / or the third face. A port or outlet may be provided in any face.

[0060] Optionally, one or more ports are located at or in adjacent regions between adjacent faces. Optionally, one or more outlets are located at or in adjacent regions between adjacent faces.

[0061] This provides the advantage of harvesting a maximum amount of material from the device, particularly when the first surface and / or the second surface are rotated to direct the material into the port, which may ensure maximum harvesting efficiency.

[0062] Optionally, the flexible side wall comprises a corrugated wall.

[0063] Optionally, the flexible side wall comprises a bellows side wall. The bellows side wall may include a series of deformable regions alternating with rigid portions. Flexible side walls, such as bellows side walls, may be held within an optionally surrounding external support frame.

[0064] This provides the advantage that the compressible wall element is movable in response to pivoting of the base, i.e., the first and second faces, and also provides the advantage of providing a compressible device that compresses and / or expands in response to an external force, promoting mixing of the contents.

[0065] Optionally, at least one of the surfaces comprises an adherent cell culture substrate or coating.

[0066] This provides the advantage that the device is suitable for adherent cells so that cells can be "passaged" between surfaces within the device.

[0067] Optionally, the adherent cell culture substrate or coating comprises an extracellular matrix.

[0068] Optionally, the adherent cell culture substrate or coating is selected from the group comprising collagen, fibronectin, vitronectin, laminin, gelatin, polylysine, cellulose, or combinations thereof.

[0069] Optionally, the base comprises polystyrene, polycarbonate, low density polyethylene, high density polyethylene, silicone, or a thermoplastic elastomer. In certain embodiments, the base comprises silicone, particularly substantially gas permeable silicone.

[0070] Optionally, at least one or each surface comprises polystyrene, polycarbonate, low density polyethylene, high density polyethylene, silicone, or a thermoplastic elastomer. In certain embodiments, the base comprises silicone, particularly substantially gas permeable silicone.

[0071] Optionally, the base comprises a composite material, such as a silicone inner wall and a substantially gas impermeable outer wall.

[0072] Optionally, the base comprises a substantially gas impermeable thermoplastic elastomer.

[0073] The aforementioned materials offer many advantages. For example, a polystyrene base or surface allows adherent cells to adhere directly to the surface, reducing the need for additional substrates or coatings. Furthermore, a silicone base provides gas permeability to the base of the culture device, preventing hypoxia during use of the culture device. Furthermore, a thermoplastic elastomer base may provide a gas-impermeable base while providing the necessary biocompatibility and mechanical integrity for the base of the device, which may be advantageous for certain materials cultured within the device.

[0074] Optionally, the base is rigid, i.e., the base comprises a hard, i.e. non-flexible, material.

[0075] Optionally, each surface is rigid or non-flexible, i.e., each surface comprises a rigid or non-flexible material. The surfaces may be rigidly arranged so that they cannot bend or fold relative to one another.

[0076] Optionally, the base comprises a first side and an opposing second side. The first side may comprise a respective culture surface. The second side may be substantially planar. The second side may comprise a block of material, such as a wedge, forming an angled shape with one or more sides. The second side may have a substantially horizontal surface from which the block of material extends axially. The block of material may be integrally formed with the second side. Optionally, the second side may comprise a base and at least one wall rising from the base in the axial direction, i.e., along the longitudinal axis. The at least one wall may form an inclined surface.

[0077] Optionally, at least one surface forms an angle with respect to the second side of the base, i.e., one or more surfaces on the first side of the base form an angle with respect to the second side of the base. Optionally, the second side of the base defines a transverse plane, i.e., lies within the transverse plane.

[0078] Optionally, the device includes a top spaced from a base by a sidewall, such that the top is located at a distal end of the sidewall and the base is located at a proximal end of the sidewall. The top may include an opening, which may be internally or externally threaded.

[0079] Preferably, the device is a culture device. Optionally, the culture device is a biological culture device, such as a cell culture device. In the case of a culture device, the first surface can be a first culture surface and / or the second surface can be a second culture surface. Additional surfaces, such as a third surface, can also form a culture surface, such as a third culture surface.

[0080] According to another aspect of the present invention, there is provided a culture device comprising a base and flexible sidewalls extending axially from the base along a central longitudinal axis, the flexible sidewalls defining an interior volume of the device, the base comprising a plurality of culture surfaces, at least one of the culture surfaces being defined by at least a portion of the base and forming an angle with respect to a transverse plane extending substantially perpendicular to the central longitudinal axis.

[0081] According to another aspect of the present invention, there is provided a system for culturing cells, the system comprising a culture device as described herein and an interface member operably coupled to an upper portion of the device.

[0082] Optionally, the interface member is threadably coupled to a threaded opening optionally formed in the top wall of the device.

[0083] Optionally, the interface member comprises one or more ports for the flow of substances into and / or out of the device, and optionally, the one or more ports comprise septum seals.

[0084] According to another aspect of the present invention, there is provided a system for culturing cells, the system comprising: a device as described herein; and an actuator configured to engage at least a portion of the device and move the device between a first configuration in which a first surface is horizontal and a second configuration in which a second surface is horizontal.

[0085] That is, "horizontal" refers to a horizontal plane, i.e., a transverse plane extending substantially perpendicular to the central longitudinal axis of the device.

[0086] Optionally, the actuator is configured to move the device and / or the base (ie, the device, the base, or the device and the base) about at least one axis, or two axes, or more than two axes.

[0087] Optionally, the actuator may be configured to move the device and / or base (i.e., the device, the base, or the device and the base) about two axes so that each face can be positioned in a horizontal plane. That is, the base may be movable about a first axis so that one or more faces can be positioned in a horizontal plane. That is, the base may be movable about a second axis so that one or more faces can be positioned in a horizontal plane. In some examples, the axes may be coaxial with adjacent regions between the faces. In some examples, the axes may be perpendicular to each other. Optionally, the base may be movable about more than one axis so that each face can be positioned in a horizontal plane. Any number of axes may be envisioned.

[0088] Optionally, the actuator is configured to engage at least a portion of the base and move the base about at least one axis in the transverse plane, thereby causing movement between the first configuration and the second configuration.

[0089] Optionally, the actuator is configured to engage at least a portion of the base and move the base about two axes so that each of the faces can be positioned in a horizontal plane. That is, the base can be moved about a first axis, thereby positioning one or more faces in a horizontal plane. That is, the base can be moved about a second axis, thereby positioning one or more faces in a horizontal plane. In some examples, the axes can be coaxial with adjacent regions between the faces. In some examples, the axes can be formed orthogonal to each other. Optionally, the base can be moved about more than one axis, thereby positioning each of the faces in a horizontal plane. Any number of axes can be envisioned.

[0090] This provides the advantage that the orientation of each culture surface can be controlled by the actuators, either manually or automatically by operating the actuators.

[0091] Optionally, the actuator is electrically coupled to the processor.

[0092] Optionally, the processor is configured to operate the actuator according to a set of predefined parameters.

[0093] Optionally, the processor may be electrically coupled to one or more sensors of the culture device.

[0094] Optionally, the processor may be configured to receive signals from one or more sensors and to actuate the actuator in response to the received signals.

[0095] Optionally, the actuator is further configured to engage at least a portion of the base and move the base along a longitudinal axis, which may be a central longitudinal axis of the culture device.

[0096] This provides the advantage that the entire base is translated, thereby allowing the contents of the culture device to be mixed by compressing and / or stretching the flexible side walls of the culture device.

[0097] Optionally, the system further comprises an interface member as described in the above aspects.

[0098] According to another aspect of the present invention, a kit of parts is provided, comprising a culture device as described herein and an interface plate configured to operably couple to the culture device, and / or an actuator as described herein, the actuator configured to engage at least a portion of the base and move the base about at least one axis in a transverse plane.

[0099] According to another aspect of the present invention, there is provided a cell culture method, the cell culture method comprising: providing a culture device having a first culture surface and a second culture surface, at least one of the first culture surface and the second culture surface being defined by at least a portion of a base and forming an angle with respect to a transverse plane extending substantially perpendicular to a central longitudinal axis; orienting the first culture surface in a plane that is substantially parallel to or coplanar with the transverse plane; Culturing cells on a first culture surface; passaging the cells from the first culture surface to the second culture surface by orienting the second culture surface in a plane that is substantially parallel to or coplanar with the transverse plane; Culturing cells on the second culture surface; include.

[0100] According to another aspect of the present invention, there is provided a cell culture method, the cell culture method comprising: providing a culture device as described herein; orienting the first culture surface in a plane that is substantially parallel to or coplanar with the transverse plane; Culturing cells on a first culture surface; orienting a second culture surface in a plane that is substantially parallel to or coplanar with the transverse plane, thereby passaging cells from the first culture surface to the second culture surface; Culturing cells on the second culture surface; include.

[0101] According to another aspect of the present invention there is provided a method of culturing biological material, the method comprising the steps of: providing a first surface and a second surface fixedly disposed relative to the first surface, the first surface and the second surface being non-coplanar; orienting the first surface in a substantially horizontal plane; culturing a biological material on the first surface; orienting the second surface in a substantially horizontal plane, thereby transferring the biological material from the first surface to the second surface; Culturing biological material on the second surface; Includes.

[0102] The advantage of this is that biological material, such as cell cultures, can be transferred or passaged between the two surfaces without the need to transfer them into a separate container, thus ensuring a continuous sterile environment, reducing operator handling and associated errors, and lending itself to automated processes.

[0103] Optionally, orienting the first surface in a substantially horizontal plane comprises rotating the device and / or base (i.e., the device, the base, or the device and the base) about a first axis. Optionally, orienting the second surface in a substantially horizontal plane comprises rotating the device and / or base (i.e., the device, the base, or the device and the base) about the first axis or a second (i.e., different) axis.

[0104] Optionally, the biological material comprises tissue or cells.

[0105] Optionally, the cells are adherent cells and each of the first and second surfaces comprises an adherent cell culture substrate or coating.

[0106] Optionally, the method further comprises introducing a medium into the device.

[0107] Optionally, the method further comprises removing the medium from the device.

[0108] Optionally, the method further comprises genetically modifying cellular material within the device.

[0109] Optionally, the method further comprises the step of introducing viruses and / or magnetic beads into the device.

[0110] Optionally, the method further comprises the step of introducing growth factors, cytokines, or the like into the device.

[0111] Optionally, the method further comprises removing biological material, such as cells, from the device.

[0112] According to another aspect of the present invention, there is provided a method for culturing adherent cells, the method comprising: Providing a device as described herein; orienting the first surface in a plane that is substantially parallel to or coplanar with the transverse plane; Culturing adherent cells on the first surface; Detaching the adherent cells from the first surface; orienting the second surface in a plane that is substantially parallel to or coplanar with the transverse plane, thereby passaging the adherent cells from the first surface to the second surface; culturing adherent cells on the second surface; Includes.

[0113] The advantage of this is that adherent cell cultures can be transferred or passaged between two culture surfaces without the need to transfer cells to a separate vessel. This method therefore ensures a continuous sterile environment, reduces human handling and associated errors, and is amenable to automated processes.

[0114] Optionally, the step of detaching the adherent cells from the first culture surface may be preceded by a step of washing the adherent cell culture.

[0115] Optionally, the washing step may include introducing a washing solution, such as a buffered saline solution, into the device, and may include removing the washing solution from the device.

[0116] Optionally, the step of detaching the adherent cells from the first surface includes introducing a dissociation reagent into the device. Optionally, the dissociation reagent includes trypsin, dispase, TrypLE®, collagenase, EDTA, or Accutase®, etc. Generally, any suitable dissociation reagent or method for detaching adherent cells may be used.

[0117] Optionally, any adherent cell type can be cultured in the device, including mesenchymal stem cells, induced pluripotent stem (IPS) cells, embryonic stem cells (ESC), cancer cell lines, human embryonic kidney (HEK) cells, HeLa cells, fibroblasts, epithelial cells, etc.

[0118] Optionally, the method further includes culturing adherent cells on a first substrate disposed on the first surface and / or culturing adherent cells on a second substrate disposed on the second surface.

[0119] Optionally, the method further comprises the step of introducing a medium into the device. Optionally, the method further comprises the step of removing the medium from the device.

[0120] Optionally, the method further comprises genetically modifying cellular material within the device.

[0121] Optionally, the method further comprises the step of introducing viruses and / or magnetic beads into the device.

[0122] Optionally, the method further comprises the step of introducing growth factors, cytokines, etc. into the device.

[0123] Optionally, the method further comprises the step of removing material, such as cells, from the device, whereby the cells may be detached from their respective culture surfaces prior to removal.

[0124] Optionally, the methods discussed herein are performed in the devices discussed herein.

[0125] Generally, the methods discussed herein can be performed in any suitable order. Furthermore, the methods discussed herein can optionally include a further step of passaging cells onto an additional culture surface, such as a third or fourth culture surface. In these cases, the method can further include a step of orienting the third, fourth, etc. surface in a substantially horizontal plane. In some examples, the step of orienting the third, fourth, etc. surface in a substantially horizontal plane includes rotating the device and / or base about a first axis or a second axis (i.e., an axis different from the first axis). In some examples, these axes can be coaxial with adjacent regions between the surfaces. In some examples, these axes can be perpendicular to each other. Any number of axes can be envisioned.

[0126] Optional features indicated with respect to the methodologies herein are applicable to all method "aspects" described herein.

[0127] According to yet another aspect, there is provided the use of a device as described herein or a system as described herein in the culture of adherent cells.

[0128] According to yet another aspect, there is provided the use of a device as described herein or a system as described herein in the cultivation of suspension cells.

[0129] According to yet another aspect, there is provided a kit of parts that includes a device as described herein and an interface member configured to be operably coupled to the top of the device.

[0130] As will be apparent to one skilled in the art, various elements, features, and functions may be applicable across all aspects of the invention, whether or not they are described as separate aspects. [Brief explanation of the drawings]

[0131] Exemplary embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0132] [Figure 1] 1 shows a side view of a culture device according to the present invention. [Figure 2] FIG. 2 shows a side view of the culture device of FIG. 1 with an interface plate attached. [Figure 3] FIG. 1 shows a side view of another culture device according to the present invention. [Figure 4] FIG. 4 shows a cross-sectional view of the culture device of FIG. 3 with an interface plate attached. [Figure 5] FIG. 5 shows a top view of the culture device of FIGS. [Figure 6] FIG. 10 shows a side view of another culture device with an interface plate attached, according to the present invention. [Figure 7] FIG. 7 shows a top view of the culture device of FIG. 6. [Figure 8] FIG. 1 shows a top view of another culture device according to the present invention. [Figure 9] 9 shows a view of the culture device along section AA of FIG. 8. [Figure 10] 9 shows a view of the culture device along cross section BB of FIG. 8. [Figure 11] 9 shows a view of the culture device along section CC of FIG. 8. [Figure 12] 1(a) and (b) show top views of another culture device according to the present invention. [Figure 13] 1(a) and (b) show top views of another culture device according to the present invention. [Figure 14]1A-1D show top views of various culture devices according to the present invention, including (a) a square base, (b) another square base, (c) a triangular base, and (d) an octagonal base. [Figure 15] 5 shows the culture device of FIGS. 3 and 4 in a first configuration in use. [Figure 16] 5 shows the culture device of FIGS. 3 and 4 in a second configuration in use. DETAILED DESCRIPTION OF THE INVENTION

[0133] The described embodiments relate to devices and methods, primarily in cell processing, particularly, but not limited to, cell and / or gene therapy. As will be appreciated by those skilled in the art, the devices and methods are readily applicable to adherent cells, suspension cells, human-derived cells, animal-derived cells, and the like. The devices and methods can be used in cell and / or gene therapy, tissue engineering, cellular agriculture (including laboratory or cell-cultured meat products), water treatment, and other similar technical fields. Additionally, the devices may be suitable for other fields, such as chemical processing, agriculture, and the like. The devices and methods are not limited to the specific applications described herein. The use of "culture device" and "culture surface" is not intended to be limiting in any way and is merely illustrative of available devices and surfaces.

[0134] In the following description, terms used for convenience only are not intended to limit the present invention. Terms such as "top," "bottom," "upper," and "lower" refer to directions in the reference drawings and indicate the orientation of the described parts in an assembled and attached state. The terms "inner," "inward," and "outer," "outward," respectively, refer to directions toward and away from a designated centerline or the geometric center (e.g., central axis) of the described element, and their specific meanings are readily apparent from the described context. Furthermore, the terms "proximal" (i.e., closer) and "distal" (i.e., farther) refer to a position relative to an object or connection point.

[0135] Furthermore, in this specification, the terms "connected," "fixed," and the like include not only a direct connection between two components without any other components therebetween, but also an indirect connection between two components with one or more other components therebetween. These terms include the words and phrases specifically mentioned above, their derivatives, and words having similar meanings.

[0136] Furthermore, unless otherwise specified, the use of ordinal adjectives such as "first," "second," and "third" merely indicates that the objects so described are different instances of the same kind and does not imply that the objects so described are in any particular order in time, space, precedence, or otherwise. Like reference numerals are used throughout to denote like features, and different instances are designated by hundreds digits.

[0137] 1 and 2 illustrate a culture device 10 including a base 12 and a flexible sidewall 14. The flexible sidewall 14 extends axially from the base 12 along a central longitudinal axis L of the culture device. The flexible sidewall 14 extends from a proximal end adjacent to and connected to the base 12 toward a distal end. The distal end includes an opening 16, the inner periphery of which may be threaded. The flexible sidewall 14 is formed as a bellows-type sidewall, with a series of deformable regions 18 sandwiched between rigid regions 20. The culture device 10 includes an optional outlet port 22 coupled to an interface plate 24, which optionally includes multiple inlet ports 26 formed as septum seals, as shown in FIG. 2, to allow materials to enter and / or exit the culture device 10.

[0138] Generally, the base 12 comprises a rigid material, and the flexible sidewall 14 comprises an elastic material. The base 12 may be composed of polystyrene, polypropylene, polycarbonate, low-density polyethylene (LDPE) or high-density polyethylene (HDPE), a thermoplastic elastomer, silicone, etc. The flexible sidewall 14 may be composed of silicone, a substantially gas-permeable or substantially gas-impermeable thermoplastic elastomer, a flexible polymer film, etc. Any combination of materials for the base 12 and the flexible sidewall 14 may be envisioned. Similarly, in an embodiment not shown, the sidewall may be composed of a rigid material.

[0139] 2, the culture device 10 includes an interface plate 24 coupled to the opening 16 thereof. The interface plate 24 may be threadedly coupled to the threaded inner surface of the opening 16. Other coupling methods, such as a push-fit or clip-fit, may also be envisioned. The interface plate 24 includes a plurality of ports 26 formed as septum seals to allow materials to pass through the interface plate 24 into and out of the culture device 10. The plurality of ports 26 may be provided with access to the culture device 10 through the use of needle-based connectors (not shown).

[0140] As shown in FIG. 2, the base 12 of the culture device 10 includes a first side 12a and an opposing second side 12b. The first side 12a defines a first culture surface 28 and a second culture surface 30 separated by an adjacent region 32. The first culture surface 28 and the second culture surface 30 are disposed at a fixed angle relative to each other, as described further herein. As shown in FIG. 1, the first culture surface 28 is inclined relative to the second side 12b of the base 12, forming a fixed angle α with the second side 12b. In this case, the second side 12b extends in a transverse plane (i.e., a horizontal plane) extending perpendicular to the central longitudinal axis L and is disposed within the transverse plane. In other words, the second side 12b is planar. Referring again to FIG. 2, the angle α between the first culture surface 28 and the second side 12b is fixed and is formed by a material block 12c. The cross section of the material block 12c is approximately triangular. In this way, the second side 12b of the base 12 is provided as a continuous, i.e., flat, surface, and the material block 12c provides an angled first culture surface 28. In other words, the material block 12c is provided as a so-called wedge, thereby providing a fixed, angled surface 28.

[0141] The first culture surface 28 and the second culture surface 30 may be the same or different materials. In some examples, particularly those involving the culture of adherent cells, the first culture surface 28 and / or the second culture surface 30 include a coating or substrate to allow cell attachment or are composed of a suitable material that allows cell attachment, such as polystyrene. An adjacent region 32 generally separates the first culture surface 28 and the second culture surface 30. The culture device 10 includes multiple optional outlet ports 22 located on the second culture surface 30 adjacent to the adjacent region 32, but may alternatively be formed in the adjacent region 32 or the first culture surface 28. While one port 22 is shown in this example, more ports or no ports at all are equally envisioned.

[0142] 3 and 4 show another culture device 100 substantially similar to the culture device 10 of FIGS. 1 and 2, respectively, and similar elements will not be described in further detail. In the example of FIGS. 3 and 4, the outlet port (reference number 22 in FIGS. 1 and 2) is not provided. Furthermore, in the example of FIGS. 3 and 4, the material block (reference number 12c in FIG. 2) is not provided. This results in the base 112 having a fixed angle β between the first culture surface 128 and a transverse plane T extending perpendicular to the central longitudinal axis L. The transverse plane T is partially defined by the base 112, particularly the second culture surface 130 and the adjacent region 132.

[0143] Figure 5 shows a top view of the culture device 10 of Figures 1-4. As shown, a base 12, 112 is provided with a first culture surface 28, 128 and a second culture surface 30, 130 separated by an adjacent region 32, 132. An optional outlet port 22 (present in Figures 1 and 2) is shown in dotted lines to indicate its optionality. The optional outlet port 22 may be located on any of the culture surfaces 28, 128, 30, 130. Similarly, multiple outlet ports (not shown) may be provided.

[0144] Figure 6 shows a side view of another incubation device 200. The incubation device 200 is substantially similar to that described in Figures 3 and 4. In particular, the incubation device 200 includes a base 212, a flexible sidewall 214, an interface plate 224, and an optional port 226, as described above.

[0145] The culture device 200 includes a first culture surface 228 separated from a second culture surface 230 by an abutment region 232. The first culture surface 228 and the second culture surface 230 are disposed at an angle to each other, as described below. In this example, the first culture surface 228 forms an angle α with respect to the transverse plane T, and the second culture surface 230 forms an angle β with respect to the transverse plane T. The transverse plane T is defined in part by the base 212, in that the abutment region 232 (i.e., the axis defined by the abutment region 232) is disposed within the transverse plane T. In this example, the angles α and β are shown as different. In other examples, they may be the same. In this particular example, α is 30 degrees and β is 45 degrees.

[0146] Figure 7 shows a top view of the culture device 200 of Figure 6. As shown, the first culture surface 228 and the second culture surface 230 of the base 212 are separated by an abutment region 232.

[0147] 6 and 7 is substantially similar to the culture device 100 of FIGS. 3 and 4, but may incorporate additional features of the culture device 10 of FIGS. 1 and 2. For example, the first culture surface 228 and / or the second culture surface 230, or the adjacent region 232, may include optional outlet ports. Additionally, the base 212 may be provided with material blocks positioned on either side of the adjacent region 232, providing angled first culture surface 228 and second culture surface 230 while maintaining a continuous, planar second surface, i.e., lower surface, of the base 212.

[0148] Figures 8-11 illustrate yet another incubation device 300. The incubation device 300 is substantially similar to the incubation device 200 described in connection with Figures 6 and 7, except that it includes one additional incubation surface (i.e., three total). For example, as shown in Figures 9-11, the incubation device 300 includes features such as a base 312, a flexible sidewall 314, an interface plate 324, and a port 326.

[0149] 8 , the culture device 300 includes a base 312 having a first culture surface 328, a second culture surface 330, and a third culture surface 334. The first culture surface 328, the second culture surface 330, and the third culture surface 334 are each disposed at an angle relative to one another, as described later in this specification. The first culture surface 328 is separated from the second culture surface 330 by a first adjacent region 332. The second culture surface 330 is separated from the third culture surface 334 by a second adjacent region 336. The third culture surface 334 is separated from the first culture surface 328 by the second adjacent region 336. The first, second, and third culture surfaces 328, 330, and 334 are each formed as a sector, i.e., in this example, the bottom surface 312 is circular. That is, each of the first, second, and third culture surfaces 328, 330, 334 is defined by two radii and one arc.

[0150] Figure 9 is a cross-sectional view taken along line AA in Figure 8, illustrating the relationship between first culture surface 328 and third culture surface 334, separated by second abutment region 336. As shown, first culture surface 328 forms a fixed angle α with transverse plane T. In this example, α is 20 degrees. As shown, third culture surface 334 forms a fixed angle γ with transverse plane T. In this example, γ is 45 degrees.

[0151] Figure 10 is a cross-sectional view taken along line BB in Figure 8, illustrating the relationship between second culture surface 330 and third culture surface 334, separated by second abutment region 336. As shown, second culture surface 330 forms a fixed angle β with transverse plane T. In this example, β is 30 degrees. Also, as discussed above with respect to Figure 9, third culture surface 334 forms a fixed angle γ with transverse plane T. In this example, γ is 45 degrees.

[0152] Figure 11 shows a cross-sectional view along line CC, i.e., second adjacent region 336, of Figure 8, illustrating the relationship between first culture surface 328 and second culture surface 330, separated by second adjacent region 332. As shown, and as described above with respect to Figure 9, first culture surface 328 forms a fixed angle α with transverse plane T. In the illustrated example, α is 20 degrees. As described above with respect to Figure 10, second culture surface 330 forms a fixed angle β with transverse plane T. In the illustrated example, β is 30 degrees.

[0153] As described above, the base 312 of the incubation device 300 is formed from three angled or inclined cylindrical sectors, thereby forming the respective incubation surfaces 328, 330, 334. The areas of the respective incubation surfaces 328, 330, 334 increase progressively from the first incubation surface 328 to the third incubation surface 334, with an area ratio of 1:3:6 (i.e., first incubation surface: second incubation surface: third incubation surface). In a specific example, the area of ​​the first incubation surface 328 is 25 cm 2 , the area of ​​the second culture surface 330 is 75 cm 2 , the area of ​​the third culture surface 334 is 150 cm 2 The "area" in this context refers to the planes that form angles α, β, and γ with the transverse plane T, respectively, and the area that defines the "area," i.e., the area of ​​the culture surface that extends within the plane of the culture surface, defines the area in which cells, tissues, or other materials can be cultured.

[0154] 12(a) and 12(b) show another culture device 400 having a base 412 including three culture surfaces: a first culture surface 428, a second culture surface 430, and a third culture surface 434. The first culture surface 428 is separated from the second culture surface 430 by a first adjacent region 432. The second culture surface 430 is separated from the third culture surface 434 by a second adjacent region 436. As shown in FIG. 12(a), the first adjacent region 432 and the second adjacent region 436 each extend across a chord of the generally circular base 412 and are parallel to one another. The first, second, and third culture surfaces 428, 430, and 434 each have approximately equal areas, with the area ratio of the first, second, and third culture surfaces 428, 430, and 434 being approximately 1:1:1. 12(b), in another example, the first adjacent region 432 extends across a chord of the generally circular base 412, and the second adjacent region 436 extends across a diameter of the generally circular base 412. Thus, the first adjacent region 432 and the second adjacent region 436 extend parallel to one another. The first, second, and third culture surfaces 428, 430, 434 each have progressively larger areas, with the area ratio between the first, second, and third culture surfaces 428, 430, 434 being approximately 1:2:3, or in another example, 1:3:6.

[0155] In either example, at least two of the culture surfaces 428, 430, 434 form fixed angles with respect to the transverse plane, as in the previous examples. In a specific example, the first culture surface 428 and the third culture surface 434 form fixed angles with respect to the transverse plane, and the second culture surface 430 is substantially parallel to or coplanar with the transverse plane. The first and third culture surfaces 428, 434 can have equal or different fixed angles with respect to the transverse plane.

[0156] 13(a) and 13(b) show a culture device 500 having a base 512 including four culture surfaces (first culture surface 528, second culture surface 530, third culture surface 534, and fourth culture surface 538). Four adjacent regions (first adjacent region 540, second adjacent region 542, third adjacent region 544, and fourth adjacent region 546) are also provided. The first culture surface 528 is separated from the second culture surface 530 by the first adjacent region 540. The second culture surface 530 is separated from the third culture surface 534 by the second adjacent region 542. The third culture surface 534 is separated from the fourth culture surface 538 by the third adjacent region 544. The fourth culture surface 538 is separated from the first culture surface 528 by the fourth adjacent region 546.

[0157] The difference between Figures 13(a) and 13(b) is the point at which adjacent regions 540, 542, 544, and 546 intersect. In Figure 13(a), adjacent regions 540, 542, 544, and 546 each intersect at a central origin O, which is a point along the central longitudinal axis of the device. In the example of Figure 13(a), the areas of each of the culture surfaces 528, 530, 534, and 538 are equal. Alternatively, each of the adjacent regions 540, 542, 544, and 546 intersect at a non-center point P along an axis other than the central longitudinal axis of the device. In the example of Figure 13(b), the areas of each of the culture surfaces 528, 530, 534, and 538 are different.

[0158] In each example, at least three of the culture surfaces 528, 530, 534, 538 form fixed angles with respect to the transverse plane, similar to that described in the previous example. In a particular example, the first culture surface 528, the second culture surface 530, and the third culture surface 534 each form a fixed angle with respect to the transverse plane, while the fourth culture surface 538 is substantially parallel to, or coplanar with, the transverse plane. The first, second, and third culture surfaces 528, 530, 534 can form equal or different angles with respect to the transverse plane.

[0159] Figures 14(a) through 14(d) illustrate different cross-sectional shapes of the bases 612, 712, 812, and 912 of the culture device, each having an abutment region 632, 732, 832, and 932 separating a first culture surface 628, 728, 828, and 929 from a second culture surface 630, 730, 830, and 930. Figure 14(a) illustrates a substantially square base 612 including an abutment region 632 extending between opposing sides of the square base 612. Figure 14(b) illustrates a substantially square base 712 including an abutment region 732 extending between adjacent sides of the square base 712. Figure 14(c) illustrates a substantially triangular base 812 including an abutment region 832 extending between adjacent sides of the triangular base 812. 14(d) illustrates a generally octagonal base 912 that includes abutment regions 932 extending between opposing corners of the generally octagonal base 912. As will be appreciated by those skilled in the art, the base 912 may have any shape or cross-sectional profile, and the abutment regions 932 may extend between any suitable edges, sides, corners, etc. of such base 912.

[0160] In the example of Figures 14(a) through 14(d), at least one or both of the first culture surface 628, 728, 828, 928 and the second culture surface 630, 730, 830, 930 form an angle with respect to the transverse plane T, as described herein. The angle formed may be any angle contemplated herein.

[0161] The culture devices of the present invention shown in Figures 1 through 14 can be used to passage cells as follows. In particular, because the angles between the culture surfaces (or the angles between particular culture surfaces and the transverse plane T) are fixed, moving the base (i.e., moving the transverse plane T at a predetermined angle) allows cells to pass between the culture surfaces, so that each culture surface is arranged in a predetermined order in a horizontal plane. While certain exemplary protocols described below refer to the use of the culture device 100 of Figures 3 and 4, such use may equally apply to any of the other culture devices described herein.

[0162] [Example 1: Suspension cell culture] In one example, a suspension cell culture, i.e., a suspension of a desired type of cells in an appropriate medium, is introduced into the cell culture device 100 shown in Figures 3 and 4. In particular, the cell culture may be loaded into the volume of the culture device 100 from an external container having a needle-based connector (not shown). The needle-based connector may pierce a septum seal in the external container and may also pierce one of the ports 126 of the interface plate 124, thereby fluidly connecting the external container and the culture device 100. Thus, the cell culture is introduced into the culture device 100.

[0163] Before or after the introduction of the suspension cell culture, an external actuator (not shown) can rotate the entire base 112 so that the first culture surface 128 is substantially horizontal, i.e., flat or coplanar with the transverse plane T. This is shown in FIG. 15. The external actuator (not shown) can pivot or rotate the base about an axis substantially aligned with the adjacent region 132. Note that the flexible sidewalls 114 allow the base 112 to pivot, as shown in FIG. 15.

[0164] Cells in the cell culture settle on base wall 112, i.e., first culture surface 128. Optionally, base 112 can be agitated by an external drive, such as by rotating about an axis defined by abutment region 132, so that all cells are moved to and settle on first culture surface 128. Alternatively, the entire culture device 100 can be tilted to cause the cells to settle and settle on first culture surface 128.

[0165] The cells in the culture are allowed to grow, achieving exponential cell growth. During the growth process, appropriate substances, such as growth factors, fresh medium, proteins, magnetic beads, antibodies, and viruses, can be added to the cell culture. These substances can be introduced from an external container equipped with a needle-type connector, similar to the introduction of the cell culture described above. During growth, the base 112, i.e., the first culture surface 128 and the second culture surface 130, can be periodically rotated about an axis defined by the adjacent region 132 to mix and / or resuspend the cell culture. Generally, the periodic rotation about the axis defined by the adjacent region 132 induces turbulence within the cell culture device 100, promoting mixing and / or resuspension of the contents. The periodic rotation about the axis defined by the adjacent region 132 can be performed at any suitable angle, such as +10 degrees to -10 degrees. The cell culture is allowed to grow until a desired cell density is reached.

[0166] Optionally, a medium exchange step is provided. In particular, a portion of the cell culture medium, i.e., depleted or consumed medium, is removed from the culture device 100 and replaced with fresh medium. The medium can be removed through port 126 of the interface plate 124. Fresh medium is introduced through port 126 of the interface plate 124 using an external container with a needle-based connector, similar to the introduction of the cell culture described above. Similarly, as will be understood by those skilled in the art, a medium exchange step can be provided after cell passaging, as described below. In some examples, medium exchange steps can be provided both before and after cell passaging, as described below.

[0167] The culture device 100, specifically its base 112, is pivoted about an axis defined by the adjacent region 132. In particular, an external actuator (not shown) pivots the entire base 112 so that the second culture surface 130 is substantially horizontal, i.e., flat or flush with the transverse plane T. This is shown in FIG. 16 . In this manner, cells are transferred, or passaged, from the first culture surface 128 to the second culture surface 130 by gravity, until all cells are present on the second culture surface 130. The cell culture is then allowed to grow until a desired cell density is achieved. During growth, the base 112 can be periodically pivoted about the axis defined by the adjacent region 132 to mix and / or resuspend the cell culture. Generally, the periodic pivoting about the axis defined by the adjacent region 132 can induce turbulence within the cell culture device 100, facilitating mixing and / or resuspension of the contents. The periodic rotation about the axis defined by the abutment region 132 may be performed at an angle of +10 degrees to -10 degrees.

[0168] Optionally, further transfer or passaging of cells may be performed, i.e., from second culture surface 130 to first culture surface 128. In this case, the process described above is performed in reverse, i.e., the entire base 112 is returned from the position of FIG. 16 to the position of FIG. 15. This may result in further mixing or passaging and may promote oxygenation of the cell culture. The cell culture is allowed to grow until the desired cell density is reached.

[0169] Additionally, the cell culture may be mixed and / or resuspended by pivoting the base 112 about the axis defined by the abutment region 132. Thus, after mixing and / or resuspension, the cells may settle on either the first or second culture surface 128, 130, depending on which culture surface is positioned in the horizontal plane.

[0170] Finally, once the cell culture has matured and reached a desired density, the cell culture is harvested through port 126 in interface plate 124. Alternatively, if an optional port is provided in the base of the culture device, the cells may be harvested through such port.

[0171] [Example 2: Adherent cell culture] In another example, an adherent cell culture, i.e., adherent cells in an appropriate medium, is introduced into the cell culture device 100 shown in Figures 3 and 4. The cell culture device 100 includes a coating or substrate on each of the first and second culture surfaces 128, 130 to allow cells to adhere. In particular, the cell culture may be introduced into the volume of the culture device 100 from an external container that includes a needle-based connector (not shown). The needle-based connector may pierce a septum seal in the external container and pierce one of the ports 126 in the interface plate 124, thereby fluidly connecting the external container and the culture device 100.

[0172] Before or after the introduction of the adherent cell culture, an external actuator (not shown) can rotate the entire base 112 so that the first culture surface 128 is substantially horizontal, i.e., flat or coplanar with the transverse plane T. This is shown in FIG. 15. The external actuator (not shown) can pivot or rotate the base about an axis substantially aligned with the adjacent region 132. Note that the flexible sidewalls 114 allow the base 112 to pivot as shown in FIG. 15.

[0173] Cells in the cell culture may be settled on the base wall 112, ie, first culture surface 128, particularly on its coating or substrate.

[0174] The cell culture is grown through adhesion to the first culture surface 128, achieving exponential cell growth. The cells adhere to the first culture surface 128 and can be grown thereon for a period of time, for example, 10 minutes to 24 hours, preferably 6 to 12 hours. During growth, appropriate substances such as growth factors, fresh medium, proteins, magnetic beads, antibodies, viruses, etc. can be added to the cell culture. These substances can be introduced from an external container equipped with a needle-type connector, similar to the cell culture introduction method described above. The cell culture is allowed to grow until it reaches a desired cell density.

[0175] Optionally, a medium exchange step is performed before passaging cells from the first culture surface 128 to the second culture surface 130. Specifically, a portion of the cell culture medium, i.e., the depleted or spent medium, is removed from the culture device 100 and replaced with fresh medium. The medium can be removed through port 126 of the interface plate 124. Fresh medium is introduced through port 126 of the interface plate 124 using an external container with a needle-based connector, similar to the introduction of the cell culture described above. As will be understood by those skilled in the art, a medium exchange step can also be performed after passaging the cells, as described below. In some examples, a medium exchange step can be performed both before and after passaging the cells, as described below.

[0176] Before transferring, i.e., passaging, the adherent cells from the first culture surface 128 to the second culture surface 130, the cell culture is washed. Specifically, all or part of the cell culture medium is removed from the culture device 100 through one of the ports 126 of the interface plate 124. Next, a wash solution is introduced into the culture device 100. The wash solution may be a buffered saline solution, such as phosphate-buffered saline. Generally, any suitable wash solution may be used to ensure removal of substances such as serum, calcium, and magnesium that may inhibit the action of the dissociation reagent described below. The culture device 100 is gently agitated, for example, by rocking, to wash the cell culture. This agitation may be performed by moving the base 112 with an external actuator (not shown). The wash solution is then expelled from the culture device 100 through one of the ports 126 of the interface plate 124.

[0177] After washing and before passaging the adherent cells, a dissociation reagent is added to the culture device 100. The dissociation reagent may be preheated to 37°C before addition. The dissociation reagent may be trypsin, TrypLE®, or another reagent that dissociates the adherent cells from the coating or substrate on the first culture surface 128. The culture device 10 is gently agitated, for example by rocking, to completely detach the cells from the first culture surface 128. This agitation may also be performed by moving the base 112 with an external actuator (not shown). The culture device 100 may be incubated at room temperature for a period of time.

[0178] As the adherent cells detach from the first culture surface 128, the base 112 pivots about the axis defined by the adjacent region 132. Specifically, an external actuator (not shown) causes the entire base 112 to pivot so that the second culture surface 130 is substantially horizontal, i.e., flat or flush with the transverse plane T. This is shown in FIG. 16. In this manner, the cells are transferred, or passaged, from the first culture surface 128 to the second culture surface 130 by gravity, and all cells reside on the second culture surface 130. At this stage, fresh cell culture medium can be added to the culture device through one of the ports 126 of the interface plate 124. The adherent cells then adhere to the coating or substrate on the second culture surface 130. The cells are allowed to adhere and grow for a specified period of time, e.g., 10 minutes to 24 hours, preferably 6 to 12 hours. Once the cells have adhered for the specified period of time, the cell culture medium containing the dissociation reagent is removed through one of the ports 126 of the interface plate 124. Fresh cell culture medium is then introduced through port 126 of interface plate 124 using an external container with a needle-based connector, similar to the cell culture introduction method described above, to allow cells to grow on the coating or substrate on second culture surface 130. During growth, appropriate substances such as growth factors, fresh medium, proteins, magnetic beads, antibodies, viruses, etc. may be added to the cell culture. These substances may be introduced through an external container with a needle-type connector, similar to the cell culture introduction method described above. The cell culture is allowed to grow until it reaches a desired cell density.

[0179] Finally, once the cell culture has matured and reached the desired density, it is harvested through port 122 of interface plate 124. Alternatively, if an optional port is provided in the base of the culture device, the cells may be harvested through such port. In particular, the washing and dissociation steps described above can be applied to cell cultures present on a coating or substrate on second culture surface 130, thereby ensuring that adherent cells are properly dissociated from the coating or substrate. The base 112 is then moved or pivoted by an actuator (not shown) about the axis defined by adjacent region 132 to ensure that all cells are detached from the coating or substrate. The cells are then harvested as described above.

[0180] While the exemplary protocol described above describes two culture surfaces, one skilled in the art can apply the teachings above to any number of culture surfaces. In particular, for three, four, or more culture surfaces, cells can be passaged from the first culture surface to the second and then to the third culture surface by similarly moving the base. For adherent cells, a separation step as described above is required between each passage.

[0181] Additionally, the exemplary protocols described above may be performed on any of the devices described herein. Additionally, the exemplary protocols may be performed in any order, for example, suspension or adherent cells may be cultured first on second culture surface 130 and then passaged to first culture surface 128.

[0182] Furthermore, while the use of the device described above has been described with respect to moving the base to allow for material transfer (or "passaging"), it is equally envisioned that the entire device may be moved or tilted so that a continuous surface may be positioned horizontally. This is particularly true in instances where the side walls are rigid.

[0183] Those skilled in the art will appreciate that the above-described embodiments are presented by way of example only and in a limiting sense only, and that various changes and modifications may be made without departing from the scope of the present invention as defined by the appended claims. Various changes may be made to the detailed designs described above, such as variations in shape, size, material, arrangement, assembly, etc.

[0184] In certain embodiments, the invention may be defined by one or more of the following clauses.

[0185] [Section 1] a base (12, 112, 212, 312, 412, 512, 612, 712, 812, 912) having a first surface (28, 128, 228, 328, 428, 528, 638, 728, 828, 928) and a second surface (30, 130, 230, 330, 430, 530, 630, 730, 830, 930) fixedly disposed relative to the first surface; and a sidewall (14, 114, 214, 314) extending from said base to define an interior volume of the device; A device (10, 100, 200, 300) comprising: The device, wherein the first surface and the second surface are non-coplanar.

[0186] [Section 2] 2. The device of clause 1, wherein the sidewall is a compressible sidewall.

[0187] [Section 3] The device of clause 1 or clause 2, wherein the first surface forms a first angle (α) with a transverse plane (T) extending substantially perpendicular to a central longitudinal axis (L) of the device.

[0188] [Section 4] The device of paragraph 3, wherein the first angle is between about 1 degree and about 45 degrees, preferably between about 5 degrees and about 30 degrees.

[0189] [Section 5] The device of any one of clauses 1 to 4, wherein the second surface forms a second angle (β) with a transverse plane (T) extending substantially perpendicular to a central longitudinal axis (L) of the device.

[0190] [Section 6] The device according to paragraph 5, wherein the second angle is between about 1 degree and about 45 degrees, preferably between about 5 degrees and about 30 degrees.

[0191] [Section 7] The device of any one of clauses 1 to 6, wherein the ratio of the area of ​​the first surface to the area of ​​the second surface is from about 1:9 to about 1:1.

[0192] [Section 8] The device of clause 7, wherein the ratio is about 1:3.

[0193] [Section 9] 9. The device of any one of clauses 1 to 8, wherein the base further comprises a third surface (334, 434, 534) fixedly disposed relative to each of the first surface and the second surface, and wherein the first surface, the second surface, and the third surface are non-coplanar.

[0194] [Section 10] 10. The device of clause 9, wherein the third surface forms a third angle (γ) with a transverse plane (T) extending substantially perpendicular to a central longitudinal axis (L) of the device.

[0195] [Section 11] 11. The device according to paragraph 10, wherein the third angle is between about 1 degree and about 45 degrees, preferably between about 5 degrees and about 30 degrees.

[0196] [Section 12] 12. The device of any one of clauses 9 to 11, wherein the area ratio between the first surface, the second surface, and the third surface is about 1:2:3 or about 1:3:6.

[0197] [Section 13] The device of any one of clauses 1 to 12, wherein the base further comprises at least one port (22).

[0198] [Section 14] The device of any one of clauses 1 to 13, wherein at least one surface comprises an adherent cell culture substrate or coating.

[0199] [Section 15] 15. The device of clause 14, wherein each surface comprises an adherent cell culture substrate or coating.

[0200] [Section 16] 16. The device of clause 14 or clause 15, wherein the adherent cell culture substrate or coating is selected from the group comprising collagen, fibronectin, vitronectin, laminin, gelatin, polylysine, cellulose, or a combination thereof.

[0201] [Section 17] 17. The device of any one of clauses 1 to 16, wherein the base and / or at least one surface comprises polystyrene, polycarbonate, low-density polyethylene, high-density polyethylene, silicone, or a thermoplastic elastomer.

[0202] [Section 18] The device of any one of clauses 1 to 17, wherein the device is a cellular device.

[0203] [Section 19] A device according to any one of clauses 1 to 18; an actuator configured to engage at least a portion of the device and move the device between a first configuration in which the first surface is horizontal and a second configuration in which the second surface is horizontal; A system comprising:

[0204] [Section 20] 1. A method for culturing biological material, comprising: providing a first surface (28, 128, 228, 328, 428, 528, 638, 728, 828, 928) and a second surface (30, 130, 230, 330, 430, 530, 630, 730, 830, 930) fixedly disposed relative to the first surface, the first surface and the second surface being non-coplanar; orienting the first surface in a substantially horizontal plane; culturing biological material on the first surface; orienting the second surface in a substantially horizontal plane, thereby transferring biological material from the first surface to the second surface; culturing a biological material on the second surface; The method comprising:

[0205] [Section 21] 21. The method of clause 20, wherein the biological material comprises tissue or cells.

[0206] [Section 22] 22. The method of clause 21, wherein the cells are adherent cells and the first surface and the second surface each comprise an adherent cell culture substrate or coating.

[0207] [Section 23] 20. Use of a device according to any one of clauses 1 to 18 or a system according to claim 19 in culturing adherent cells.

[0208] [Section 24] 20. Use of a device according to any one of clauses 1 to 18 or a system according to claim 19 in the cultivation of suspension cells.

[0209] [Section 25] A device according to any one of claims 1 to 18; an interface member configured to be coupled to an upper portion of the device; A parts kit comprising: [Explanation of symbols]

[0210] 10, 100, 200, 300 culture devices 12, 112, 212, 312, 412, 612, 712, 812, 912 base 12c Material Block 14, 114, 214, 314 flexible side walls 16 Opening 28, 128, 228, 328, 428, 528, 638, 728, 828, 928 First surface, first culture surface 18 Deformable Area 20 hard area 22, 122 outlet ports 24, 124, 224, 324 interface plates 26, 126, 326 inlet ports 30, 130, 230, 330, 430, 530, 630, 730, 830, 930 Second surface, second culture surface 132, 232, 332, 336, 540, 542, 544, 546, 632, 732, 832, 932 adjacent areas 334, 434, 534 Third Side

Claims

1. A culture device (10, 100, 200, 300) comprising: a base (12, 112, 212, 312, 412, 512, 612, 712, 812, 912) having a first surface (28, 128, 228, 328, 428, 528, 638, 728, 828, 928) and a second surface (30, 130, 230, 330, 430, 530, 630, 730, 830, 930) fixedly disposed relative to the first surface; and a flexible sidewall (14, 114, 214, 314) extending from the base to define an interior volume of the culture device; In a culture device (10, 100, 200, 300) comprising: A culture device, wherein the first surface and the second surface are non-coplanar.

2. The culture device of claim 1 , wherein the sidewall is a compressible sidewall.

3. The culture device of claim 1 or 2, wherein the first surface forms a first angle (α) with a transverse plane (T) extending substantially perpendicular to a central longitudinal axis (L) of the culture device.

4. The culture device of claim 3, wherein the first angle is between about 1 degree and about 45 degrees, preferably between about 5 degrees and about 30 degrees.

5. The culture device according to any one of claims 1 to 4, wherein the second surface forms a second angle (β) with a transverse plane (T) extending substantially perpendicular to a central longitudinal axis (L) of the culture device.

6. The culture device of claim 5, wherein the second angle is from about 1 degree to about 45 degrees, preferably from about 5 degrees to about 30 degrees.

7. The culture device of any one of claims 1 to 6, wherein the ratio of the area of ​​the first surface to the area of ​​the second surface is from about 1:9 to about 1:

1.

8. The culture device of claim 7 , wherein the ratio is about 1:

3.

9. The culture device of any one of claims 1 to 8, wherein the base further comprises a third surface (334, 434, 534) fixedly positioned relative to each of the first surface and the second surface, and the first surface, the second surface, and the third surface are non-coplanar.

10. The culture device of claim 9, wherein the third surface forms a third angle (γ) with a transverse plane (T) extending substantially perpendicular to a central longitudinal axis (L) of the culture device.

11. The culture device of claim 10, wherein the third angle is from about 1 degree to about 45 degrees, preferably from about 5 degrees to about 30 degrees.

12. The culture device of any one of claims 9 to 11, wherein the area ratio between the first surface, the second surface, and the third surface is about 1:2:3 or about 1:3:

6.

13. The culture device of any one of claims 1 to 12, wherein the base further comprises at least one port (22).

14. The culture device of any one of claims 1 to 13, wherein at least one surface comprises an adherent cell culture substrate or coating.

15. The culture device of claim 14 , wherein each surface comprises an adherent cell culture substrate or coating.

16. 16. The culture device of claim 14 or 15, wherein the adherent cell culture substrate or coating is selected from the group comprising collagen, fibronectin, vitronectin, laminin, gelatin, polylysine, cellulose, or a combination thereof.

17. The culture device of any one of claims 1 to 16, wherein the base and / or at least one surface comprises polystyrene, polycarbonate, low density polyethylene, high density polyethylene, silicone, or a thermoplastic elastomer.

18. The culture device according to any one of claims 1 to 17, wherein the culture device is a cell culture device.

19. A culture device according to any one of claims 1 to 18; an actuator configured to engage at least a portion of the culture device and move the culture device between a first configuration in which the first surface is horizontal and a second configuration in which the second surface is horizontal; A system comprising:

20. 1. A method for culturing biological material, comprising: providing a first surface (28, 128, 228, 328, 428, 528, 638, 728, 828, 928) and a second surface (30, 130, 230, 330, 430, 530, 630, 730, 830, 930) fixedly disposed relative to said first surface, said first surface and said second surface being non-coplanar; orienting the first surface in a substantially horizontal plane; culturing a biological material on the first surface; orienting the second surface in a substantially horizontal plane, thereby transferring biological material from the first surface to the second surface; culturing a biological material on the second surface; The method comprising:

21. 21. The method of claim 20, wherein the biological material comprises tissue or cells.

22. 22. The method of claim 21, wherein the cells are adherent cells and the first surface and the second surface each comprise an adherent cell culture substrate or coating.

23. Use of the culture device according to any one of claims 1 to 18 or the system according to claim 19 in culturing adherent cells.

24. Use of the culture device according to any one of claims 1 to 18 or the system according to claim 19 in the culture of suspension cells.

25. A culture device according to any one of claims 1 to 18, an interface member configured to be coupled to a top of the culture device; A parts kit comprising:

Images