Cell culture arrangement, device and method of use

Abstract

A cell culture device is disclosed comprising a fixed bed arranged within a housing, wherein the fixed bed comprises a continuous corrugated porous medium comprising a top portion, a bottom portion, a front portion, a rear portion, a right side and a left side, and a plurality of folds folded on the right side and the left side, and wherein the continuous corrugated porous medium also has vertical fluid flow channels along the longitudinal axis between adjacent folds.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 326,085 (filing date: March 31, 2022), incorporated by reference. Background Technology

[0003] Cells can be cultured in a fixed bed, where they are attached to a porous carrier. However, an improved fixed bed is required.

[0004] The present invention is provided to improve upon at least some of the disadvantages of the prior art. These and other advantages of the present invention will become apparent from the description below.

[0005] One aspect of the present invention provides a cell culture array comprising: (a) a housing having a first end and a second end and a longitudinal axis; and (b) at least one fixed bed arranged within the housing, wherein the at least one fixed bed comprises a continuous pleated porous medium having a plurality of folds folded on an upper end, a lower end, a front end, a rear end, a right side, a left side, and a right side and a left side, and the continuous pleated porous medium has a vertical fluid flow channel along the longitudinal axis between adjacent folds.

[0006] In some embodiments of a cell culture array, at least one fixed bed comprises at least one removable sampling element comprising a porous medium arranged within a continuous pleated porous medium.

[0007] In another embodiment, a cell culture device comprises: (a) a cell culture array of one embodiment; (b) a pump body arranged at a second end of a housing, wherein the pump body comprises a central cavity; (c) an impeller arranged in the central cavity of the pump body; and (d) a cell culture vessel having a central chamber, wherein the cell culture array is arranged in the central chamber.

[0008] In another embodiment, the cell culture device comprises: (a) a cell culture array of one embodiment; (b) a pump assembly arranged at a second end of a housing, wherein the pump assembly comprises a pump outer body and a pump body connected to the pump outer body, and the pump body comprises a central cavity; (c) an impeller arranged in the central cavity of the pump body; and (d) a cell culture vessel having an open first vessel end and a closed second vessel end, and a vessel sidewall connected to the open first vessel end and the closed second vessel end, wherein the cell culture vessel has a central chamber; and the cell culture array and the pump assembly are arranged within the central chamber.

[0009] A method for cell culture according to one embodiment of the present invention comprises: a step of introducing a cell culture medium and cells into a cell culture device of one embodiment; and a step of operating a magnetic impeller. In some embodiments, the method further comprises a step of harvesting cultured cells. Brief explanation of the drawing

[0010] FIGS. 1a to 1d are drawings showing representative diagrams of a "dry" fixed bed comprising a wrinkled porous cell culture medium according to an embodiment of the present invention. FIG. 1a shows an isometric view (the bed has a rectangular configuration), FIG. 1b shows a top view, and FIGS. 1c and 1D show enlarged views of the "1C" and "1D" portions of FIG. 1b, respectively. FIGS. 2a and 2d are drawings showing representative drawings of a "dry" fixed bed comprising a wrinkled porous cell culture medium according to another aspect of the present invention. FIG. 2a shows an isometric view (the bed has a rectangular configuration), FIG. 2b shows a top view, and FIGS. 2c and 2d show enlarged views of the "2C" and "2D" portions of FIG. 2b, respectively. FIG. 3a is a diagram showing "self-spacing" due to tangential flow through a fixed bed and tangential fluid flow of cell culture liquid (liquid culture medium) between pairs of legs of each fold, and also shows the fold height; FIG. 3b is a diagram showing an isometric view of a corrugated fixed bed having a rectangular configuration according to another aspect of the present invention, and also shows an exemplary fixed bed height. FIGS. 4a through 4d are drawings illustrating the use of scrap material as a "sacrificial layer" to create the fixed bed illustrated in FIG. 1a. FIG. 4a shows the insertion of scrap material on both sides of a porous cell culture medium; FIG. 4b shows a corrugated porous cell culture medium having corrugated scrap material on both sides of the culture medium; FIG. 4c shows an enlarged view of section "E" illustrated in FIG. 4b. FIG. 4d shows the fixed bed after the removal of the scrap material. FIGS. 5a through 5d are drawings showing a cell culture apparatus comprising a fixed bed containing a corrugated porous cell culture medium within a bed housing according to another aspect of the present invention, wherein the cell culture array is arranged within a vessel providing a cell culture apparatus according to another aspect of the present invention. FIG. 5a shows an exploded view of the cell array and the cell culture apparatus; FIG. 5b shows an isometric assembly view of the cell culture apparatus. FIG. 5c shows a perspective assembled view of the cell culture apparatus for transport / storage and also shows a shipping cover, FIG. 5d shows a cross-sectional assembled view for transport / storage and also shows a shipping cover and a fixed bed retainer, a stabilizing element inserted between the shipping cover and the bed retainer, and an impeller arranged within a pump body under the bed housing. FIGS. 6a through 6d are drawings showing a cell culture array comprising two separate fixed beds each containing a corrugated porous cell culture medium within a bed housing, wherein the cell culture array is arranged within a vessel providing a cell culture apparatus according to another aspect of the present invention. FIG. 6a shows an assembled cell culture array, FIG. 6b shows the assembled cell culture array and pump array shown in FIG. 6a; FIG. 6c shows an exploded view of a cell culture apparatus including an impeller arranged in a pump body below the bed housing; FIG. 6d shows an equiaxial top view of the assembled cell culture apparatus, wherein in FIG. 6c and 6d, the transport cover and stabilization element are removed so that the bioreactor lid can be placed on top before operating the apparatus. FIGS. 7a through 7g are drawings showing a cell culture array comprising two separate fixed beds, each containing a corrugated porous cell culture medium within a bed housing, wherein the cell culture array is arranged within a vessel providing a cell culture device according to another aspect of the present invention. FIG. 7a shows a cross-sectional assembly for transport / storage and also shows a transport cover, a fixed bed housing, and a stabilizing element interposed between the transport cover and the bed retainer; FIG. 7b shows a perspective assembly of a cell culture device for transport / storage and also shows the transport cover. FIG. 7c shows a perspective view of the stabilizing element after the transport cover has been removed. FIG. 7d shows an exploded view of the cell culture device after the stabilizing element has been removed and also shows removable sampling elements inserted into the corrugated porous cell culture medium of the fixed bed and an impeller arranged in a pump body below the bed housing. FIGS. 7e and 7f respectively illustrate top and bottom views of a bed housing of a cell culture device, wherein the inner walls of the pump outer body include optional ribs for separating the fixed beds from the inner walls and a retainer for holding the fixed beds of the bed housing. FIG. 7g illustrates a partial view of the cell culture device shown in FIG. 7d, showing a plurality of removable sampling elements inserted into the corrugated porous cell culture medium of the fixed beds. FIGS. 8a through 8f are drawings illustrating a cell culture apparatus comprising a plurality of individual fixed beds, each comprising a corrugated porous cell culture medium within a bed housing according to another aspect of the present invention. FIG. 8a shows a perspective view of the assembled cell culture apparatus and also shows various optional ports for sampling / determining various parameters, for example, during operation. FIG. 8b shows a cross-sectional view of the cell culture apparatus illustrated in FIG. 8a with the upper housing section removed, and also shows an upper baffle or grid plate and an impeller arranged within a pump body below the bed housing, as well as a central column S bend and a flow diverter located in the center of the fixed bed housing. FIG. 8c shows a cross-sectional view of the bed housing with the upper grid plate removed; FIG. 8d shows a top perspective view of a cell culture array without fixed beds in the fixed bed chambers. FIG. 8e shows the bottom of the bed housing and also shows the grid plate providing the bottom of the 23 fixed bed chambers; FIG. 8f shows a top perspective view of a cell culture array with fixed beds in the fixed bed chambers. FIGS. 9a through 9c are drawings showing a cell culture apparatus comprising a plurality of stackable sections according to another aspect of the present invention, wherein the stackable sections provide vertically arranged fixed bed chambers for accommodating fixed beds (a single fixed bed is accommodated within the vertically arranged stacked fixed bed chambers providing a full-length fixed bed cavity), wherein each fixed bed comprises a corrugated porous cell culture medium within a bed housing. FIG. 9a is a top perspective view (the cover is transparent) showing a cell culture array comprising a plurality of stackable sections, providing vertically arranged fixed bed chambers for accommodating fixed beds, wherein each fixed bed comprises a corrugated porous cell culture medium. FIG. 9b shows a partial cross-sectional view of the cell culture device illustrated in FIG. 9a without fixed beds within vertically arranged stacked fixed bed chambers, and also shows an impeller arranged at the bottom of the culture array and at the bottom of the housing, and FIG. 9c shows a partial cross-sectional view of the cell culture device illustrated in FIG. 9a having fixed beds within vertically arranged stacked fixed bed chambers. Specific details for implementing the invention

[0011] According to one aspect of the present invention, a cell culture array is provided comprising: (a) a housing having a first end and a second end and a longitudinal axis; and (b) at least one fixed bed arranged within the housing, wherein the at least one fixed bed comprises a continuous pleated porous medium having a plurality of folds folded on the top, bottom, front, rear, right side and left side and right side and left side, and the continuous pleated porous medium also has vertical fluid flow channels along the longitudinal axis between adjacent folds.

[0012] In some embodiments, the cell culture array comprises a plurality of detachable sampling elements comprising a porous medium inserted into a continuous pleated porous medium.

[0013] In some embodiments, the cell culture array comprises at least two fixed beds arranged within a housing, each of the at least two fixed beds comprises a continuous corrugated porous medium comprising a top portion, a bottom portion, a front portion, a rear portion, a right portion and a left portion, and a plurality of folds folded on the right portion and the left portion, and the continuous corrugated porous medium also has vertical fluid flow channels along the longitudinal axis between adjacent folds.

[0014] In some embodiments of the cell culture array, each continuous pleated porous medium has a pleat density in the range of 25% to 95%, in some embodiments, in the range of 40% to 95%, more typically in the range of 40% to 85%. According to some embodiments, the cell culture array comprises at least two fixed beds arranged within a housing, each of the at least two fixed beds comprises a continuous pleated porous medium comprising a top portion, a bottom portion, a front portion, a rear portion, a right side and a left side, and a plurality of pleats folded on the right side and the left side (each having first and second pleat legs), and the continuous pleated porous medium also has vertical fluid flow channels along the longitudinal axis between adjacent pleats.

[0015] Typically, a corrugated porous medium is oriented such that the corrugations are oriented in the horizontal xy plane and the vertical fluid flow channels are oriented in the vertical z plane.

[0016] In another embodiment, the cell culture device comprises: (a) a cell culture array of one embodiment; (b) a pump body arranged at a second end of a housing, wherein the pump body comprises a central cavity; (c) an impeller arranged in the central cavity of the pump body; and (d) a cell culture vessel having a central chamber, wherein the cell culture array is arranged in the central chamber.

[0017] In another embodiment, the cell culture device comprises: (a) a cell culture array of one embodiment; (b) a pump assembly arranged at a second end of a housing, wherein the pump assembly comprises a pump outer body and a pump body connected to the pump outer body, and the pump body comprises a central cavity; (c) an impeller arranged in the central cavity of the pump body; and (d) a cell culture vessel having an open first vessel end and a closed second vessel end, and a vessel sidewall connected to the open first vessel end and the closed second vessel end, wherein the cell culture vessel has a central chamber; and wherein the cell culture array and the pump assembly are arranged within the central chamber.

[0018] In one embodiment of a cell culture device, the pump assembly includes a pump lid attached to a second end of the housing, and the pump outer body is connected to the pump lid. Alternatively or additionally, in one embodiment of a cell culture device, the impeller includes a magnetic impeller.

[0019] Other aspects of the present invention include a method for culturing cells, a method for treating cell culture medium, and a fixed-bed bioreactor system.

[0020] A cell culture method according to one embodiment of the present invention comprises: a step of introducing a cell culture medium and cells into a cell culture device of one embodiment; a step of operating a magnetic impeller to continuously supply an aerated liquid culture medium containing nutrients and dissolved oxygen to a fixed bed; and a step of removing undesirable materials generated from biological processes, such as carbon dioxide and lactate. In some embodiments, the method further comprises a step of harvesting cultured cells.

[0021] Advantageously, the cell culture liquid (liquid culture medium) flows between the pleat legs, and accordingly, the pleats of the porous cell culture medium "self-align" to provide the desired suitable spacing between the pleat legs and adjacent pleats without spacers, while simultaneously exhibiting low back pressure and shear force.

[0022] A cell culture array, a cell culture device, and a cell culture system may have a single fixed bed containing a cell culture medium, or a plurality of fixed beds containing a cell culture medium, for example, two or more, 20 or more, 100 or more, or 240 or more fixed beds.

[0023] Each of the components of the present invention will be described in more detail below, and similar components have similar designation numbers.

[0024] With reference to the embodiments shown in FIGS. 1a through 1d (components ending in "A", "a" and "a'") and FIGS. 2a through 2d (components ending in "B", "b" and "b'"), the fixed bed (150A, 150B) comprises a wrinkled porous cell culture medium (100A, 100B) comprising a continuous wrinkled porous medium (50A, 50B), wherein the continuous wrinkled porous medium (50A, 50B) comprises an upper portion (51A, 51B), a lower portion (52A, 52b), a front portion (53A, 53B), a rear portion (54A, 54B), a right side (55A, 55B) and a left side (56A, 56B), and a plurality of folds (60A, 60B) folded on the right side and the left side, wherein the continuous The corrugated porous medium (50A, 50B) has vertical fluid flow channels (70A, 70B) between the legs of the corrugations.

[0025] Each fold has a pair of fold legs (61A, 62A; 61B, 62B), and each fold leg has a first fold leg surface (61a, 62a; 61b, 62b) and a second fold leg surface (61a', 62a'; 61b, 62b'), wherein portions of the first fold leg surface and the second fold leg surface within the fold can come into contact with each other, and portions of one first fold leg surface can come into contact with portions of the second fold leg surface of an adjacent fold, and during use, due to tangential flow, vertical fluid flow channels are formed between the fold legs providing "self-spacing" (see FIG. 3a).

[0026] In some embodiments, a fixed bed comprising a continuous corrugated porous medium within a housing has a corrugation density in the range of 25% to 95%, typically in the range of 40% to 95%, preferably in the range of 40% to 85%. Typically, the corrugated porous medium may be oriented such that the corrugations are oriented in the horizontal xy-plane and the vertical fluid flow channels are in the vertical z-plane, wherein the closed edges of the corrugations are parallel to the vertical fluid flow.

[0027] The embodiments of the fixed bed are expandable and can have any suitable fixed bed height, for example, in the range of 2 cm to 150 mm, typically in the range of 10 mm to 100 mm.

[0028] The fixed bed can have various configurations and typically has a rectangular or square configuration (see figure).

[0029] As illustrated in FIGS. 1a through 1d and FIGS. 2a through 2d (representing a “dry” medium), the folds may have wider or narrower gaps (“radii,” where radius R1 in FIG. 1c is greater than radius R2 in FIG. 2c) on the inner surfaces of the folds, and the medium provides flow channels having closed ends (formed by the folds) and open ends (without folds), and the flow channels alternate being closed at the first end and open at the second end, and being open at the first end and closed at the second end. However, although parts of the opposing surfaces appear to be in contact with each other, once the cell culture device is operated, during tangential flow, at least major parts of some opposing surfaces will spread apart from each other, thereby providing the desired suitable separation (see FIGS. 1c, FIGS. 1d, FIGS. 2c, FIGS. 2d, and FIG. 3a). Furthermore, the liquid will penetrate multiple layers, and the parts of the opposing surfaces maintaining contact will still retain cell culture functions.

[0030] In some embodiments, the porous cell culture medium may be pleated using scrap material as "sacrificial layers" during the plating process. For example, as shown in FIGS. 4a to 4d, a carrier medium (171, 172) may be placed on one side of the porous cell culture medium and pleated together (e.g., using a pleating machine), and the scrap material is sequentially removed as shown in FIG. 4d. The resulting fixation bed may have a larger radius (e.g., "R1") as shown in FIG. 1c compared to pleated fixation beds that do not use sacrificial layers (e.g., having a smaller radius "R2") as shown in FIG. 2c.

[0031] In preferred embodiments of a cell culture array in which a fixed bed is arranged within a housing, the fixed bed fold density is in the range of 40% to 85%, allowing for a homogeneous cell distribution in the bed due to cells moving along flow channels. For example, using FIG. 3b for reference, the fixed bed (150) has a fixed height of 100 mm and a fold height of 40 mm, with 66 folds per 40 mm = 76% fold density. A 100% fold density is when the combined thickness of all folds inside the housing is equal to the width of the internal cavity of the housing (e.g., when the width of the internal cavity is 40 mm), and the thickness of each leg of the fold is 0.23 mm (making the fold thickness 0.46 mm), resulting in 40 mm / 0.46 mm = 87 folds for a 100% fold density. In this configuration, the entire housing is filled with a carrier material, and there is no usable space between them. To allow flow channels, the number of folds is reduced to less than 100% of the theoretical maximum amount of folds that fit in the housing.

[0032] A continuous corrugated porous medium may have any number of corrugations. For example, a fixed bed may have corrugations in the range of 50 to 60, but may have fewer than 50 or more than 60 corrugations.

[0033] According to embodiments of the present invention, a flexible porous cell culture medium may be a woven or nonwoven porous cell culture medium or a porous membrane, and may be formed from any numerous materials known in the art, including, for example, natural or, more preferably, synthetic polymers such as polyethylene terephthalate (PET). Suitable porous cell culture media are commercially available; one nonwoven porous cell culture medium is nonwoven hydrophilized PET. The porous cell culture medium typically has a thickness in the range of 50 to 400 μm.

[0034] A porous cell culture medium may have any suitable pore structure, for example, pore size (e.g., bubble point or KL as described in U.S. Patent No. 4,340,479, or as demonstrated by capillary condensation flow pore measurement), mean flow pore (MFP) size (e.g., when characterized using a porometer, e.g., Porvair Porometer (Porvair plc, Norfolk, UK), or a porometer available under the trade name POROLUX (Porometer.com, Belgium)), pore class, pore diameter (e.g., when characterized using a modified OSU F2 test as described in U.S. Patent No. 4,925,572), or removal class medium.

[0035] The porous cell culture medium may have any desired critical wetting surface tension (CWST, as defined, e.g., in U.S. Patent 4,925,572). The CWST may be selected as known in the art, e.g., as further disclosed in U.S. Patents 5,152,905, 5,443,743, 5,472,621 and 6,074,869. The surface properties of the porous cell culture medium may be modified by wet or dry oxidation, coating or depositing a polymer or hydrogel on the surface, or by a grafting reaction (e.g., to affect cell adhesion, to affect the CWST, to include a surface charge, e.g., positive or negative charge, and / or to change the polarity or hydrophilicity of the surface). Modification includes, for example, irradiation, surface coating and / or curing with polar or charged monomers, charged polymers, and performing chemical modification to attach functional groups to the surface. The grafting reaction may be activated by exposure to an energy source such as gas plasma, vapor plasma, corona discharge, heat, Van de Graff generator, ultraviolet light, electron beam, or various other forms of radiation, or by surface etching or deposition using plasma treatment.

[0036] In some embodiments (e.g., see FIG. 7a, 7d and 7g), the cell culture array comprises a plurality of removable sampling elements (800) comprising a porous medium (801) inserted into a continuous pleated porous medium (e.g., inserted into a flow channel between the pleated legs). If desired, the sampling elements comprise a porous medium of single sheets, wherein the porous medium is the same as the porous medium used to create the continuous pleated porous medium. If desired, the removable sampling elements may include pull tabs attached to the sampling elements, or the length of the sampling element extending (protruding) beyond the continuous pleated porous medium provides the pull tabs.

[0037] Sampling elements can be used, for example, to measure the cell population density of cells attached to the sampling element. Alternatively or additionally, analyzing the sample includes one or more replicable retrovirus tests, exogenous virus tests, and sterility tests (e.g., where the sampling element having attached cells is placed in a liquid medium to analyze for exogenous microbial growth).

[0038] According to embodiments of the present invention, a cell culture array is provided comprising: (a) a housing having a first end and a second end and a longitudinal axis; and (b) at least one fixed bed arranged within the housing, wherein the at least one fixed bed comprises a continuous corrugated porous medium comprising an upper end, a lower end, a front end, a rear end, a right side, and a left side, and a plurality of folds (the folds having legs) folded on the right side and the left side (see, for example, FIG. 1a to 1d and FIG. 2a to 2d), and the continuous corrugated porous medium also comprises vertical fluid flow channels along the longitudinal axis between adjacent folds, at least one fixed bed.

[0039] As illustrated in FIGS. 5a to 5d, FIGS. 6a to 6d, FIGS. 7a to 7g, FIGS. 8a to 8f, and FIGS. 9a to 9c, a fixed bed (150) comprising a corrugated porous cell culture medium (100) is arranged within a bed housing (200) having a first end (201), a second end (202), a bed chamber (250), and a longitudinal axis (L) to provide a cell culture array (500) ( FIGS. 6a to 6d and FIGS. 7a to 7g show two fixed beds (150), wherein each of the two fixed beds (150) comprises a corrugated porous cell culture medium (100) arranged in separate bed chambers (250) within the same bed housing (200); FIGS. 8a to 8f and FIGS. 9a to FIG. 9c illustrates more than two fixed beds (150), wherein each of the more than two fixed beds (150) comprises a corrugated porous cell culture medium (100) arranged in separate bed chambers (250) within the same bed housing (200). The bed housing may be a single piece from a first end to a second end (as shown in FIG. 7a and FIG. 8b) or may comprise a plurality of segments fitted together. For example, the housings shown in FIG. 5d and FIG. 6a through 6d have a lower segment (200B) and an upper segment (200A) fluidly tightly engaged, and a similar result is provided by the vertically arranged stacked fixed bed chambers shown in FIG. 9a through 9c (stackability is provided for ease of manufacturing).

[0040] In some embodiments, as illustrated in more detail in FIG. 6c, FIG. 6d, and FIG. 8b, a bed retainer (such as a baffle or grid plate) (210) having openings (211) is arranged at a first end (201) of the housing, and in some embodiments, the bed retainer can be fastened to the housing by rotating parts of the retainer so as to be fitted into slots (212) at the first end (201), for example, as illustrated in FIG. 6d.

[0041] In the embodiments illustrated in FIGS. 7e and 7f, the bed housing (200) has inner walls (205), the inner walls (205) include optional outwardly extending ribs (206) that separate the cell culture medium (100) from the inner walls (205), the first end (201) of the bed housing includes optional inwardly facing projections (207A), and the second end (202) of the bed housing includes optional inwardly facing projections (207B) that retain the cell culture medium within the bed housing (200).

[0042] In the embodiments illustrated in FIG. 5a, FIG. 5d, FIG. 6c, FIG. 6d, FIG. 7a, and FIG. 7d, the cell culture device (1000) comprises: a cell culture array (500); a pump assembly (600) arranged at a second end (202) of a bed housing (200), wherein the pump assembly (600) comprises an optional pump cover (601) attached to the second end of the housing, a pump outer body (602) connected to the pump cover (601), and a pump body (610) connected to the pump outer body (602), wherein the pump body (610) comprises a central cavity (650); and a magnetic drive impeller (700) arranged in the central cavity (650) of the pump body (610). A cell culture vessel (900) having an open first vessel end (901) and a closed second vessel end (902), and a vessel side wall (905) connected to the open first vessel end (901) and the closed second vessel end (902), wherein the cell culture vessel (900) has a central chamber (950), and a cell culture array (500) and a pump assembly (600) are arranged in the central chamber (950).

[0043] Aspects of the cell culture device may include any number of ports and may include at least one inlet port and at least one outlet port. For example, FIG. 8a shows a perspective view of an assembled cell culture device and also shows various optional ports at the top of the device for sampling / measuring various parameters during operation, for example, as well as an inlet port and an outlet port. Using the aspect illustrated in FIG. 8a for reference, in another aspect, the top of the device (the upper surface of the first cell culture housing (910)) includes a large port for accommodating, for example, a pump assembly, and / or a recess (912) for accommodating other components.

[0044] The exemplary embodiments of the cell culture device illustrated in FIGS. 5d and 7a have a closed transport / storage cover (1101) that fluidly tightly covers an open first vessel end (901), wherein an optional stabilizing element (1110) (see FIG. 7c), such as blocking foam, is arranged between the inner surface of the closed cover (1101) and the bed retainer (210). Before using the cell culture device, the cover (1101) and the stabilizing element (1110) (if present) are removed (see FIG. 6c, FIG. 6d, and FIG. 7a), and a bioreactor lid (e.g., a stainless steel cover) is fluidly placed on top of the bed retainer.

[0045] Typically, a cell culture device has at least one structure that enables the delivery of a cell culture medium (liquid) that is essentially free of cells (e.g., through one or more connectors on a bioreactor system cover or integrated into said device).

[0046] As mentioned above, the cell culture array and / or cell culture device may have a single fixed bed or multiple fixed beds, each comprising a wrinkled porous cell culture medium.

[0047] For example, with reference to FIGS. 6c and FIGS. 7d, the cell culture apparatus (1000) comprises a cell culture array (500) comprising a first fixed bed (150) and a second fixed bed (150) arranged in separate bed chambers (250) within a bed housing (200). In other embodiments, the cell culture apparatus comprises more than two fixed beds, for example, an embodiment of the cell culture apparatus (2000) illustrated in FIG. 8f has more than 20 fixed beds in separate bed chambers (250) within a bed housing (200), and an embodiment of the cell culture apparatus (3000) illustrated in FIGS. 9a through 9c has more than 200 fixed beds in separate stacks of bed chambers (250) within a bed housing (200).

[0048] With reference to FIGS. 8a through 8f, an illustrated embodiment of a cell culture apparatus (2000) comprises a cell culture array (500) comprising more than two fixed beds (150) arranged within a bed housing (200), for example, more than 10 fixed beds (23 fixed beds (150) are illustrated in separate bed chambers (250), for example, see FIG. 8f). Each bed chamber (250) comprises a first chamber end (275) and a second chamber end (276), wherein the bed housing has a grid plate (209) at the second end of the bed housing. If desired, bed spacers (enabling fluid to be delivered along the spacers to the beds) may be arranged in any embodiment of the cell culture array according to the present invention, the bed housing and / or each bed chamber (e.g., the spacers may be arranged below the beds (150)), wherein the spacers are held within the apparatus by a grid plate (209) at the second chamber end / second bed housing end. The grid plate has at least one slot (209A) (three are shown) that enables fluid flow arranged at each second chamber end. In some embodiments, the use of spacers is preferred so that the same bed housing can be used to accommodate fixed beds of various lengths.

[0049] In an embodiment of the cell culture device (2000) illustrated in FIGS. 8a through 8f, the device comprises a pump body (610) having a central cavity (650) arranged below a second end (202) of a bed housing (200), wherein a self-driven impeller (700) is arranged in the central cavity (650) of the pump body (610), and a flow diverter (613) (having an S-bend on the impeller leading to the central column) serving as an outlet toward a central column (614) provides an inlet for liquid in the center of the impeller coming out of an outer chamber (950), passing through fixed beds, and coming down toward the impeller, wherein the bed housing is arranged within a cell culture vessel (900) having a first (upper) cell culture housing (910) and a second (lower) cell culture housing (911), and the second cell culture housing is open to the first The cell culture vessel has a container end (901), an open second container end (902'), and a container sidewall (905') connected to the open first container end and the second container end, and the cell culture vessel has a central chamber (950), wherein the cell culture array is arranged in the central chamber.

[0050] With reference to FIGS. 9a through 9c, an illustrated embodiment of a cell culture device (3000) comprises a cell culture array (500) comprising more than two fixed beds (150) arranged within a bed housing (200) (e.g., more than 200 fixed beds (150) in separate bed chambers (250) are illustrated). Each bed chamber (250) comprises a first chamber end (275), a second chamber end (276), and a grid plate (209) at the second chamber end / second bed housing end. The grid plate has at least one slot (209) that enables fluid flow, arranged at each second chamber end. In an illustrated embodiment of the cell culture apparatus (3000), the cell culture array comprises a plurality of stackable sections, and the stackable sections provide vertically arranged fixed bed chambers that accommodate fixed beds (as illustrated in FIG. 9b, a single fixed bed is accommodated in vertically arranged stacked fixed bed chambers that provide a full-length fixed bed cavity), and each fixed bed comprises, according to another embodiment of the present invention, a corrugated porous cell culture medium within a bed housing.

[0051] In an embodiment of the cell culture device (3000) illustrated in FIGS. 9a to 9c, the device comprises a pump body (610) having a central cavity (650) arranged below a second end (202) of a bed housing (200), wherein a magnetic impeller (700) is arranged in the central cavity (650) of the pump body (610), and the bed housing is arranged within a cell culture vessel (900) having an open first vessel end (901), an open second vessel end (902'), and a vessel side wall (905') connected to the open first vessel end and the second vessel end, and the cell culture vessel has a central chamber (950), wherein a cell culture array is arranged in the central chamber.

[0052] Typically, a bioreactor system according to embodiments of the present invention may be operated as known in the art (see, for example, iCELLis® disposable fixed-bed bioreactor system including iCELLis® nanosystem and iCELLis® 500 system (Pall Corporation, Port Washington, NY, USA)), and may include at least one, preferably at least two, sensors, such as sensors for dissolved oxygen, pH, and temperature, as used in commercially available fixed-bed bioreactor systems (e.g., iCELLis® disposable fixed-bed bioreactor system including iCELLis® nanosystem and iCELLis® 500 system (Pall Corporation, Port Washington, NY, USA)).

[0053] The bed housing may be made of any suitable impermeable material, including any rigid impermeable thermoplastic material compatible with the fluid to be processed. For example, the housing may be made of a metal such as stainless steel or a polymer. In some embodiments, the housing is a polymer, such as plastic, or in some embodiments, acrylic, polypropylene, polystyrene, polyethylene, polyethylene terephthalate, or polycarbonate resin.

[0054] All references, including publications, patent applications, and patents cited herein, are individually and expressly indicated as being incorporated into this specification by reference, and are incorporated into this specification by reference to the same extent as the whole is described herein.

[0055] Unless otherwise specified in this specification or clearly contradicted by the context, the use of singular terms and “at least one” and similar descriptors in the context describing the invention (particularly in the context of the following claims) shall be interpreted to include both singular and plural forms. Unless otherwise specified in this specification or clearly contradicted by the context, the use of the term “at least one” following a list of one or more items (e.g., “at least one of A and B”) shall be interpreted to mean one item selected from the listed items (A or B) or to mean a combination of two or more of the listed items (A and B). The terms “comprising,” “having,” “including,” and “containing” shall be interpreted as open-ended terms (i.e., meaning “including but not limited thereto”) unless otherwise stated. The description of ranges of values ​​in this specification is intended to serve as a simplified method of individually referring to each individual value within the range, unless otherwise indicated in this specification, and each individual value is incorporated into this specification as if it were individually described in this specification. All methods described in this specification may be performed in any suitable order, unless otherwise indicated in this specification or clearly contradictory in the context. Any use of any example or exemplary expression (e.g., “like”) provided in this specification is intended merely to better describe the invention and does not impose limitations on the scope of the invention unless otherwise claimed. No expression in this specification shall be interpreted as indicating that any unclaimed element is essential to the practice of the invention.

[0056] Preferred embodiments of the present invention, including the best mode known to the inventors for carrying out the present invention, have been described herein. Variations of these preferred embodiments may be apparent to a person skilled in the art when reading the foregoing description. The inventors expect that a person skilled in the art will make appropriate use of such variations, and the inventors intend that the present invention may be practiced differently from as expressly described herein. Accordingly, the present invention includes all variations and equivalents of the subject matter described in the claims appended to this specification, as permitted by applicable law. Furthermore, in all possible variations, any combination of the foregoing elements is included in the present invention unless otherwise indicated in this specification or otherwise clearly contradicted by the context.

Claims

Claim 1 A cell culture arrangement comprising: (a) a housing having a first end and a second end and a longitudinal axis; and (b) at least one fixed bed arranged within the housing, wherein the at least one fixed bed comprises a continuous pleated porous medium having a top end, a bottom end, a front end, a rear end, a right side and a left side, and a plurality of pleats folded on the right side and the left side, and further comprises a plurality of removable sampling elements in which the continuous pleated porous medium has vertical fluid flow channels along the longitudinal axis between adjacent pleats, and the continuous pleated porous medium has a plurality of removable sampling elements in which the porous medium is inserted into the vertical fluid flow channels. Claim 2 In claim 1, the continuous pleated porous medium is a cell culture array having a pleat density in the range of 25% to 95%. Claim 3 The cell culture array according to claim 1 or 2, wherein the cell culture array comprises at least two fixed beds arranged within the housing, and each of the at least two fixed beds comprises a continuous corrugated porous medium comprising a top portion, a bottom portion, a front portion, a rear portion, a right side and a left side, and a plurality of folds folded on the right side and the left side, and further wherein the continuous corrugated porous medium has vertical fluid flow channels along the longitudinal axis between adjacent folds. Claim 4 A cell culture array according to claim 1 or 2, wherein the plurality of detachable sampling elements comprise pull tabs. Claim 5 A cell culture device comprising: (a) a cell culture array according to claim 1 or 2; (b) a pump body arranged at the second end of the bed housing, wherein the pump body comprises a central cavity; (c) an impeller arranged in the central cavity of the pump body; and (d) a cell culture vessel having a central chamber, wherein the cell culture array is arranged in the central chamber. Claim 6 A cell culture method comprising the following steps: a step of placing a cell culture medium and cells into a cell culture device of claim 5; and a step of operating the impeller. Claim 7 In claim 6, the cell culture method further comprises the step of harvesting cultured cells. Claim 8 A cell culture method according to claim 7, further comprising the step of separating the plurality of detachable sampling elements and analyzing the cell sample.

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