Cell culture bottle

Abstract

A cell culture vial is provided having a vial body defining a cell culture medium receiving cavity. The bottle includes an elongated neck defining an opening to the cavity, a bottom, and a sidewall extending from the elongated neck to the bottom of the bottle. The side wall includes a circumferential beveled portion that may or may not be machined into a facet, the beveled portion extending from the outermost edge of the bottle body to the bottom of the bottle body at a constant angle relative to the central axis of the bottle to define a side wall angle.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 613,402 filed on December 21, 2023, U.S. Provisional Application No. 63 / 494,656 filed on April 6, 2023, and U.S. Provisional Application No. 63 / 478,003 filed on December 30, 2022, and is a continuation-in-part of U.S. Application No. 29 / 869,448 filed on December 30, 2022, the contents of which are hereby expressly incorporated herein by reference as if fully set forth herein. Technical Field

[0002] The present invention relates generally to laboratory ware and, more particularly, to cell culture flasks. Background Art

[0003] Shake flasks, also referred to as cell culture flasks, are typically used to culture organisms in a laboratory environment. In this regard, a certain amount of organisms (e.g., any one of prokaryotic cells, mammalian cells, yeast cells, or insect cells) are placed in a cell culture flask containing a cell culture medium so that these organisms can grow into significantly more organisms as quickly as possible. The growth of an organism or cell depends on the cell culture medium and thus the organism is exposed to sufficient levels of oxygen. That is, cell growth increases as the cell culture medium is aerated, thereby achieving continuous oxygen exchange and carbon dioxide removal therefrom. In order to improve ventilation and oxygen transfer (i.e., oxygen transfer rate ("OTR")) into the cell culture medium, a laboratory shaker is typically used to shake shake flasks. For this reason, shaking the shake flask creates a vortex that exposes more liquid levels to the oxygen in the headspace of the culture flask.

[0004] Typically, oxygen transfer occurs through two liquid levels inside the shake flask: the main liquid level of the cell culture medium and the liquid film formed on the wetted side walls of the shake flask. In this regard, the OTR in the shake flask is determined by the size and geometry of the culture flask, the shaking speed, the liquid addition volume, and the environmental conditions. With respect to the shake flask geometry, baffles can be used to generate turbulence to improve ventilation and oxygen transfer during shaking. Furthermore, increasing the shaking speed can also increase the ventilation of the cell culture medium, thereby increasing oxygen transfer. However, although baffles and shaking speed can improve ventilation during the shaking operation, baffles in particular may cause cells to experience higher levels of shear stress (e.g., hydrodynamic stress) that may impair cell viability. In addition, strongly perturbed baffles may cause the cell culture medium to foam, thereby hindering oxygen transfer.

[0005] During cell culture and growth, proper agitation is crucial for cell viability and must be balanced with the homogeneity of the cell suspension, particularly for shear-sensitive cell types such as mammalian and insect cells. Consequently, sharp features within shake flasks, such as steep baffles with sharp edges, can easily stress and damage cells over a wide range of shaker speeds. Therefore, there is a need for a cell shake flask that can aerate cell culture medium for rapid and efficient culture of organisms, particularly those that are shear-sensitive, without the undesirable effects of shear stress and foaming. Summary of the Invention

[0006] The present invention overcomes the above-mentioned and other disadvantages and drawbacks of cell culture flasks.While the present invention will be discussed in conjunction with certain embodiments, it should be understood that the invention is not limited to the specific embodiments described herein.

[0007] Thus, in one aspect, a cell culture flask is provided having a body defining a cell culture medium receiving cavity. In one embodiment, the body comprises: an elongated neck defining an opening to the cavity; a bottom; and a sidewall extending from the elongated neck to the bottom of the body, the sidewall comprising a circumferentially inclined portion extending at a constant angle relative to a central axis of the flask from an outermost periphery of the body to the bottom of the body to define a sidewall angle.

[0008] In certain embodiments, the culture bottle having a circumferentially sloped portion further comprises at least one baffle formed in the bottle body so as to extend in a radially inward direction relative to the central axis of the bottle to form a recess in the culture bottle. In some embodiments, the at least one baffle comprises six equally spaced baffles distributed around the circumference of the bottle body. In other embodiments, the at least one baffle comprises a first pair of baffles spaced 60° apart from each other around the central axis of the bottle and a second pair of baffles spaced 60° apart from each other around the central axis of the bottle, wherein the first pair of baffles are diametrically opposed to the second pair of baffles around the central axis of the bottle.

[0009] In another embodiment, the bottle body includes: a slender neck defining an opening to the cavity; a bottom; a side wall extending from the slender neck to the bottom of the bottle body, the side wall including a circumferential facet portion extending from the outermost periphery of the bottle body to the bottom of the bottle body, the circumferential facet portion being defined by a plurality of facets distributed around the circumference of the bottle body.

[0010] In certain embodiments, the culture bottle having a circumferential facet portion further comprises at least one baffle formed in a surface of one of the plurality of facets and a surface of the bottom of the bottle body so as to extend in a radially inward direction relative to the central axis of the bottle. In some embodiments, the at least one baffle comprises six baffles equally spaced apart and distributed around the circumference of the bottle body, with each baffle formed on a surface of a different one of the plurality of facets and a surface of the bottom. In other embodiments, the at least one baffle comprises a first pair of baffles spaced 60° apart from each other around the central axis of the bottle so as to be formed on a surface of a different one of the plurality of facets and the bottom, and a second pair of baffles spaced 60° apart from each other around the central axis of the bottle so as to be formed on a surface of a different one of the plurality of facets and the bottom, wherein the first pair of baffles are diametrically opposed to the second pair of baffles around the central axis of the bottle.

[0011] In some embodiments, a laboratory shaker is provided in conjunction with the cell culture flasks described herein.

[0012] In another aspect, a method of culturing cells is provided, comprising providing a cell culture flask as described herein, introducing a cell culture medium and one or more cells into the cavity; and culturing the cells under conditions that support growth and / or expansion of the one or more cells.

[0013] In another embodiment, a method for expressing a protein of interest is provided, comprising providing a cell culture flask as described herein, introducing cell culture medium and one or more cells configured to express the protein of interest into a chamber; and culturing the cells under conditions that support expression of the protein of interest. In some embodiments, the method further comprises isolating the protein of interest from the cell culture medium and / or the one or more cells.

[0014] In another embodiment, a method for producing a viral vector is provided, comprising providing a cell culture flask as described herein, introducing cell culture medium and one or more cells configured to express the viral vector into a chamber; and culturing the cells under conditions that support expression of the viral vector. In some embodiments, the method further comprises isolating the viral vector from the cell culture medium and / or the one or more cells.

[0015] Various additional features and advantages of the present invention will become more readily apparent to those skilled in the art after reading the following detailed description of one or more illustrative embodiments in conjunction with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments of the invention and, together with the general description given above and the detailed description given below, serve to describe one or more embodiments of the invention.

[0017] Figure 1 is a front view of a cell culture flask according to one embodiment of the present invention.

[0018] Figure 2 It is along Figure 1 Cross-section of the shake flask taken along the midline 2-2.

[0019] Figure 3 yes Figure 1 - Figure 2 Magnified cross-section of the bottom of the shake flask, showing detail of the small baffles.

[0020] Figure 4 yes Figure 1 - Figure 3 A bottom perspective view of the bottom of a shake flask showing the bottom separated from the shake flask.

[0021] Figure 5 yes Figure 4 A partial bottom view of the bottom of the shake flask showing details of the small baffles.

[0022] Figure 6 yes Figure 1 - Figure 5 Bottom view of the shake flask.

[0023] Figure 7A is with Figure 6 Similar views showing the first and second pairs of baffles.

[0024] Figure 7B is a bottom view of a shake flask with small baffles according to one embodiment of the present invention.

[0025] Figures 8A to 8C is a bottom view of a shake flask with large baffles according to an embodiment of the present invention.

[0026] Figure 9 is a front view of a cell culture flask according to another embodiment of the present invention.

[0027] Figure 10 It is along Figure 9 Cross-section of the shake flask taken along the midline 10-10.

[0028] Figure 11 yes Figure 9 - Figure 10 An enlarged cross-section of the bottom of a shake flask showing detail of a facet.

[0029] Figure 12 yes Figure 9 - Figure 11 A bottom perspective view of the bottom of the shake flask of FIG. 1 is shown separated from the shake flask.

[0030] Figure 13 yes Figure 12 A partial bottom view of the bottom of the shake flask, showing the facet details.

[0031] Figure 14 yes Figure 12 Bottom view of the shake flask.

[0032] Figure 15 is a front view of a cell culture flask according to another embodiment of the present invention.

[0033] Figure 16 It is along Figure 15 Cross-section of the shake flask taken along the midline 16-16.

[0034] Figure 17 yes Figure 15 - Figure 16 Magnified cross-section of the bottom of the shake flask, showing detail of the small baffles.

[0035] Figure 18 yes Figure 15 - Figure 17 A bottom perspective view of the bottom of the shake flask of FIG. 1 is shown separated from the shake flask.

[0036] Figure 19 yes Figure 18 A bottom view of a partial bottom portion of a shake flask showing details of the small baffles.

[0037] Figure 20 yes Figure 15 Bottom view of the shake flask.

[0038] Figure 21A is with Figure 20 Similar views showing the first and second pairs of baffles.

[0039] Figure 21B is a bottom view of a shake flask with small baffles according to one embodiment of the present invention.

[0040] Figures 22A to 22C is a bottom view of a shake flask with large baffles according to an embodiment of the present invention.

[0041] Figure 23 is a schematic diagram showing a cell culture flask on a laboratory shaker inside an incubator.

[0042] Figure 24 is a front view of a cell culture flask according to one embodiment of the present invention.

[0043] Figure 25 It is along Figure 1 Cross-sectional view of the shake flask taken along line 24-24.

[0044] Figure 26 yes Figure 24 A magnified cross-section of the bottom of a 25-well shake flask showing details of the sloped surface.

[0045] Figure 27 yes Figure 24 - Figure 26 A bottom perspective view of the bottom of the shake flask of FIG. 1 is shown separated from the shake flask.

[0046] Figure 28 yes Figure 27 A partial bottom view of the bottom of a shake flask.

[0047] Figure 29 yes Figure 24 - Figure 28 Bottom view of the shake flask.

[0048] Figure 30 Graph depicting the growth performance of Expi293 cells cultured in faceted bottom flasks at varying final volumes and control 30 mL cultures.

[0049] Figure 31 is a graph depicting the growth performance of ExpiSf9 cells cultured in slanted-bottom and glossy-bottom flasks at a final volume of 2 liters.

[0050] Figure 32 is a graph depicting the growth performance of ExpiCHO cells cultured in slanted bottom flasks at a final volume of 2 liters and a control volume of 30 mL.

[0051] Figure 33 is a bar graph depicting antibody expression levels in transfected Expi293 cells cultured in a final volume of 2.25 liters and a control volume of 30 mL.

[0052] Figure 34 is a bar graph depicting GFP expression levels in baculovirus-infected Expi293 cells cultured in a final volume of 2 liters and a control volume of 30 mL.

[0053] Figure 35 is a bar graph depicting protein expression levels in transfected ExpiCHO cells cultured following the Max Titer expression protocol from a final volume of 2 liters and a control volume of 30 mL.

[0054] Figure 36 is a bar graph depicting protein expression levels in transfected ExpiCHO cells cultured following a standard expression protocol from a final volume of 2 liters and a control volume of 30 mL. DETAILED DESCRIPTION

[0055] Referring now to the drawings, and in particular to Figure 1 and Figure 2 , shows a cell culture flask 10 according to a first embodiment of the present invention. The cell culture flask 10 includes a body 12 that defines a cavity 14 for receiving a cell culture medium for culturing one or more organisms (i.e., cells). As will be described in further detail below, the cell culture flask 10 is configured to be placed on a laboratory shaker and agitated at various oscillation frequencies and motions to cause a target protein to be expressed in the cavity 14. The body 12 includes an elongated neck 16 that defines an opening 18 leading to the cavity 14; a bottom 20 and a sidewall 22 extending from the elongated neck 16 to the bottom 20. The cell culture flask 10 also includes at least one baffle 24 formed in the body 12, the baffle being configured to increase oxygen exchange during agitation of the cell culture medium contained in the cell culture flask 10, particularly for shear stress-sensitive cell types, to thereby increase cell titer and protein expression while minimizing cell shear and foaming, as will be described further below.

[0056] An exemplary cell culture bottle 10 can be a 5 liter (L) bottle, for example, having a minimum filling volume of 5 L below the elongated neck 16. The cell culture bottle 10 can have a total cavity 14 filling volume of 5.5 L. However, the preferred working volume of the cell culture bottle 10 can be in the range of 2.5 L to 3.5 L. In either case, embodiments of the present invention can also be applied to the design and manufacture of smaller or larger cell culture bottles (e.g., 10 L), and are particularly suitable for culture bottles having a filling volume in the range of 125 mL to 28,000 mL, for example. To this end, the drawings are not intended to be limiting.

[0057] Continue to refer Figure 1 and Figure 2 The elongated neck 16 of the body 12 defines an opening 18 leading to the cavity 14, through which cell culture medium and cells can be introduced into and removed from the cell culture bottle 10. The elongated neck 16 includes a threaded portion 26 configured to threadably receive a closure device (not shown), and a collar 28 defining a contact surface for the closure device. The elongated neck 16 may also include a groove 30 configured to receive an O-ring (not shown) for forming a seal between the elongated neck 16 and the closure device. The closure device is configured to close the opening 18 to prevent, for example, spillage of the cell culture medium contained in the cell culture bottle 10. The closure device can be a vent cap that allows air exchange (e.g., allowing oxygen to enter the cavity and carbon dioxide to leave the cavity). To this end, the threaded portion 26 can include, for example, 83B threads configured to receive an 83B threaded closure device.

[0058] refer to Figure 2 and Figure 4 The bottom 20 of the bottle body 12 includes a bottom surface 32 and a central recess 34 that curves gently upward from the bottom surface 32 and into the cavity 14. The contour of the central recess 34 serves to guide the vortex of the cell culture medium over the at least one baffle 24 during shaking of the cell culture bottle 10. Figure 4 As shown in FIG, the bottom surface 32 comprises a generally flat annular disk having a circumferential outer edge 36 defining an outer diameter of the bottom surface 32 and a circumferential inner edge 38 defining an inner diameter of the bottom surface 32. To this end, the bottom surface 32 is configured to support the cell culture flask 10 in an upright position on a support surface, such as a laboratory shaker.

[0059] like Figure 1 - Figure 3 As shown in FIG, the sidewall of the bottle body 12 includes a first circumferential bevel portion 40 that extends upward from the bottom 20 of the bottle body 12 to the outermost peripheral edge 42 of the bottle body 12 in a radially outward direction relative to the central axis A1 of the cell culture bottle 10. The outermost peripheral edge 42 of the cell culture bottle 10 is defined as the portion of the culture bottle 10 where the diameter of the bottle body 12 is the largest. In any case, the first bevel portion 40 extends radially outward and upward at a constant angle relative to the central axis A1 (i.e., the vertical axis) of the cell culture bottle 10 to define a sidewall angle θ1, as shown in FIG. Figure 3 As shown in . The side wall angle θ1 may be, for example, in the range between 30° and 60°. In a preferred embodiment, the side wall angle θ1 is 45°.

[0060] The sidewall 22 of the bottle body 12 further includes a second circumferential bevel portion 44 extending from the outermost periphery 42 of the bottle body 12 to a shoulder 46 of the bottle body 12. The shoulder 46 and the second bevel portion 44 of the sidewall 22 may be substantially symmetrical about the central axis A1 of the cell culture bottle 10. Figure 2 As shown in FIG, the second beveled portion 44 extends radially inward from the outermost peripheral edge 42 of the body 12 to a shoulder 46 relative to the central axis A1 of the cell culture flask 10. Specifically, the second beveled portion 44 may extend toward the central axis A1 of the cell culture flask 10 at an angle between 2° and 5° from vertical. In the exemplary embodiment of the cell culture flask 10 shown, the second beveled portion 44 extends radially inward at an angle of 3.8° from vertical. The shoulder 46 extends upward from the second beveled portion 44 to the elongated neck 16 at an angle between 15° and 20° from the horizontal (i.e., a horizontal plane transverse to the central axis A1 of the cell culture flask 10). In the exemplary embodiment of the cell culture flask 10 shown, the shoulder 46 extends from the second beveled portion 44 to the elongated neck 16 at an angle of 16.164° from the horizontal.

[0061] The overall height of the cell culture flask 10 (i.e., the distance measured between the bottom 20 of the body 12 and the top of the elongated neck 16) can be 10.8 inches. Thus, the cell culture flask 10 can be effectively placed in a standard benchtop incubator (e.g., a laboratory shaker and a lid or hood) with a laboratory shaker and a lid or hood. Figure 23 ), while providing clearance between the opening 18 of the elongated neck 16 and the lid for a pipette or other tool to be used to add / remove fluids, such as cell culture medium and cells, to / from the cell culture flask 10. To this end, the elongated neck 16 may have an inner diameter (ID) of 2.57 inches and a height of 3.3 inches to accommodate, for example, a 50 mL serological pipette.

[0062] refer to Figure 2 - Figure 5 The cell culture bottle 10 further includes at least one baffle 24 formed in the bottle body 12. In the exemplary embodiment shown, the cell culture bottle 10 includes four baffles that are symmetrically spaced about the central axis A1 of the cell culture bottle 10. Figure 2 - Figure 3 As shown in FIG, each baffle 24 is formed in the body 12 so as to extend in a radially inward direction relative to the central axis A1 of the cell culture bottle 10 to form a recess 48 in the body 12. Specifically, each baffle 24 is generally V-shaped to define a pair of flat sidewalls 50 that are joined together along edges 52 that define the peak of each baffle 24 within the cavity 14 of the body 12 (e.g., Figure 2 ).like Figure 5 As shown in FIG, each baffle 24 is generally triangular in cross-section, with edges 52 forming the vertices of the triangle. Notably, edges 52 are curved (i.e., rounded) in cross-section to facilitate smooth transfer of cell culture medium across baffles 24, thereby minimizing shear stress and cell damage during agitation of the cell culture medium.

[0063] like Figure 2 and Figure 3 As shown in FIG, a pair of side walls 50 of each baffle 24 extends between the first sloped portion 40 of the side wall 22 and the bottom 20 of the bottle body 12 such that the edge 52 extends in a radially inward direction relative to the central axis A1 of the cell culture bottle 10. In this regard, the edge 52 extends from a first terminal end 54 located on the first sloped portion 40 of the side wall 22 and a second terminal end 56 located on the bottom surface 32 of the bottom 20 of the bottle body 12. More specifically, as shown in FIG. Figure 3As shown in FIG, the edge 52 of each baffle is angled relative to the central axis A1 (i.e., the vertical axis) of the cell culture flask 10 to define a baffle angle θ2. The baffle angle θ2 can be, for example, in the range of 45° to 70° (or 20° to 45° from a horizontal reference plane). In a preferred embodiment, the baffle angle θ2 is 60° (or 30° from a horizontal reference plane).

[0064] Now refer to Figure 3 - Figure 5 In the embodiment, each baffle side wall 50 is generally triangular in shape to define a first leg edge 58 and a second leg edge 60, with the hypotenuse being a common side 52 shared by the pair of side walls 50. Each first leg edge 58 extends along the first bevel portion 40 of the side wall 22 and between the first terminal end 54 of the edge 52 and the outer edge 36 of the bottom surface 32. Each second leg edge 60 extends along the bottom surface 32 and between the outer edge 36 of the bottom surface 32 and the second terminal end 56 of the edge 52. Figure 4 and Figure 5 As best shown in FIG, each baffle 24 is generally V-shaped in cross section due to the angular relationship between the paired side walls 50. More specifically, as shown in FIG. Figure 5 As shown in FIG, the pair of side walls 50 are angled relative to each other to form a baffle side wall angle θ3 therebetween. The baffle side wall angle θ3 may, for example, be in the range of 90° to 110°. In a preferred embodiment, the baffle side wall angle θ3 is 100°. For this reason, it is important to have a wide baffle side wall angle θ3 (i.e., 100°) to minimize the steepness of the baffles, thereby reducing shear stress and cell damage during agitation of the cell culture medium.

[0065] like Figure 4 and Figure 6 As best shown in FIG. 1 , each baffle 24 extends a greater distance along the first beveled portion 40 of the bottle body 12 than along the bottom surface 32. Thus, the length of each first leg 58 is greater than the length of each second leg 60. In this regard, the first beveled portion 40 of the sidewall 22 includes a length L, which is measured between the outer edge 36 of the bottom surface 32 and the outermost peripheral edge 42 of the bottle body 12. Each first leg 58 may extend a distance from the outer edge 36 of the bottom surface 32 such that the first terminal end 54 of the leg 52 is positioned within an area of the first beveled portion 40 of the sidewall 22 that is between 40% and 60%, and preferably 45% and 50%, of the length L of the circumferential beveled portion 40 of the sidewall 22, as measured from the outer edge 36 of the bottom surface 32 of the bottle body 12. Thus, the positioning of the first terminal end 54 of the leg 52 along the first beveled portion 40 of the sidewall 22 influences the baffle angle θ2.

[0066] Continue to refer Figure 4 and Figure 6, the bottom surface 32 includes a width W, which is measured as the distance between the inner edge 38 and the outer edge 36 of the bottom surface 32 of the bottle body 12. In this regard, each second leg 60 extends a distance from the outer edge 36 of the bottom surface 32 such that the second terminal end 56 of the leg 52 is positioned within an area of the bottom surface 32 that is between 50% and 70%, and preferably 60% and 65%, of the width W of the bottom surface 32, as measured from the outer edge 36 of the bottom surface of the bottle body 12. To this end, the positioning of the second terminal end 56 of the leg 52 along the bottom surface 32 also affects the baffle angle θ2.

[0067] Now refer to Figure 7A The cell culture flask 10 includes a first pair 64 or set of baffles 24 and a second pair 66 or set of baffles 24, the second pair 66 being diametrically opposed to the first pair 64 of baffles about the central axis A1 of the cell culture flask 10. The baffles 24 forming the first pair 64 of baffles 24 are spaced 60° apart from one another about the central axis A1 of the cell culture flask 10, and the baffles 24 forming the second pair 66 of baffles 24 are also spaced 60° apart from one another about the central axis A1 of the cell culture flask 10. To this end, the baffles 24 forming the first and second pairs 64, 66 of baffles 24 can be spaced farther apart or closer together about the central axis A1 of the cell culture flask 10, such as within a range of 50° to 70°.

[0068] Figure 7B A cell culture flask 10a having six baffles 24 according to an embodiment of the present invention is shown. As shown, the baffles 24 are spaced apart in a symmetrical arrangement about the central axis A1 of the cell culture flask 10a, with the baffles 24 being equally spaced in 60° increments about the central axis A1 of the cell culture flask 10a. To this end, the cell culture flask 10a may include fewer or more baffles 24 spaced at different increments about the central axis A1 of the cell culture flask 10a, such as four baffles 24 spaced 90° apart about the central axis A1 of the cell culture flask 10a.

[0069] Now refer to Figure 8A - Figure 8C , wherein like numerals represent like features, illustrates additional embodiments of cell culture flasks 10b and 10c according to the present invention and will now be described. The primary difference between these embodiments of cell culture flasks 10b and 10c and the cell culture flask 10 of the previously described embodiment is the placement of baffles 24b. Notably, baffles 24b are larger than baffles 24 of the previously described embodiment. Figure 8A - Figure 8C The baffle 24b can be called a "large" baffle 24b, and Figure 1 - Figure 7BThe baffles 24 of the embodiment shown in FIG. 2 can be referred to as "small" baffles 24. In this regard, the side 52b of each large baffle 24b is longer in length so that the first terminal end 54b of the side 52b is positioned farther from the outer edge 36 of the bottom surface 32 along the first sloped portion 40 of the bottle body 12. As shown, each first leg side 58b can extend a distance from the outer edge 36 of the bottom surface 32 so that the first terminal end 54b of the side 52b is positioned on the first sloped portion 40 of the sidewall within an area between 85% and 99%, and preferably between 95% and 99%, of the length L of the circumferential sloped portion 40 of the sidewall 22, as measured from the outer edge 36 of the bottom surface 32 of the bottle body 12. Similarly, each second leg edge 60b extends a distance from the outer edge 36 of the bottom surface 32 such that the second terminal end 56b of the edge 52 is positioned on the bottom surface 32 within an area between 85% and 99%, and preferably between 95% and 99%, of the width W of the bottom surface 32 as measured from the outer edge 36 of the bottom surface 32 of the bottle body 12. Due to the size of the larger baffle 24b, its baffle angle θ 2b The baffle angle θ2 is steeper than that of the previously described embodiments, as shown in FIG. Figure 8C In this regard, the baffle angle θ 2b is 52° (or 38° from the horizontal reference plane).

[0070] Figure 8A A cell culture flask 10b is shown having four large baffles 24b symmetrically spaced about the central axis of the cell culture flask, according to an embodiment of the present invention. In this regard, the cell culture flask 10b includes a first pair or group of large baffles 24b 64b and a second pair or group of large baffles 24b 66b diametrically opposed to the first pair 64b of baffles 24b about the central axis A1 of the cell culture flask 10b. The baffles 24b forming the first pair 64b of baffles 24b are spaced 60° apart from one another about the central axis A1 of the cell culture flask 10b, while the baffles 24b forming the second pair 66b of baffles 24b are also spaced 60° apart from one another about the central axis A1 of the cell culture flask 10b. To this end, the baffles 24b forming the first and second pairs 64b, 66b of baffles 24b may be spaced farther or closer to each other around the central axis A1 of the cell culture flask 10, for example, within a range of 50° to 70°.

[0071] Figure 8B A cell culture flask 10c including six large baffles 24b according to an embodiment of the present invention is shown. As shown, the baffles 24b are spaced apart in a symmetrical arrangement about the central axis A1 of the cell culture flask 10c. Specifically, the baffles 24b are equally spaced in 60° increments about the central axis A1 of the cell culture flask 10c.

[0072] Now refer to Figure 24 - Figure 29 , which is compared with the above Figure 1 - Figure 6 With reference to the embodiment of the cell culture flask 10 described above, like numerals denote like features. A cell culture flask 10i according to another embodiment of the present invention is illustrated and will now be described. The primary difference between this embodiment of the cell culture flask 10i and the previously described embodiment of the cell culture flask 10 is that the body 12i includes a circumferential beveled portion 40i and lacks at least one baffle. As described in further detail below, the circumferential beveled portion 40i provides minimal turbulence during agitation of the cell culture medium contained in the cell culture flask 10i, enabling the effective cultivation of certain cell types that are sensitive to shear stress.

[0073] refer to Figure 24 and Figure 25 The cell culture bottle 10i includes a body 12i defining a cavity 14i for receiving a cell culture medium for culturing one or more cells, and an elongated neck 16 defining an opening 18 leading to the cavity 14i. The body 12i also includes a bottom 20i and a sidewall 22i extending from the elongated neck 16 to the bottom 20i. Figure 27 As best shown in FIG, the bottom 20i of the bottle body 12i includes a bottom surface 32i and a central recess 34 that curves gently upward from the bottom surface 32i and into the cavity 14i. To this end, the bottom surface 32i includes a generally flat annular disk having a circumferential outer edge 36i defining the outer diameter of the bottom surface 32i and a circumferential inner edge 38 defining the inner diameter of the bottom surface 32i. Figure 25 As shown in FIG, the sidewall 22i includes a peripheral bevel portion 40i and a second peripheral bevel portion 44 extending from an outermost peripheral edge 42 of the bottle body 12i to a shoulder 46 of the bottle body 12i.

[0074] refer to Figure 25 and Figure 27 The bottom 20i of the bottle body 12i includes a bottom surface 32 and a central recess 34 that curves gently upward from the bottom surface 32 and into the cavity 14i. The contour of the central recess 34 serves to create a vortex in the cell culture medium during shaking of the cell culture bottle 10. Figure 27 As shown in FIG, the bottom surface 32 comprises a generally flat annular disk having a circumferential outer edge 36i defining an outer diameter of the bottom surface 32 and a circumferential inner edge 38 defining an inner diameter of the bottom surface 32. To this end, the bottom surface 32 is configured to support the cell culture flask 10 in an upright position on a support surface, such as a laboratory shaker.

[0075] like Figure 24 - Figure 26As shown in FIG, the sidewall of the bottle body 12i includes a first circumferential bevel portion 40i that extends upward from the bottom 20i of the bottle body 12i in a radially outward direction relative to the central axis A1 of the cell culture bottle 10i to the outermost peripheral edge 42 of the bottle body 12i. The outermost peripheral edge 42 of the cell culture bottle 10i is defined as the portion of the culture bottle body 12i in the culture bottle 10 where the diameter is the largest. In any case, the first bevel portion 40i extends radially outward and upward at a constant angle relative to the central axis A1 (i.e., the vertical axis) of the cell culture bottle 10i to define a sidewall angle θ1, as shown in FIG. Figure 26 As shown in . The side wall angle θ1 may be, for example, in the range between 30° and 60°. In a preferred embodiment, the side wall angle θ1 is 45°.

[0076] refer to Figure 2 - Figure 5 , the cell culture flask 10 does not include at least one baffle 24 formed in the flask body 12. In some embodiments, the lack of baffles may be particularly advantageous for shear stress-sensitive cell types to minimize shear stress and cell damage during agitation of the cell culture medium. Compared to cell culture flasks with baffles, the cell culture flask 10i having only a bevel significantly reduces the risk of foaming. Using a culture flask 10i having only a bevel thus increases cell titer and protein expression while minimizing cell shear and foaming.

[0077] refer to Figure 27 and Figure 29 , the bottom surface 32i includes a width W, which is measured as the distance between the inner edge 38 and the outer edge 36 of the bottom surface 32i of the bottle body 12i.

[0078] Now refer to Figure 9 - Figure 14 , which is compared with the above Figure 1 - Figure 7A 10 , wherein like numerals denote like features, a cell culture flask 10d according to another embodiment of the present invention is shown and will now be described. The primary difference between this embodiment of the cell culture flask 10d and the previously described embodiment of the cell culture flask 10 is that the body 12d includes a circumferential faceted portion 70 instead of the circumferential beveled portion 40. In this regard, the circumferential faceted portion 70 extends radially upward and outward between the bottom 20d of the body 12d and the outermost peripheral edge 42 of the body 12d. The circumferential faceted portion 70 is defined by a plurality of facets 72 distributed around the circumference of the body 12d. As described in further detail below, the circumferential faceted portion 70 provides sufficient turbulence, which is required to increase oxygen exchange during agitation of the cell culture medium contained in the cell culture flask 10d for the effective cultivation of certain cell types that are sensitive to shear stress.

[0079] refer to Figure 9 - Figure 10 The cell culture bottle 10d includes a body 12d defining a cavity 14d for receiving a cell culture medium for culturing one or more cells, and an elongated neck 16 defining an opening 18 leading to the cavity 14d. The body 12d also includes a bottom 20d and a sidewall 22d extending from the elongated neck 16 to the bottom 20d. Figure 12 As best shown in FIG, the bottom 20d of the bottle body 12d includes a bottom surface 32d and a central recess 34 that curves gently upward from the bottom surface 32d and into the cavity 14d. To this end, the bottom surface 32d includes a generally flat annular disk having a circumferential outer edge 36d defining the outer diameter of the bottom surface 32d and a circumferential inner edge 38 defining the inner diameter of the bottom surface 32d. Figure 10 As shown in FIG, the side wall 22d includes a peripheral faceted portion 70 and a second peripheral beveled portion 44 extending from the outermost peripheral edge 42 of the bottle body 12d to the shoulder 46 of the bottle body 12d.

[0080] refer to Figure 10 and Figure 11 The circumferential facet portion 70 extends upward from the bottom 20d of the bottle body 12 to the outermost peripheral edge 42 of the bottle body 12d in a radially outward direction relative to the central axis A1 of the cell culture bottle 10d. Specifically, the angle of the circumferential facet portion 70 is defined by the angle of each facet 72 relative to the central axis A1 (i.e., the vertical axis) of the cell culture bottle 10d. Figure 11 As shown in FIG, each facet 72 is angled at a constant angle relative to the central axis A1 of the cell culture flask 10d to define a facet angle θ4. The facet angle θ4 can, for example, be within a range of 30° to 60° (or 30° to 60° from a horizontal reference plane). In a preferred embodiment, the facet angle θ4 is 45°. To this end, the facets 72 provide a flat surface that supports the cell culture flask 10d at an angle (equal to the facet angle θ4) relative to the support surface on which the cell culture flask 10d is placed, thereby facilitating the recovery of cells from the cell culture flask 10d.

[0081] refer to Figure 12 - Figure 14 The cell culture flask 10d includes 12 facets 72, which are symmetrically arranged around the central axis A1 of the cell culture flask 10d (e.g., Figure 14 ). The 12 facets 72 layout is particularly suitable for culturing cells that are sensitive to shear stress, however, the cell culture bottle 10d can include fewer or more facets 72 as needed. Figure 12As best shown in FIG. 1 , each facet 72 defines a bottom edge 74 along the circumferential outer edge 36d of the bottom surface 32d, a rounded edge 76 at the outermost peripheral edge 42 of the body 12d, and a pair of side edges 78 extending between the bottom edge 74 and the rounded edges 76. The facet bottom edges 74 collectively define the circumferential outer edge 36d of the bottom surface 32d. In any case, shaking the cell culture flask 10d with a laboratory shaker causes a vortex of cell culture fluid to flow across the facets 72 and, in particular, across the side edges 78. To this end, the side edges 78 form low points or valleys between each facet 72, which generate turbulence during shaking of the cell culture flask 10d with a laboratory shaker. Compared to cell culture flasks with baffles, a cell culture flask 10d having only facets significantly reduces the risk of foaming.

[0082] Now refer to Figure 15 - Figure 21A , which is compared with the above Figure 9 - Figure 14

[0026] Referring now to the embodiment of the cell culture flask 10 described above, like numerals denote like features. A cell culture flask 10e according to another embodiment of the present invention is shown and will now be described. The cell culture flask 10e of this embodiment differs primarily from the cell culture flask 10d of the previously described embodiment in that the cell culture flask 10e further includes at least one baffle 24 formed in the body 12e. The baffle is configured to increase oxygen exchange during agitation of the cell culture medium contained in the cell culture flask 10e, particularly for shear-sensitive cell types, to thereby increase cell titer and protein expression while minimizing cell shearing and foaming.

[0083] Figure 15 - Figure 20 A cell culture flask 10e is shown having four "small" baffles 24 formed in the flask body 12e, as described above with respect to the embodiment of the present invention. Figure 1 - Figure 7A For example, the embodiment of the cell culture bottle 10 is as follows. Figure 16 and Figure 17 As shown in FIG, the pair of side walls 50 of each baffle 24 extend between the circumferential face portion 70e of the side wall 22e and the bottom surface 32e of the bottle body 12e such that the edge 52 extends in a radially inward direction relative to the central axis A1 of the cell culture bottle 10e. In this regard, the edge 52 extends from a first terminal end 54 located on the face 72 of the circumferential face portion 70e of the side wall 22e and a second terminal end 56 located on the bottom surface 32e of the bottle body 12e. Figure 18 As shown in , the first terminal ends 54 of the edges 52 may be approximately centered between the sides 78 of the respective facets 72. Figure 17As shown in FIG, the side 52 of each baffle 24 is angled relative to the central axis A1 (i.e., the vertical axis) of the cell culture flask 10 to define a baffle angle θ2. The baffle angle θ2 can be, for example, in the range of 45° to 70° (or 20° to 45° from a horizontal reference plane). In a preferred embodiment, the baffle angle θ2 is 60° (or 30° from a horizontal reference plane).

[0084] refer to Figure 18 and Figure 19 Due to the angled relationship between the paired side walls 50, each baffle 24 is generally V-shaped in cross section. More specifically, as Figure 19 As shown in FIG, the pair of side walls 50 are angled relative to each other to form a baffle side wall angle θ3 therebetween. The baffle side wall angle θ3 may be, for example, in the range of 90° to 110°. In a preferred embodiment, the baffle angle θ3 is 100°.

[0085] Now refer to Figure 20 The circumferential facet portion of the sidewall, and in particular each facet 72, includes a length L1 measured as the distance between the base 74 and the rounded edge 76 of each facet 72. Each first leg 58 may extend from the base 74 (or the outer edge 36e of the bottom surface 32e) a distance such that the first terminal end 54 of the leg 52 is positioned on the facet 72 within an area between 40% and 60%, and preferably 45% and 50%, of the length L1 of the facet 72, as measured from the base 74 of the facet 72. Similarly, the bottom surface 32e includes a width W1 measured as the distance between the circumferential inner edge 38 and the outer edge 36e of the bottom surface 32e of the bottle body 12e. In this regard, each second leg 60 extends a distance from the outer edge 36 of the bottom surface 32e (or the bottom edge 74 of the edge face 72) so that the second terminal end 56 of the leg 52 is positioned on the bottom surface 32e in an area between 50% and 70% and preferably 60% and 65% of the width W1 of the bottom surface 32e, as measured from the outer edge 36 of the bottom surface 32e of the bottle body 12e).

[0086] refer to Figure 21A The cell culture flask 10e includes four baffles 24, namely a first pair or set of baffles 24 64e and a second pair or set of baffles 24 66e, which are diametrically opposed to the first pair 64e of baffles 24 about the central axis A1 of the cell culture flask 10e. The first pair 64e of baffles 24 are spaced 60° apart from each other about the central axis A1 of the cell culture flask 10e, while the second pair 66e of baffles 24b are spaced 60° apart from each other about the central axis A1 of the cell culture flask 10e.

[0087] Figure 21BA cell culture flask 10f having six baffles 24 according to another embodiment of the present invention is shown. As shown, the baffles 24 are spaced symmetrically about the central axis A1 of the cell culture flask 10f, with the baffles 24 being equally spaced in 60° increments about the central axis A1 of the cell culture flask 10f. To this end, the cell culture flask 10f may include fewer or more baffles 24 spaced at different increments about the central axis A1 of the cell culture flask 10f, such as four baffles 24 spaced 90° apart about the central axis A1 of the cell culture flask 10f.

[0088] Now refer to Figure 22A - Figure 22C , which is compared with the above Figure 15 - Figure 21A With reference to the embodiment of the cell culture flask 10e described above, like numerals denote like features. Other cell culture flasks 10g and 10h according to the present invention are shown and will now be described. The main difference between these cell culture flasks 10g and 10h and the cell culture flask 10e described above is the use of large baffles 24b, similar to the above description of the embodiment of the cell culture flask 10e. Figure 8A - Figure 8C 1. As shown, each first leg edge 58b extends from the bottom edge 74 of the face 72 (or the outer edge 36g of the bottom surface 32g) a distance such that the first terminal end 54b of the edge 52b is positioned within an area of each face 72 between 85% and 99%, and preferably between 95% and 99%, of the length L1 of the face 72, as measured from the bottom edge 74 of the face 72. Similarly, each second leg edge 60b extends from the bottom edge 74 of the face 72 (or the outer edge 36g of the bottom surface 32g) a distance such that the second terminal end 56b of the edge 52b is positioned within an area of the bottom surface 32g between 85% and 99%, and preferably between 95% and 99%, of the width of the bottom surface 32g, as measured from the bottom edge 74 of the face 72. To this end, the baffle angle θ 2b is 52° (or 38° from the horizontal reference plane), such as Figure 22C As shown in .

[0089] refer to Figure 22AThe baffles 24b are symmetrically spaced apart from one another about the central axis A1 of the cell culture flask 10g. In this regard, the cell culture flask 10g includes a first pair or group of large baffles 24g, each comprising 64g, and a second pair or group of large baffles 24g, each comprising 66g, each comprising 66g, each comprising 64g, each comprising 66g, each comprising 66g, each comprising 66g, each comprising 64g, each comprising 66g, each comprising 66g, each comprising 66g, each comprising 66g, each comprising 64g, each comprising 66g, each comprising 66g, each comprising 60° ...

[0090] Figure 22B A cell culture flask 10h according to another embodiment of the present invention is shown having six large baffles 24b symmetrically spaced about the central axis A1 of the cell culture flask 10h. Specifically, the baffles 24b are equally spaced in 60° increments about the central axis A1 of the cell culture flask 10h. To this end, each baffle 24b is formed on a surface of a different one of the plurality of facets 72 and on a surface of the bottom 32h.

[0091] The cell culture flask 10-10i can be molded using a blow molding process and can be formed from polyethylene terephthalate glycol (PETG). However, the cell culture flask 10-10i can also be formed using other manufacturing techniques, such as printing the cell culture flask 10-10i from a digital model using a three-dimensional (3D) printer. Furthermore, the cell culture flask 10-10i can be molded from a variety of radiation-sterilizable transparent plastics, such as polycarbonate (PC), polystyrene (PS), polyethylene terephthalate (PET), various acrylic acid-based polymers (ACRs), polymethylpentene (PMP), or any other suitable transparent plastic that is optically clear to allow for quick visual inspection of the cell culture medium and cells, such as to observe the volume of liquid added, foaming, medium degradation and contamination, pH changes, etc. The sidewall 22-22i of each cell culture bottle 10-10i can be provided with a volume scale that can be clearly read from the outside of the bottle in specific units (e.g., metric units) when the cell culture bottle 10a-10h is empty. For example, for a 5L cell culture bottle, the volume scale can be 0.5L to 5L, with 0.5L increments.

[0092] Figure 23An exemplary laboratory shaker 80 is shown located within an incubator 82 for agitating and culturing cells within an exemplary cell culture flask 10. However, the cell culture flask 10 may be any of the embodiments 10 to 10i described above. The incubator 82 may be a Thermo Fisher Reach-In CO2 incubator (Model 3950), and the laboratory shaker may be a Thermo Fisher MAX Q16HP, a Thermo Scientific MaxQ 416HP, or a Thermo Scientific MaxQ2000, all commercially available from the assignee of the present invention. As shown, the cell culture flask 10 contains a volume of liquid 84 (i.e., cell growth medium and cells), and the laboratory shaker 80 is configured to orbitally agitate the cell culture flask 10 at various oscillation frequencies up to 150 rpm, for example, to allow expression of a target protein into the cavity 14 of the cell culture flask 10. To this end, orbital agitation of the cell culture flask 10 causes the liquid 84 (i.e., cell culture medium and cells) contained within the cell culture flask 10 to rotate and flow upward along the sidewall 22 of the cell culture flask 10.

[0093] The present invention also contemplates a method for culturing cells using the above-mentioned cell culture flask 10-10i. In certain embodiments of the above-mentioned cell culture flask 10-10i, the specific cell lines targeted for culture and tested for culture are as follows: Expi293 suspension-adapted human embryonic kidney (HEK), for growth in Gibco Expi293 expression medium; adapted to ExpiSf TM ExpiSf of Sf9 insect cells grown in CD medium TM Non-engineered derivative; ExpiCHO has been screened and derived from a non-engineered subclone isolated from CHO-S Chinese Hamster Ovary (CHO) cells. However, the cell culture flasks can be used to culture prokaryotic cells, mammalian cells (such as CHO cells), yeast cells, or insect cells. Reference Figure 23 The method includes providing a cell culture flask 10-10i and introducing a certain amount of cell culture medium (e.g., Gibco Expi293 expression medium) and a certain amount of cells (e.g., Expi293) into the cavity 14 of the cell culture flask 10. The cells are cultured under conditions that support cell growth and / or expansion, including, for example, shaking the cell culture flask at a shaking frequency of 50 rpm to 150 rpm using a laboratory shaker 80, to express the target protein into the cavity 14. The method also includes isolating the target protein from the cell culture medium and / or one or more cells.

[0094] In some embodiments, a method for expressing a target protein by culturing cells is provided, wherein the cells are configured to express the target protein under conditions that support the expression of the target protein using the above-mentioned cell culture flask 10-10i. In some embodiments, the target protein may include a recombinant protein. In some embodiments, the target protein includes a viral protein, such as but not limited to a lentiviral protein or an adeno-associated viral protein. In some embodiments, the method further includes isolating the target protein from the cell culture medium and / or one or more cells. Exemplary protein expression systems for use with the cell culture flasks provided herein include but are not limited to: ExpiCHO TM Expression System, FreeStyle TM MAX CHO Expression System, Expi293 TM Expression System, FreeStyle TM 293 Expression System, FreeStyle TM MAX 293 Expression System and ExpiSf TM expression system (all from Thermo Fisher Scientific).

[0095] In some embodiments, a method for producing viral particles is provided by culturing cells under conditions that support the production of viral particles from the cells in a culture flask as described herein. In some embodiments, the cells are transfected with a recombinant viral vector, including but not limited to a lentiviral vector or an adeno-associated viral vector, and then cultured in a culture flask. In some embodiments, the cells are infected with recombinant viral particles, including but not limited to lentiviral particles or adeno-associated viral particles, and then cultured in a culture flask. In some embodiments, the method further comprises isolating the viral particles from the cell culture medium and / or cells after the culture period. Exemplary viral production systems for use with the cell culture flasks provided herein include, but are not limited to, AAV-MAX Helper-Free AAV Production System (Thermo Fisher Scientific) and LV-MAX TM Lentiviral Production System (Thermo Fisher Scientific). Example

[0096] The following examples illustrate certain specific embodiments of the present invention and are not intended to limit the scope of the present invention. The embodiments herein are further described by the following examples and detailed schemes. However, the examples are only intended to illustrate examples and should not be construed as limiting the scope of the present invention. The contents of all references, published patents and patent applications cited throughout this application are incorporated herein by reference. Example 1

[0097] Cell growth performance was evaluated for cultures of several different cell lines using the exemplary 5-liter slanted-bottom, non-baffled culture flasks and 5-liter ridged-bottom, non-baffled culture flasks provided herein, which are referred to in this example as "slanted-bottom culture flasks" and "ridged-bottom culture flasks," respectively.

[0098] Expi293 suspension-adapted human embryonic kidney cell line TM The cells were 0.5×10 6 The density of cells / mL was inoculated into Expi293 TM Expression medium (Thermo Fisher Scientific) and cultured under standard culture conditions. Starting from this very low cell density and doubling every 24 hours, the culture will typically reach its peak density after about 6 days of culture. Expi293 cell growth and viability were assessed in culture volumes ranging from 2 to 3.5 liters in 5-liter culture flasks. Figure 30 Example showing the growth performance of Expi293 cells in a ridged bottom flask, with over 1.0 × 10 viable cells by day 6 post-seeding 7 Expi293 cell growth performance in 2 to 3.5 liter cultures was similar to that of cells grown in a 30 ml culture volume.

[0099] ExpiSf9 insect cells adapted to high-density suspension growth TM The cells were 0.5×10 6 The density of cells / mL was inoculated into ExpiSf9 TM CD medium (Thermo Fisher Scientific) and cultured under standard culture conditions according to the manufacturer's recommendations. Starting from this very low cell density and doubling every 24 hours, the culture will typically reach its peak density after about 6 days of culture. ExpiSf9 cell growth and viability were assessed in culture volumes ranging from 2 to 3.5 liters in 5-liter culture flasks. Figure 31 Examples showing the growth performance of ExpiSf9 cells in 2 L cultures in slanted-bottom and ridged-bottom flasks, with viable cells reaching approximately 1.8 × 10 cells by approximately day 6 after seeding. 7 The highest density was 10 cells / ml.

[0100] ExpiCHO Chinese hamster ovary cells for high-density suspension culture TM The cells were 0.3×10 6 The cells were seeded at a density of 10 cells / mL in ExpiCHO TMCulturing cells in Expression Medium (Thermo Fisher Scientific) under standard culture conditions. Starting at this very low cell density and doubling every 18-20 hours, cultures typically reach peak density 5 days after inoculation. ExpiCHO cell growth and viability were assessed in culture volumes ranging from 2 to 3.5 liters in 5-liter ribbed-bottom flasks. Figure 32 Example showing ExpiCHO cell growth performance in 2 L cultures in slanted-bottom and ridged-bottom flasks, with viable cells reaching approximately 1.7 × 10 cells by approximately day 6 post-inoculation. 7 The highest density was 10 cells / ml. Example 2

[0101] Using exemplary 5-liter beveled bottom, non-baffled culture bottles and 5-liter ridged bottom, non-baffled culture bottles as provided herein, cultures were evaluated for recombinant protein expression from several cell types. These culture bottles are referred to in this example as "beveled bottom culture bottles" and "ridged bottom culture bottles," respectively. Protein yields (titers) from large culture volume culture bottles were compared with small-scale control culture bottles with a final volume of 30 ml.

[0102] Using Expi293 TM Expression system (Thermo Fisher Scientific), Expi293 TM The cells were 3×10 6 The density of cells / mL was inoculated into Expi293 TM Expression medium and transfected with antibody expression vector plasmid DNA according to the manufacturer's instructions. The next day, according to the manufacturer's instructions, TM Transfection enhancer was added to the transfected cell cultures. After 6 days of culture, the titer of antibodies expressed and secreted into the culture medium was determined.

[0103] Recombinant protein production was evaluated in Expi293 culture volumes ranging from 2 to 2.5 L in 5-L bevel-bottom and faceted-bottom flasks. Protein production results from large-volume cultures were comparable to small-scale control flasks. Figure 33 An example of recombinant antibody production in a 2.25 liter final volume of Expi293 culture is shown.

[0104] To use ExpiSf9 TM Assess protein production in cells using Bac-to-Bac TM Recombinant baculovirus containing GFP encoding DNA was prepared using the ExpiSf TMExpression system (Thermo Fisher Scientific), ExpiSf9 TM The cells were 5×10 6 The density of cells / mL was inoculated into ExpiSf TM CD medium and add ExpiSf to the culture according to the manufacturer's instructions. TM The next day, cells were infected with a baculovirus stock containing GFP-encoding DNA. Three days later, the cultures were harvested and GFP protein titers were quantified using a plate reader.

[0105] Recombinant protein production was evaluated in ExpiSf9 culture volumes ranging from 2 to 2.5 L in 5-L slanted-bottom and faceted-bottom flasks. Protein production results from large-volume cultures were comparable to small-scale control flasks. Figure 34 An example of GFP production in a 2 liter final volume of ExpiSf9 culture is shown.

[0106] Using ExpiCHO TM Expression system (Thermo Fisher Scientific), Ex-piCHO TM The cells were 6×10 6 The cells were inoculated at a density of 10 cells / mL in ExpiCHO TM Expression medium and transfected with antibody expression vector plasmid DNA according to the manufacturer's instructions. The next day, ExpiCHO TM Enhancer and ExpiCHO TM Feed is added to the transfected cell cultures and after 8-14 days of culture, the titer of the antibody expressed and secreted into the culture medium is determined.

[0107] When following the manufacturer's ExpiCHO TM For the Max Titer protocol, the incubation temperature was lowered from 36.5°C to 32°C on day 1 after transfection, and another volume of ExpiCHO was added to the culture on day 5 after transfection. TM Feed and harvest cells on day 14. Figure 35 Shown is an example of recombinant antibody production in ExpiCHO culture in a final volume of 2 liters following the Max Titer protocol. Figure 36 Shown are examples of recombinant antibody production in 2 liters of final volume ExpiCHO culture following a variant of the standard protocol. When using slanted-bottom flasks, protein production results in large-scale cultures of transfected cells were similar to those in small-scale control flasks. Surprisingly, the use of slanted-bottom flasks provided herein overcomes the unsuccessful protein production observed after CHO cell transfection in large volumes when using other culture vessels.

[0108] Although the present invention has been illustrated by describing various embodiments thereof, and although the embodiments have been described in considerable detail, it is not intended that the scope of the appended claims be restricted or in any way limited to such details. Thus, the various features discussed herein may be used alone or in any combination. Additional advantages and modifications will readily become apparent to those skilled in the art. Therefore, the invention in its broader aspects is not limited to the specific details and illustrative examples shown and described. Therefore, changes may be made to such details without departing from the scope of the general inventive concept.

Claims

1. Cell culture flask, including: A bottle body defining a cavity for receiving a cell culture medium and comprising: an elongated bottleneck defining an opening to the cavity; bottom; The side wall extends from the elongated neck to the bottom of the bottle body, and the side wall includes a circumferential slope portion, which extends from the outermost periphery of the bottle body to the bottom of the bottle body at a constant angle relative to the central axis of the bottle to define a side wall angle.

2. The cell culture flask according to claim 1, characterized in that The side wall angle is in the range of 30° to 45°.

3. The cell culture flask according to claim 1, characterized in that The base includes a bottom surface having a width, the width being measured as the distance between a circumferential outer edge of the bottom surface and a circumferential inner edge of the bottom surface.

4. The cell culture flask according to claim 3, characterized in that The bottom surface includes a central recess.

5. The cell culture flask according to any one of claims 1 to 4, characterized in that: The bottle further includes at least one baffle formed in the bottle body so as to extend in a radially inward direction relative to a central axis of the bottle to form a recess in the bottle.

6. The cell culture flask according to claim 5, characterized in that: The at least one baffle defines an edge extending from a first terminal end on the circumferential sloped portion of the sidewall to a second terminal end on the bottom of the bottle body.

7. The cell culture flask according to claim 6, characterized in that: The side of the at least one baffle is angled relative to the central axis of the bottle to define a baffle angle.

8. The cell culture flask according to claim 7, characterized in that: The baffle angle is in the range of 45° to 70°.

9. The cell culture flask according to claim 6, characterized in that: The at least one baffle includes a first side wall and a second side wall, each extending from a circumferential sloped portion of the side wall and a bottom of the bottle body to an edge of the baffle in a radially inward direction relative to a central axis of the bottle.

10. The cell culture flask according to claim 9, characterized in that: The first sidewall is angled relative to the second sidewall to define a baffle sidewall angle.

11. The cell culture flask according to claim 10, characterized in that: The baffle side wall angle is in the range of 90° to 110°.

12. The cell culture flask according to claim 6, characterized in that: The at least one baffle defines a triangle having a cross-sectional shape with edges forming vertices of the triangle.

13. The cell culture flask according to claim 9, characterized in that The first side wall and the second side wall are each triangular in shape, with the sides of the baffle forming the hypotenuse of each side wall.

14. The cell culture flask according to claim 13, characterized in that The first side wall and the second side wall each include a first support leg and a second support leg, wherein the first support leg extends along the circumferential inclined portion of the side wall to a first terminal end of the edge located thereon, and the second support leg extends along the bottom to a second terminal end of the edge located thereon.

15. The cell culture flask according to claim 14, characterized in that: The length of each first leg is greater than the length of each second leg.

16. The cell culture flask according to claim 6, characterized in that The circumferential bevel portion of the side wall includes a length measured as the distance between the bottom of the bottle body and the outermost peripheral edge of the bottle body, wherein the first terminal end of the edge is located in an area on the circumferential bevel portion of the side wall between 40% and 60% of the length of the circumferential bevel portion of the side wall measured from the bottom of the bottle body.

17. The cell culture flask according to claim 16, characterized in that The bottom includes a bottom surface having a width, the width being measured as the distance between a circumferential outer edge of the bottom surface and a circumferential inner edge of the bottom surface, wherein the second terminal end of the edge is located in an area on the bottom surface between 50% and 70% of the width of the bottom surface measured from the circumferential outer edge of the bottom surface.

18. The cell culture flask according to claim 6, characterized in that The circumferential bevel portion of the side wall includes a length measured as the distance between the bottom of the bottle body and the outermost peripheral edge of the bottle body, wherein the first terminal end of the edge is located in an area on the circumferential bevel portion of the side wall between 85% and 90% of the length of the circumferential bevel portion of the side wall measured from the bottom of the bottle body.

19. The cell culture flask according to claim 18, characterized in that The bottom includes a bottom surface having a width, the width being measured as the distance between a circumferential outer edge of the bottom surface and a circumferential inner edge of the bottom surface, wherein the second terminal end of the edge is located in an area on the bottom surface between 85% and 99% of the width of the bottom surface measured from the circumferential outer edge of the bottom surface.

20. The cell culture flask according to claim 5, characterized in that The at least one baffle comprises six baffles that are equally spaced apart and distributed around the circumference of the bottle body.

21. The cell culture flask according to claim 5, characterized in that The at least one baffle comprises a first pair of baffles spaced 60° apart from each other around the central axis of the bottle and a second pair of baffles spaced 60° apart from each other around the central axis of the bottle, wherein the first pair of baffles are diametrically opposed to the second pair of baffles around the central axis of the bottle.

22. The cell culture flask and laboratory shaker combination according to any one of claims 1 to 21, wherein the circumferential slope portion is configured to stir the cell growth medium in the flask during operation of the laboratory shaker.

23. Cell culture flask, including: A bottle body defining a cavity for receiving a cell culture medium and comprising: an elongated bottleneck defining an opening to the cavity; bottom; The side wall extends from the elongated neck to the bottom of the bottle body, the side wall comprising a circumferential facet portion extending from the outermost periphery of the bottle body to the bottom of the bottle body, the circumferential facet portion being defined by a plurality of facets distributed around the circumference of the bottle body.

24. The cell culture flask according to claim 23, characterized in that Each of the plurality of facets extends from an outermost periphery of the bottle body to a bottom of the bottle body at a constant angle relative to a central axis of the bottle to define a facet angle.

25. The cell culture flask according to claim 24, characterized in that Each facet angle is in the range of 30° to 60°.

26. The cell culture flask according to claim 23, characterized in that The base includes a bottom surface having a circumferential outer edge defining an outer diameter of the bottom surface and a circumferential inner edge defining an inner diameter of the bottom surface.

27. The cell culture flask according to claim 26, characterized in that The bottom surface includes a central recess.

28. The cell culture flask according to any one of claims 23 to 27, characterized in that: The culture bottle further includes at least one baffle formed in a surface of one facet of the plurality of facets and a surface of the bottom of the bottle body so as to extend in a radially inward direction relative to a central axis of the bottle.

29. The cell culture flask according to claim 28, characterized in that The at least one baffle forms a recess in the bottle body.

30. The cell culture flask according to claim 28, wherein The at least one baffle defines an edge extending from a first terminus on the edge face to a second terminus on the bottom of the bottle body.

31. The cell culture flask according to claim 30, characterized in that The side of the at least one baffle is angled relative to the central axis of the bottle to define a baffle angle.

32. The cell culture flask according to claim 31, characterized in that The baffle angle is in the range of 45° to 70°.

33. The cell culture flask according to claim 30, characterized in that The at least one baffle includes a first side wall and a second side wall, each extending in a radially inward direction relative to the central axis from a circumferential land portion of the side wall and the bottom of the bottle body to an edge of the baffle.

34. The cell culture flask according to claim 33, characterized in that The first sidewall is angled relative to the second sidewall to define a baffle sidewall angle.

35. The cell culture flask according to claim 34, characterized in that The baffle side wall angle is in the range of 90° to 110°.

36. The cell culture flask according to claim 30, characterized in that The at least one baffle forms a triangle having a cross-sectional shape with sides forming vertices of the triangle.

37. The cell culture flask according to claim 33, wherein The first side wall and the second side wall are each triangular in shape, with the sides of the baffle forming the hypotenuse of each side wall.

38. The cell culture flask according to claim 37, characterized in that The first side wall and the second side wall each include a first support edge and a second support edge, wherein the first support edge extends along the circumferential edge portion of the side wall to a first terminal end of the edge located thereon, and the second support edge extends along the bottom to a second terminal end of the edge located thereon.

39. The cell culture flask according to claim 38, characterized in that The length of each first leg is greater than the length of each second leg.

40. The cell culture flask according to claim 30, wherein The facet includes a length measured as the distance between the bottom of the bottle body and the outermost periphery of the bottle body, wherein the first terminal end of the edge is located in an area on the facet between 40% and 60% of the facet length measured from the bottom of the bottle body.

41. The cell culture flask according to claim 40, characterized in that The bottom includes a bottom surface having a width, the width being measured as the distance between a circumferential outer edge of the bottom surface and a circumferential inner edge of the bottom surface, wherein the second terminal end of the edge is located in an area of the bottom surface between 50% and 70% of the bottom surface width measured from the circumferential outer edge of the bottom surface.

42. The cell culture flask according to claim 30, wherein The facet includes a length measured as the distance between the bottom of the bottle body and the outermost periphery of the bottle body, wherein the first terminal end of the edge is located in an area on the facet between 85% and 99% of the facet length measured from the bottom of the bottle body.

43. The cell culture flask according to claim 42, characterized in that The bottom includes a bottom surface having a width, the width being measured as the distance between a circumferential outer edge of the bottom surface and a circumferential inner edge of the bottom surface, wherein the second terminal end of the edge is located in an area of the bottom surface between 85% and 90% of the bottom surface width measured from the circumferential outer edge of the bottom surface.

44. The cell culture flask according to claim 28, wherein The at least one baffle comprises six baffles that are equally spaced and distributed around the circumference of the bottle body, and each baffle is formed on a surface of a different one of the plurality of facets and a surface of the bottom.

45. The cell culture flask according to claim 28, wherein The at least one baffle includes a first pair of baffles, which are spaced 60° apart from each other around the center axis of the bottle so as to be formed on the surface of a different facet of the plurality of facets and the surface of the bottom, and a second pair of baffles, which are spaced 60° apart from each other around the center axis of the bottle so as to be formed on the surface of a different facet of the plurality of facets and the surface of the bottom, wherein the first pair of baffles are diametrically opposed to the second pair of baffles around the center axis of the bottle.

46. The cell culture flask and laboratory shaker combination according to any one of claims 23 to 45, wherein the circumferential faceted portion is configured to agitate the cell growth medium within the flask during operation of the laboratory shaker.

47. A method of culturing cells, comprising: Providing a cell culture flask according to any one of claims 1 to 21 and 23 to 45; introducing cell culture medium and one or more cells into the chamber; as well as The cells are cultured under conditions that support the growth and / or expansion of the cell or cells.

48. The method according to claim 47, wherein The incubation steps were performed on a shaker.

49. The method according to claim 48, characterized in that The shaker shook the culture flask at a shaking frequency ranging between 50 rpm and 150 rpm.

50. The method according to any one of claims 47 to 49, wherein: The one or more cells are mammalian cells.

51. The method according to claim 50, characterized in that The mammalian cells are human embryonic kidney (HEK) cells or Chinese hamster ovary (CHO) cells.

52. A method for expressing a target protein, comprising: Providing a cell culture flask according to any one of claims 1 to 21 and 23 to 45; introducing cell culture medium and one or more cells configured to express a protein of interest into the chamber; Cultivate cells under conditions that support expression of the target protein; as well as The protein of interest is optionally isolated from the cell culture medium and / or the one or more cells.

53. The method according to claim 52, characterized in that The incubation steps were performed on a shaker.

54. The method according to claim 53, wherein The shaker shook the culture flask at a shaking frequency ranging between 50 rpm and 150 rpm.

55. The method according to any one of claims 52 to 54, characterized in that: The one or more cells are mammalian cells.

56. The method according to claim 55, characterized in that The mammalian cells are human embryonic kidney (HEK) cells or Chinese hamster ovary (CHO) cells.

57. A method for producing a viral vector, comprising: Providing a cell culture flask according to any one of claims 1 to 21 and 23 to 45; introducing cell culture medium and one or more cells configured to express the viral vector into the chamber; culturing the cells under conditions that support expression of the viral vector; and Optionally, the viral vector is isolated from the cell culture medium and / or from one or more cells.

58. The method according to claim 57, wherein The incubation steps were performed on a shaker.

59. The method according to claim 58, characterized in that The shaker shook the culture flask at a shaking frequency ranging between 50 rpm and 150 rpm.

60. The method according to claim 57, wherein The cells are human embryonic kidney (HEK) cells or Chinese hamster ovary (CHO) cells.

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