WO2015191429A1

WO2015191429A1 - Incubator flask assembly for cell culture

Info

Publication number: WO2015191429A1
Application number: PCT/US2015/034646
Authority: WO
Inventors: Noah B. PEFAUR
Original assignee: Abbvie, Inc.
Priority date: 2014-06-09
Filing date: 2015-06-08
Publication date: 2015-12-17

Abstract

Embodiments of the present invention feature a device having a base element and a flask element wherein said flask element has a volume greater than about 25 milliliters for growing cell cultures and the device fits in standard multiple well processing equipment.

Description

INCUBATOR FLASK ASSEMBLY FOR CELL CULTURE

Related Applications

This international application claims priority from U.S. Provisional Application No. 62/009,378 filed June 9, 2014, the contents of which are hereby incorporated by reference in its entirety.

Field of the Invention

Embodiments of the present invention relate generally to devices and apparatus for cell culture.

Background of the Invention

Laboratory equipment for performing cell culture, for example, cell culture shaking apparatus is expensive and often limited to specialized consumable articles, due to the size and/or shape of features that receive such articles. One such specialized consumable article is a multiple well plate. The typical multiple well plate has a base with dimensions of 12.8 centimeters by 8.6 centimeters and a height of approximately 3.5 to 4.5 centimeters. A deep well multiple well plate has the same base dimensions and a height of approximately 2.5 to 4.0 centimeters.

Typical multiple well microtiter plates are not suited for larger scale cell culture because the wells are often too small, leading to reduced growth, viability, and protein production levels when compared to cell cultures grown in shake flasks. However, certain equipment for receiving such multiple plates, such as incubators, shakers, robotic liquid handling systems, and samplers cannot use, flasks, vials and the like because they often lack the ability to grasp rounded objects, or orient those objects on a platform. These physical constraints impede the ability to automate scale up production of cell cultures on robotics platforms, and process cell culture products once produced. Additionally, the use of flasks, vials, etc., for cell culture places constraints on throughput, as stacking flasks in a shaking incubator is impractical due to their footprint, shape, and height. Summary of the Invention

Embodiments of the present invention are directed to devices and methods for performing cell culture in equipment for receiving multiple well plates where a larger volume of cell culture is required. As used herein, the term "cell culture" refers to the process of growing cells outside their natural environment and, in particular, to growing eukaryotic cells of multicellular organisms under controlled conditions. One embodiment of the device of the present invention has a flask element and a base element. The flask element has one or more walls defining a vessel having an opening, interior bottom surface and one or more interior side surfaces and at least one exterior surface. At least one of the interior bottom surface and the interior side surface is constructed and arranged to support a cell culture. And, the vessel has a volume of greater than about 25 milliliters. The opening has a cross sectional area and the interior bottom surface has a cross sectional area and the cross sectional area of the interior bottom surface is greater than the cross sectional area of the opening. The base element is affixed to the exterior surface of the flask element to hold the flask element in an operating position within an incubator shaking device. The base element is constructed and arranged to have a footprint corresponding to a multiple well plate, with the base element and flask element constructed and arranged, in an embodiment, to have a height not exceeding a deep well multiple well plate. The flask element affixed to the base element is for containing cell cultures in shaking type incubators designed for multiple well plates. For example, without limitation, the base element has dimensions of approximately

12.0 -13.5 centimeters in length and about 8.0 to 9.0 centimeters in width. As used herein, the term "footprint of a multiple well plate" refers to such dimensions. And, the flask element and the base element have a total height of about 3.5 to 4.5 centimeters. As used herein, the term "height of a deep well multiple well plate" refers to such dimensions. These dimensions approximate the dimensions of 96 well plates and multiples of 96 well plates. As used herein, the term "affixed" means attached to or held by and also means incorporated as a unitary structure therewith.

One further device features a flask element having one or more walls defining a vessel having an opening, bottom surface, one or more interior side surfaces and an exterior surface section and a frusto-conical section with a top opening. At least one of the interior bottom surface and the interior side surface is constructed and arranged to support a cell culture. One embodiment of the flask element features an exterior surface that is constructed and arranged to cooperate with a base element. That is, the exterior surface is received by a base element to hold the flask in operating position within a multiple well device.

One embodiment features a flask element that is constructed and arranged to be removably affixed to the base element. The flask element can be removed from the base element for special processing. For example, it may be desirable to autoclave or sterilize the flask element apart from the base element. One embodiment of the flask element features an exterior surface that cooperates with tab elements on the base element which tab elements hold the flask element in the operating position. The flask element may be affixed to one or more base elements.

One embodiment of the flask element features a bottom surface having a depression for collecting fluids. The fluids collected in the depression are extracted by extracting tools such as pipettes and the like which can enter the opening and extend into the depression.

One embodiment of the flask element features a bottom surface having a circular form with a diameter of about eight centimeters. The opening, having a cross sectional area less than the cross sectional area of the interior bottom surface is about 30% of the diameter of the interior bottom surface, or about 1.0 to 2.5 centimeters. The at least one side surface is generally cylindrical, although other shapes may be used, having a height of about 1.0 to about 3.0 centimeters. One embodiment features at least one wall having a frusto-conical section having an opening. One embodiment features a frusto-conical section having a height of about 1.0 to about 3.0 centimeters. The total working volume of the sections of the flask defined by the one or more interior surface and bottom surface is about 25 to about 150 milliliters. A further embodiment features a total working volume of about 40 to about 60 milliliters.

One embodiment features a breathable filter which is interposed between the interior of the vessel at or about the opening and the exterior of the vessel for the exchange of gases and to isolate cell cultures contained by the vessel from contaminants. One embodiment of a breathable filter is a breathable tape affixed to the exterior surface of the vessel.

One embodiement features raised baffles in the base of the flask portion. One embodiment features a flask element or portion thereof integral to the base element as in integrally molded as a single piece or permanently fixed to the each other by fusion or glues or interfitting locking tabs, snap fittings and the like.

A further embodiment features a base element. One embodiment features a base element having a housing for affixing to the exterior wall of a flask element to hold the flask element in an operating position within an incubator shaking device. The base element constructed and arranged to have a footprint corresponding to a multiple well plate and the base element and flask element constructed and arranged to have a height not exceeding a deep well multiple well plate. The base element is for holding a flask element containing a cell culture affixed in an operating position in shaking type incubators designed for multiple well plates.

One embodiment features a housing comprised of a molded plastic, cellulosic materials, or metal frameworks and combinations thereof. As used herein, metal frameworks comprise racks and wire structures or plate-like metal forms. Cellulosic materials comprise cardboard and the like. One embodiment of a housing made of cellulosic materials is disposable. One embodiment of a housing has a substantially flat folded nonworking storage position and an expanded working position for holding a flask element.

One embodiment of the base element features a housing having one or more side walls and one or more mounting walls or finger elements projecting inward from said side walls for holding a flask element.

One embodiment features a base element with cooperating top and bottom features to allow the base elements to be stacked in an interfitting assembly structure.

A further embodiment of the invention is directed to a method of performing cell culture. The method comprises the steps of providing a device comprising a flask element and a base element. The flask element has one or more walls defining a vessel having an opening, interior bottom surface and at least one interior side surface and at least one exterior surface. The opening has a cross sectional area and the interior bottom surface has a cross sectional area and the cross sectional area of the interior bottom surface is greater than the opening cross sectional area. At least one of the interior bottom surface and the interior side wall is constructed and arranged to support a cell culture. And, the vessel has a volume of about 25 to 150 milliliters. The base element is affixed to the exterior surface of the flask element to hold the flask element in an operating position within an incubator shaking device. The base element is constructed and arranged to have a footprint corresponding to a multiple well plate, with the base element and flask element constructed and arranged to have a height not exceeding a deep well multiple well plate. The flask element is affixed to the base element for containing cell cultures in shaking type incubators designed for multiple well plates, and the method further comprises the step of growing a cell culture on at least one of the interior bottom surface and interior side surface.

One embodiment of the present invention features the further step of placing a breathable filter element over the opening, for example, without limitation, one breathable filter element is a breathable tape.

Embodiments of the present method feature the use of the device received in an operating position in processing equipment for multiple well plates. Further embodiment feature the exchange, or replacement of a first flask element with a second flask element or a first flask element that has been processed apart from the base element.

These and other features and advantages of the present invention will be apparent to those skilled in the art upon viewing the drawings which are summarized briefly in the text below and reading the detailed description of the invention that follows.

Brief Description of the Drawings Figure 1 depicts a cutaway plan view of a device embodying features of the present invention.

Figure 2 depicts a top view of a device embodying features of the present invention.

Figure 3 depicts a slightly elevated exploded view of a device embodying features of the present invention. Detailed Description of the Invention

Embodiments of the present invention will be described in detail with respect to devices and methods for performing cell culture in equipment for receiving multiple well plates, and in particular, 96 well plates. Those skilled in the art will recognize that the description herein can be modified and altered and therefore the present description should not considered limiting but as an exemplification of the present invention.

Turning now to Figure 1 , a device, generally designated by the numeral 11 , embodying features of the present invention is depicted. The device 11 has two major elements, a flask element 15 and a base element 17.

As seen in Figure 2, the flask element 15 has one or more walls 21 defining a vessel 23 having an opening 25, interior bottom surface 27 and one or more interior side surfaces 29 and at least one exterior surface 31. The opening 25 has a cross sectional area and the interior bottom surface 27 has a cross sectional area; and the cross sectional area of the interior bottom surface 27 is greater. At least one of the interior bottom surface 27 and the interior side surface 29 is constructed and arranged to support a cell culture. The transition from bottom surface 27 to side surface 29 may be gradual or markedly different. As depicted, the bottom surface 27 and side surface 29 has an interior edge 33.

Interior bottom surface 27 has a depression 35 for collecting fluids. Depression 35 may comprise an indentation or, as depicted, slope in the interior bottom surfaces 27 which form a low point in the middle of the flask element 15. The fluids collected in the depression 35 are extracted by extracting tools such as pipettes and the like [not shown] which can enter the opening 25 and extend into the depression 35.

The at least one wall 21 defining vessel 23 has a cylindrical section 39 and a frusto- conical section 41 forming part of the interior side surfaces 29 extending to the opening 25. In the alternative, the side wall 21 can be cylindrical with a top substantially planar wall [not shown] having an opening 25 or all frustoconcial.

As depicted, the frusto-conical section 41 has a height of about 1.0 to about 3.0 centimeters. The cylindrical section 39 of side wall 21 has a height of about one to three centimeters. A further embodiment features a total working volume of about 40 to about 60 milliliters. The one or more walls 21 define a vessel 23 having a volume of greater than about

25 milliliters to about 150 milliliters, and, preferably, about 40 to about 60 milliliters. For example, where the bottom surface is circular and has a diameter of about 8 centimeters and the one side surface extends upward approximately about 2.5 centimeters, the vessel 23 will have a volume of about 100 to about 110 milliliters. The base element 17 may comprise a wire or plate-like form or, as depicted, a housing 51 affixed to the exterior surface 31 of the flask element 15 to hold the flask element 15 in an operating position within an incubator shaking device. The housing 51 is constructed and arranged to have a footprint corresponding to a multiple well plate, with the base element 17 and flask element 15 constructed and arranged to have a height not exceeding a deep well multiple well plate.

For example, without limitation, turning now to Figure 1, the base element 17 has dimensions of approximately about 12.0 to about 13.5 centimeters in length represented by line AA and about 8.0 to about 9.0 centimeters in width as represented by line BB. As used herein, the term "footprint of a multiple well plate" refers to such dimensions.

Turning now to Figure 2, the flask element 15 and the base element 17 have a total height of about 3.5 to about 4.5 centimeters as represented by line CC. As used herein, the term "height of a deep well multiple well plate" refers to such dimensions. These dimensions approximate the dimensions of 96 well plates and multiples of 96 well plates.

The flask element 15 has an exterior surface 31 that is constructed and arranged to cooperate with a base element 17. For example, without limitation, the exterior surface 31 is received by housing 51 of a base element 17 to hold the flask element 15 in operating position within a multiple well apparatus.

Flask element 15 is permanently affixed to the base element 17 or in the alternative is constructed and arranged to be removably affixed to the base element 17. As a permanently affixed structure, flask element 15 and the housing 51 depicted in Figure 2 can be made as a unitary molded structure, or flask element 15 is glued or potted into a housing 51 by conventional means.

An embodiment featuring a flask element 15 removably affixed to base element 17, is depicted in Figure 3. As depicted, base element 17 has a housing 51 comprising a bottom piece 59 and a top plate 53. Top plate 53 fits about the exterior surface 31 of flask element 15 about the opening 25. Flask element 15 has a lip 55 which cooperates with a matching opening 57 in top plate 53. Top plate 53 snap fits into bottom piece 59 to hold flask element 15 in operating position. Flask element 15 can be removed from the base element 17 for special processing by opening the top plate 53 from the housing 51. For example, it may be desirable to autoclave or sterilize the flask element 15 apart from the base element 17.

Other means for releasably affixing flask element 15 to housing 35 may readily be applied. For example, the exterior surface 31 of flask element 15 may cooperate with tab elements or flanges on the base element 15 which tab elements or flanges [not shown] hold the flask element in the operating position. Top plate 53 may be hinged to bottom piece 59 and fold down in the manner of a clam-shell or egg-carton.

Releasably securing the flask element 15 to the base element 17 allows the base element 17 and flask element 15 to receive different respective f ask elements 15 and base elements 17. One or more of the base element 17 and flask element 17 can be considered disposable or reusable apart from the other. For example, the base element 17 can be made of plastic, metals or cellulosic materials. Cellulosic materials such as cardboard and polymeric materials such as expanded foam plastic (Styrofoam®) can be readily folded into a collapsed form for storing and unfolded into a more three dimensional form for holding a flask element 15 in an operating position. Flask element 15 is preferably made of glass or plastics suitable for cell culture.

As seen in Figure 1, base element 17 has a top surface 65 and, in Figure 2, a bottom surface 67. The bottom surface 67 has legs or pins 69, which are received in cooperating holes 71 in the top surface 65 when one base element 17 is placed on top of another base element 17 to provide a stable stack. Other features similar to the cooperating pin and hole described can be readily substituted, such as cooperating lip and rim and the like.

A breathable filter is interposed between the interior of the vessel 23 at or about the opening 25 and the exterior of the vessel 31 for the exchange of gases and to isolate cell cultures contained by the vessel from contaminants. One breathable filter is a breathable tape 61 affixed to the exterior surface 31 at the opening 25 or across the lip 55 of flask element 15 as seen in Figure 3.

Thus, the flask element 15 and the base element 17 of device 11 have been described as such relate to each other. Now, the manner of using the device 11 will be described as a method of performing cell culture. The method comprises the steps of providing a device 11 comprising a fiask element 15 and a base element 17. As used herein, the term "providing" means having in one's possession to start the process. The fiask element 15 has one or more walls 21 defining a vessel 23 having an opening 25, interior bottom surface 27 and one or more interior side surfaces 29 and at least one exterior surface 31. At least one of the interior bottom surface 27 and the interior side surface 29 is constructed and arranged to support a cell culture. The opening 25 has a cross sectional area and the interior bottom surface 27 has a cross sectional area, and the cross sectional area of the interior bottom surface 27 is greater than that of the opening 25. The vessel has a volume of greater than about 25 milliliters. The base element 17 is affixed to the exterior surface 31 of the flask element 15 to hold the flask element 15 in an operating position within an incubator shaking device. The base element 17 is constructed and arranged to have a footprint corresponding to a multiple well plate, with the base element 17 and flask element 15 constructed and arranged to have a height not exceeding a deep well multiple well plate. The flask element 15 is affixed to the base element 15 for containing cell cultures in shaking type incubators designed for multiple well plates. The method further comprises the step of growing a cell culture on at least one of the interior bottom surface 27 and interior side surface 29.

One embodiment of the present method comprises the further step of placing a breathable filter element, such as breathable tape 61, over the opening 25. The device 11 is received in an operating position in processing equipment for multiple well plates. Upon completion of the cell culture operation, the f ask element 15 is separated from the base element 17 where it is not permanently affixed. The flask element 15 and the base element 17 are further processed.

Equivalents The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein.

Claims

1. A device for growing a cell culture comprising: a. a fiask element having one or more walls defining a vessel having an opening, an interior bottom surface and at least one interior side surface and at least one exterior surface, wherein said at least one of said interior bottom surface and said interior side surface is constructed and arranged to support a cell culture, said vessel having a volume of greater than about 25 milliliters and said opening having an opening cross sectional area and said interior bottom surface having a bottom surface cross sectional area in which said bottom cross sectional are is greater than said opening cross sectional area; b. a base element affixed to said exterior wall of said flask element to hold said flask element in an operating position within an incubator shaking device, said base element constructed and arranged to have a footprint corresponding to a ANSI/SLAS standard multiple well plate, said base element and flask element constructed and arranged to have a height not exceeding a deep well multiple well plate; c. said flask element affixed to said base element for containing cell cultures in shaking type incubators designed for multiple well plates.

2. The device of claim 1, wherein at least one of said base element and flask element has means for receiving breathable tape over said opening.

3. The device of claim 1, wherein said base element has a housing.

4. The device of claim 3, wherein said housing is formed from one or more materials selected from the group comprising plastic, metal, and cellulosic materials.

5. The device of claim 3, wherein said housing is has four side walls and said flask element is fitted to one or more mounting walls projecting inward from said side walls.

6. The device of claim 3, wherein said housing has interfitting top and bottom features, said interfitting top and bottom features allowing a plurality of housings to be stacked.

7. The device of claim 3, wherein said f ask element is snap fitted into said housing, to allow a succession of different flask elements to be received in said base element.

8. The device of claim 1, wherein said interior bottom surface has a depression for receiving fluid withdrawal devices.

9. The device of claim 1, wherein said one or more walls define a vessel with circular bottom with a diameter of about eight centimeters and a height of about four centimeters.

10. A flask for growing a cell culture comprising one or more walls defining a vessel having an opening, interior bottom surface and at least one interior side surface and at least one exterior surface, at least one of said interior bottom surface and said interior side surface constructed and arranged to support a cell culture, said opening having a cross sectional area and said interior bottom surface having a cross sectional area with said cross sectional area of said interior bottom surface greater than said cross sectional area of said opening, said vessel having a volume of greater than about 25 milliliters; said exterior surface constructed and arranged to be received by a base element to hold said vessel in an operating position within an incubator shaking device, said base element constructed and arranged to have a footprint corresponding to a multiple well plate, said base element and flask element constructed and arranged to have a height not exceeding a deep well multiple well plate; said vessel affixed to said base element for containing cell cultures in shaking type incubators designed for multiple well plates.

11. The flask of claim 10, wherein said interior bottom surface has a depression for receiving fluid withdrawal devices.

12. The flask of claim 10, wherein said one or more walls define a vessel with circular bottom with a radius of about diameter of about eight centimeters and a height of about four centimeters.

13. The flask of claim 10, wherein said at least one side surface has a cylindrical section.

14. The flask of claim 13, wherein said at least one wall having a frusto-conical section having said opening.

15. The flask of claim 14, wherein said frusto-conical section has a height of about 1.0 to 3.0 centimeters and said cylindrical section has a height of about 1.0 to 2.0 centimeters.

16. A base element for a flask for growing a cell culture comprising one or more walls defining a vessel having an opening, interior bottom surface and at least one interior side surface and at least one exterior surface, at least one of said interior bottom surface and said interior side surface constructed and arranged to support a cell culture, said opening having a cross sectional area and said interior bottom surface having a cross sectional area with said cross sectional area of said interior bottom surface greater than said cross sectional area of said opening, said vessel having a volume of greater than about 25 milliliters; said exterior surface constructed and arranged to be received by said base element to hold said vessel in an operating position within an incubator shaking device, said base element constructed and arranged to have a footprint corresponding to a multiple well plate, said base element and flask element constructed and arranged to have a height not exceeding a deep well multiple well plate; said vessel affixed to said base element for containing cell cultures in shaking type incubators designed for multiple well plates.

17. The base element of claim 16 comprising a housing.

18. The base element of claim 17, wherein said housing of said base element is formed at least one of the merterials selected from the group consisting of plastic, metals and cellulosic materials.

19. The base element of claim 17, wherein said housing has at least one side wall and said flask element is fitted to one or more mounting fingers projecting inward from said at least one side wall.

20. The base element of claim 17, wherein said housing has cooperating a top piece and bottom piece holding said vessel.

21. The base element of claim 17, wherein said housing has top surface and a bottom surface having cooperating stacking features.

22. The base element of claim 16, wherein said flask is releasably affixed to allow a succession of different flask elements to be received in or on said base element.

23. A method for growing a cell culture comprising the steps of: a. providing a device having a flask element and a base element, said flask element having one or more walls defining a vessel having an opening, interior bottom surface and at least one interior side surface and at least one exterior surface, said opening having a cross sectional area and said interior bottom surface having a cross sectional area and said cross sectional area of said interior bottom surface greater than said cross sectional area of said opening, at least one of said interior bottom surface and said interior side surface constructed and arranged to support a cell culture, said vessel having a volume of greater than about 25 milliliters; said base element having affixed to said exterior wall of said flask element to hold said flask element in an operating position within an incubator shaking device, said base element constructed and arranged to have a footprint corresponding to a multiple well plate, said base element and flask element constructed and arranged to have a height not exceeding a deep well multiple well plate; said flask element affixed to said base element for containing cell cultures in shaking type incubators designed for multiple well plates; b. growing a cell culture on at least one of said interior bottom surface and interior side surface.

24. The method of claim 23, further comprising affixing breathable tape over said opening.

25. The method of claim 23, further comprising the step of placing said device in an operating position in processing equipment for a multiple well plate.

26. The method of claim 23, wherein said flask element is removed from the base element and said second flask element is affixed to said base element to allow the base element to be used with a plurality of flask elements.

27. The method of claim 23, wherein said flask element is removed from the base element and a second base element is affixed to said flask element, to allow said flask element to be used with a plurality of base elements.