HK1217725B - Multilayer tissue culture vessel - Google Patents

Description

Multilayer tissue culture container

The invention is a divisional application of an invention patent application with the application date of 2010, 7 and 22 months, the application number of 201010289380.7 and the name of 'multilayer tissue culture container'.

Technical Field

The present invention relates to laboratory vessels. More particularly, the present invention relates to multi-layered tissue culture vessels.

Background

The culture of cells (e.g., eukaryotic cells) is used for a variety of purposes, including basic research and high throughput screening. However, culturing cells in a sterile environment is laborious and expensive. Therefore, there is a need to find more efficient and cheaper laboratory vessels for cell culture.

Disclosure of Invention

The invention discloses a container for culturing cells, which comprises: a base including a floor, an upwardly extending wall at least partially bounding the floor of the base; a top including a base, a downwardly extending wall at least partially bounding the base of the top; a tubular neck having an opening defined therein; and one or more shelves, wherein each shelf comprises a base, an upwardly extending wall at least partially bounding the base of the shelf. The upwardly extending wall of the first shelf is contiguous with the downwardly extending wall of the top, with the first shelf being located between the bottom and the top. The base of each shelf has at least one aperture formed therein. The bottom, top and one or more shelves collectively define an enclosed space for culturing cells. The tubular neck extends from a container with an enclosed space accessible through an opening in the tubular neck. Advantageously, such a vessel provides high volume cell culture in a manner that increases efficiency and reduces cell culture expense.

The vessel of the present invention increases the total culture area per surface area of the bottom surface of the culture vessel. The vessel also increases the percentage of cells recovered per surface area of the bottom surface of the culture vessel. Thus, such a container provides an efficient high volume cell culture approach. Such containers may be used in a manual and/or automatic manner. Exemplary embodiments include, but are not limited to, containers that retain the general bottom surface of a standard BD Falcon T-175 flask, and are therefore compatible with automated cell culture systems, such as Automation partnerchip's SelecT^TMAnd Compact^TMAn automated cell culture system. However, it is not limited toThe container may be expanded or contracted in height compared to a standard bottle, such as a BD Falcon T-175 bottle. In particular, the stacked arrangement of the shelves in the vessel allows the height to be varied so that the surface area of the cultured cells can be expanded.

In addition, the design of the vessel of the present invention minimizes the number of operations required to fill and remove the medium, thereby increasing the efficiency of culturing cells and reducing the chance of contamination of the vessel at each operation. Preferably, the container comprises a sufficiently large opening to enable the pipette to access the back wall of the container, thereby improving good laboratory practice conditions for good cell culture techniques. Exemplary embodiments may include, but are not limited to, containers in which the opening is large enough to allow access to the back wall for a 10mL pipette, and even a 50mL pipette.

The design of the vessel of the present invention also reduces the amount of media and "hanging" cells along the walls and corners of the vessel interior and allows for efficient removal of media and cells. The vessel design also allows for efficient distribution of liquids (e.g., media, Phosphate Buffered Saline (PBS), pancreatin) to different cell layers, thereby reducing the amount of liquid required, facilitating more equal distribution of liquid, and facilitating more equal cell distribution upon seeding. In this manner, a more consistent distribution of nutrients, nutrient consumption rates, cell growth rates, and division rates can be achieved at the time of cell harvest, thereby enabling the culture of more uniform cell growth and/or differentiation, and an overall healthier cell population. Such containers also provide a large cost savings in terms of the media and digestive agents (e.g., pancreatin) required for cell growth and dissociation, respectively. In summary, the container of the present invention reduces the labor and expense associated with culturing cells.

These and other features of the present invention can be best understood from the following specification and drawings, the following of which is a brief description.

Drawings

Fig. 1 is a perspective view of a container formed in accordance with the present invention.

Fig. 2 is a cross-sectional view taken along line 2-2 of fig. 1.

Fig. 3 is a top view of the container.

Fig. 4 is a cross-sectional view taken along line 4-4 of fig. 3.

Fig. 5 is a side view of the container.

Fig. 6 is a perspective view of a base useful with the present invention.

Fig. 7 is a cross-sectional view taken along line 7-7 of fig. 6.

Fig. 8 is a perspective view of a top usable with the present invention.

Fig. 9 is a cross-sectional view taken along line 9-9 of fig. 8.

Fig. 10 is a cross-sectional view taken along line 10-10 of fig. 8.

Fig. 11 is a perspective view of a shelf useful in the present invention.

Fig. 12 is a cross-sectional view taken along line 12-12 of fig. 11.

Fig. 13 is a perspective view of a container formed in accordance with the present invention.

Fig. 14 is a cross-sectional view taken along line 14-14 of fig. 13.

Fig. 15 is a perspective view of a shelf useful in the present invention.

FIG. 16 is a schematic showing the equilibration of cell culture media in a vessel formed in accordance with the present invention.

Fig. 17 is a schematic showing the entry of a pipette into a container formed in accordance with the present invention.

FIG. 18 is a schematic showing the layering of cell culture media in a vessel formed according to the present invention.

FIG. 19 is a top view of a shelf useful in the present invention.

Fig. 20 is a cross-sectional view taken along line 20-20 of fig. 19.

Fig. 21 and 22 are enlarged views of a portion 21 and a portion 22 of fig. 20, respectively.

Fig. 23 and 24 depict different constraining wall configurations.

Fig. 25-27 depict a lid useful with the present invention.

Detailed Description

Referring to the drawings, a vessel 10 for cell culture is depicted. Those skilled in the art will appreciate that the container 10 may be used with a variety of cell cultures. The container 10 generally includes a bottom 12, a top 14, a tubular neck 16 having a defined opening 18 therein, and one or more shelves 20 intermediate the bottom 12 and the top 14. The bottom 12, top 14 and shelves 20 together define an enclosed space 22 for culturing cells. A tubular neck 16 extends from the vessel 10 and the enclosed space 22 is accessible through an opening 18 in the tubular neck 16.

The base 12 is generally dish-shaped with a base 24 that extends upwardly from a wall 26, the wall 26 at least partially bounding the base 24. Preferably, the wall 26 completely forms the perimeter boundary of the base 24.

One or more shelves 20 may be employed depending on the overall desired size of the container 10. The greater the number of shelves 20, the greater the cell culture capacity of the container 10. Excessive sizing may be undesirable due to gas flow and distribution limitations. Embodiments utilizing two or 4 shelves 20 are contemplated. However, other numbers of shelves may be used. Each shelf 20 is generally dish-shaped having a base 28 and walls 30 extending upwardly therefrom that at least partially bound the base 28. Preferably, on each shelf 20, the wall 30 completely forms the perimeter boundary of the base 28. In addition, at least one aperture 32 is formed through the base 28 of each shelf 20. Preferably, each shelf 20 includes at least two apertures 32, one gas delivery aperture 32a and one flow aperture 32 b. As discussed further below, the gas flow apertures 32a allow gas to be delivered through the shelves 20 to flow through the vessel 10. The flow apertures 32b may be used to balance the cell culture medium entering the vessel 10 so that the cell culture medium is distributed between the various support layers of the vessel 10 defined by the bottom 12 and the shelves 20. Although a dual aperture arrangement for each shelf 20 is preferred, each shelf 20 may be provided with a single aperture 32 that serves both gas delivery and balancing functions. Further, more than two apertures 32 may be utilized for one or both of the gas delivery function and the balancing function. Preferably, the gas delivery holes 32a and the flow holes 32b of each shelf 20 are spatially separated.

The top 14 is generally inverted dish-shaped with a base 34 and a wall 36 extending downwardly from the base, the wall at least partially bounding the base 34. Preferably, the tubular neck 16 is integrally formed with the top 14 so as to extend therefrom. Alternatively, the opening may be formed in the top 14 and the tubular neck 16 formed as a separate element and attached to the top 14 using any known technique, such as welding or adhesive. In addition, openings may be formed that extend from the top 14 and into one or more shelves 20 to which the tubular neck 16 is attached. The opening 18 in the tubular neck 16 can be oval (including circular) or semi-circular and can be formed by a constant or variable profile along the length of the tubular neck 16 (e.g., a portion of the opening 18 can be oval and a portion can be semi-circular along the length of the tubular neck 16).

The bottom 12, top 14, and one or more shelves 20 are arranged in a stacked manner to collectively form the container 10. Preferably, these elements are formed from a thermoplastic material that is compatible with the cells of interest. Polystyrene may be used. Those skilled in the art will appreciate that other materials may also be employed. These elements may be formed transparent, tinted (e.g., light blue) or colored (e.g., amber). In addition, various portions of the bottom 12, top 14, and shelves 20 may be modified or treated to enhance specific conditions. For example, one or more biological agents may be applied to one or more portions of the bottom 12, top 14, and/or shelves 20, including, but not limited to, extracellular matrix or components thereof, such as laminin (laminin), fibronectin, and collagen, in any combination. Additionally, or alternatively, synthetic reagents may be used. The surface may also be pretreated, for example by tissue culture treatment or protoplast polymerization. Those skilled in the art will appreciate that these different treatments or modifications can be used in various combinations and utilized depending on the intended purpose.

The bottom 12, top 14 and shelves 20 are adjacently connected to form the container 10. Specifically, the wall 36 of the top 14 abuts the wall 30 of the shelf 20, the shelf 20 being adjacent the top 14. All of the shelves 20 are located between the bottom 12 and the top 14. Adjacent shelves 20 are arranged in a stacked manner and abut the wall 30 of a lower stacked shelf 20 that abuts an adjacent shelf 20 of a higher stack. The bottom 12 abuts the shelf 20 adjacent to the bottom 12, which would be the lowest stacked shelf 20 if multiple shelves 20 were employed, with the wall 26 of the bottom 12 abutting the adjacent shelf 20. Any known technique, such as welding (e.g., ultrasonic welding), bonding, and/or mechanical joining (e.g., interlocking tongues and tongues) may be utilized in any combination to provide a liquid-tight seal at their interface. A sealing member, such as a gasket, may be interposed between adjacent elements, such as between the top 14 and an adjacent shelf 20. The gasket material may be selected to be air permeable, but liquid impermeable. Further, one or more ventilation openings 37 may be formed in the bottom 12, the top 14, and/or one or more shelves 20. A breathable/liquid impermeable membrane 39 may be provided that extends across one or more of the vent openings 37.

The bottom 12, top 14 and shelves 20 may be made in different shapes to provide different surface areas for cell culture. Accordingly, the container 10 may be formed in different sizes and different shapes, such as a flask as shown in the figures. Alternatively, the container 10 may have a rectangular shape, or form other polygonal or other shapes. Preferably, the container 10 includes a first end 38, the first end 38 being formed to support the container 10 in an upright position such that the shelves 20 are upwardly aligned. This is a preferred state for loading the cell culture fluid to reach equilibrium. More preferably, the first end 38 is located on the container 10 opposite the opening 18 of the tubular neck 16. The first end 38 may be planar or a locus of points (focus) that collectively define a support plane.

The bottom 12, top 14 and shelves 20 form the container 10. The walls 26, 30, 36 of the bottom 12, top 14 and shelves 20, respectively, and the bases 24, 28 of the bottom 12 and top 14, respectively, define portions of the exterior surface of the container 10.

To achieve equilibrium, it is preferred that the flow aperture 32b be located adjacent the first end 38. In this manner, as shown in FIG. 16, cell culture medium C is placed in container 10, container 10 is supported by first end 38, and cell culture medium C can pass through flow apertures 32b and the space (V) divided between bottom 12, shelf 20, and top 14₁、V₂、V₃… …) to achieve an equilibrium state. From this equilibrium state, the container 10 is horizontally disposed and supported by the base 12, as shown in fig. 18. The separate portions of cell culture medium C are thus distributed on the support substrate defined by the base 12 or shelves 20.

Preferably, the cell culture medium C is equilibrated to an equal volume between the bottom 12, the shelves 20, and the top 14. Equal spacing, such as the spacing z between the respective bases 24, 28 and 34 of the bottom 12, top 14 and shelves 20, may divide an equal volume between the layers, particularly adjacent the first end 38 of the container 10.

As shown in fig. 13-14, the base 34 of the top 14 may be formed generally planar in shape throughout. As shown in fig. 14, to allow good pipette access, the base 34 of the top 14 is separated from the base 28 of the adjacent rack 20 by portions that are larger than the z-spacing between the bases 28 of the racks 20, or the base 24 of the bottom 12 and the base 28 of the adjacent rack 20. This increased spacing (spacing q) allows the pipette to traverse the entire length of the container 10 and contact the first end 38, as shown in fig. 17. Thus, after cell culture, the pipette channel can be used for efficient cell culture medium and cell recovery when the container 10 is supported by the first end 38. However, the increased spacing between the base 34 of the top 14 and the base 28 of an adjacent shelf 20 may result in an enlarged space being formed between the top 14 and the adjacent shelf 20 as compared to the space formed between each shelf 20 and the space formed between the bottom 12 and its adjacent shelf 20.

Preferably, the base 34 of the top 14 is formed from first, second and third portions 42, 44, 46, respectively, that reduce the space formed between the base 34 of the top 14 proximate the first end 38 and the base 28 of an adjacent shelf 20. Specifically, referring to FIG. 18, the first portion 42 is positioned a distance x from the base 28 of an adjacent shelf 20 and the second portion 44 is positioned a distance y from the base 28 of an adjacent shelf 20, the distance x being greater than the distance y. A second portion 44 extends from the first end 38 to partially enclose the space adjacent the first end 38. Preferably, the distance y is equal to the spacing z provided between each pair of adjacent shelves 20, and between the base 12 and the adjacent shelf 20. It is further preferred that the second portion 44 has a predetermined length L extending from the first end 38 that is equal to or greater than the length required to accommodate the target balance space. As shown in FIG. 16, in this manner, as the cell culture medium C reaches equilibrium, the second portion 44 co-extends or exceeds the height of the cell culture medium C (equilibrated to space V)₁、V₂、V₃… …). Preferably, the height of the cell culture medium C is lower than the third portion 46 at equilibrium. The height of the cell culture medium C above the second portion 44, contacting the third portion 46, may result in unequal volumes resulting from equilibration. With the arrangement of the first, second and third portions 42, 44, 46, good pipette access may be provided to the first end 38, including access to the space near the second portion 44 near the first end 38, with the pipette passing through a higher region near the first portion 42. This arrangement also allows for equal volume balancing between the layers. The first and second portions 42, 44 may each form a plane, may be arranged in parallel, although other shapes (e.g., arcuate) are also useful.

the third portion 46 of the substrate 34 extends between and joins the first and second portions 42, 44. preferably, the third portion 46 forms a planar surface, although other shapes, such as an arcuate profile, may be formed. advantageously, the third portion 46 may define a suitable surface for printing or carrying other indicia, such as a bar code. for the planar surface defined by the second portion 44, the third portion 46 is disposed at an included angle α in the range of about 10-90 degrees, more preferably in the range of about 10-30 degrees, more preferably at an included angle α of about 20 degrees.the third portion 46, which is disposed at an angle to the second portion 44, provides a tapered surface that directs the flow of cell media C toward the first end 38 when the container 10 is supported by the first end 38. although an included angle α of 90 degrees is possible, a preferred included angle α is less than 90 degrees to prevent any cell media or cells from remaining at the juncture of the first and third portions 42, 26.

As shown in fig. 1, with the application of the second portion 44, a recess 48 may be defined in the container 10. The wall 36 of the top 14 may partially form a boundary of the pocket 48. The recess 48 provides a handle function in the exposed area of the wall 36 to allow a user to grasp the container therethrough.

As shown in fig. 2, the gas delivery holes 32a are preferably aligned in a straight line to define a gas flow passage 40 in the enclosed space 22. Preferably, the gas flow passage 40 extends to the vicinity of the opening 18 defined in the tubular neck 16. Gas flow passages 40 through the shelves 20 allow gas flow to various portions of the enclosed space 22.

As discussed above, and as shown in FIG. 18, in the use state of cell culture, the vessel 10 is placed supported by the base 12. In this position, the cell culture medium C is disposed across the surface of the base 24, 28 of the respective bottom 12 or shelf 20 due to the nature of its liquid. With respect to the bottom 12, the walls 26 provide a liquid constraint to keep the cell media C above the base 24. In the case of the rack 20, in order to provide a restriction to prevent the tissue cell culture medium C from passing through any of the holes 32(32a, 32b), a restriction wall 50 may be provided along the edge of each hole 32 as a dam. Preferably, the height of the wall 30 of the shelf 20 is higher than the height of the constraining wall 50. In this manner, an open space may be maintained above each layer of cell culture medium C between the shelves 20.

The base 12 and the shelves 20 may be configured for processing different volumes of cell culture medium C, although it is preferred that each layer accommodate the same volume within the same container 10. The base 24 and the walls 26 of the base 12 define a space on the base 12 for receiving the cell culture medium C. The base 28, walls 30 and constraining walls 50 of each shelf 20 define a space above the respective shelf for receiving cell culture medium C. The base 12 and the shelves 20 may be configured to each accommodate a volume of cell culture medium C in the range of 4-50 mL. Because the base 12 does not have the apertures 32 formed therein, the surface area of the base 12 may be greater than the surface area of each shelf 20; thus, the height of the layer of cell culture medium C on the bottom 12 can be slightly less than the height of the layer of cell culture medium C on each shelf 20. The spacing z is set to take into account the amount of volume of each layer of cell culture medium C to ensure sufficient headspace above the layer of cell culture medium C for proper gas distribution.

A phenomenon that the cell culture medium C is drawn to the corresponding well 32 through the restriction wall 50 by capillary action is noticed. As shown in fig. 11, 15 and 19, the constraining wall 50 is preferably curved along an arcuate longitudinal axis. The arcuate shape resists capillary attraction and provides an anti-wicking effect to the constraining wall 50. The apertures 32(32a, 32b) may form an arcuate shape with the constraining wall 50 disposed along an edge thereof. Further, as shown in fig. 11, 15, and 19, the holes 32 may have various configurations. Specifically, the gas delivery apertures 32a may be formed extending across a portion of the side edges 52 of the shelves 20 (fig. 11 and 15) or along the entire length of the side edges 52 of the shelves 20 (fig. 19).

The constraining walls 50 may be provided with other anti-wicking features, such as being disposed at an angle relative to the base 28 of the respective shelf 20 (fig. 21); provided with an arcuate cross-section (fig. 23); and/or include a non-smooth surface 53 facing away from the respective aperture 32. As shown in fig. 20-24, the non-smooth surface 53 may include various surface discontinuities or protrusions, such as bumps, pits, roughened areas, striations, and the like. These anti-capillary features are intended to disrupt capillary attraction. Additionally, or alternatively, the constraining walls 50 may be fabricated or prepared to have hydrophobic portions to repel the cell culture medium C. For example, the free edge 54 where the constraining wall 50 terminates may be made hydrophobic. Other portions of the constraining wall 50 may be treated the same. To further enhance this effect, portions of the base 28 of the shelf 20 may be made hydrophilic to enhance retention of the cell culture medium C on the base 28 of the shelf 20. These various anti-capillary features may be used in various combinations.

the vessel 10 may be provided with additional features or variations, for example, with reference to FIGS. 2 and 25, the tubular neck 16 may be provided at a different angular orientation relative to the vessel 10. As shown in FIG. 2, the tubular neck 16 extends along a longitudinal axis that is disposed at an included angle β with respect to the plane defined by the base 28 of the uppermost shelf 20. the included angle β may range from about 0 to 90 degrees. As shown in FIG. 2, the tubular neck 16 extends from the side of the top 14 at an included angle of 0 degrees. As shown in FIG. 25, the tubular neck 16 has a vertical orientation and extends from the base 34 of the top 14 at an included angle of 90 degrees. for this arrangement, the pipette channel may be provided through the gas flow channel 40 to a space located adjacent the base 24 of the bottom 12 for removal of cell culture medium or cells.

Preferably, the container 10 may be formed to be stacked with other containers. To achieve such stacking, the top portion 14 preferably defines an upper support surface that is parallel to the support surface defined by the bottom portion 12. In this manner, two or more containers 10 may be stacked with the bottom 12 of the upper stacked container being supported by the top 14 of the lower stacked container 10. For added stability, a ledge 56 may be defined on the outer surface of the base 12. Accordingly, the wall 36 of the top 14 may be formed to protrude slightly from the base 34. The wall 36 may be formed to nestingly receive the flange 56 to provide lateral stability in the stacked configuration. It should be noted that the wall 36 of the top 14, particularly by protruding from the base 34, may be configured to bear primarily the weight of the upper stack of containers 10. Instability problems caused by the presence of the recess 48 can thus be avoided.

The base 28 of each shelf 20 is preferably formed to be planar. Alternatively, the substrate 28 may be formed with corrugations, waves, or other configurations to increase surface area. For any configuration, preferably, the layer of cell culture medium C supported by the shelves 20 is disposed parallel to the vessel 10 supported by the base 12. The base 24 of the base 12 may likewise be corrugated, wavy, or otherwise formed to have an increased surface area. For any configuration, it is preferred that the layer of cell culture medium C supported by the base 12 be parallel to the other layers of cell culture medium C.

The container 10 may be provided as an assembly with a corresponding cap 58, the cap 58 being formed to fit over the tubular neck 16 to seal the opening 18. Any known arrangement that allows assembly may be used, such as a friction fit, an interference fit, a threaded connection, or a bayonet mount. The lid 58 may be formed solid so as to be non-vented. Optionally, the cover 58 may be provided with one or more vent holes 60, which may include a breathable/liquid impermeable membrane 61. In addition, the cap 58 may include one or more adjustable valves 62 that allow selective sterile connection between the enclosed space 22 and external fixtures. In addition, the cap 58 may include one or more tube connectors 64 to allow direct and continuous communication with one or more supply tubes T (e.g., gas supply tubes).

In use, the container 10 is placed over the first end 38 and a sufficient amount of cell culture medium C is introduced into the enclosed space 22 through the opening 18 to provide a target volume for the base 12 and each of the shelves 20. A pipette may be used to introduce cell culture medium C, the pipette being inserted through opening 18 into enclosed space 22 between top 14 and adjacent shelf 20. The introduced cell culture medium C collects in the space adjacent the first end 38 and equilibrates between the flow apertures 32b to provide a separate volume of cell culture medium C corresponding to the bottom 12 and each of the shelves 20. Once equilibrium is reached, the vessel 10 is adjusted to be supported by the base 12, and the cell culture medium C is spread across the base 12 and each of the shelves 20. A cap 58 may be fitted over the tubular neck 16 to seal the opening 18. Thereafter, the container 10 may be placed in an incubator. For shipping, the container 10 is preferably held upright with cell culture medium C accumulated in the enclosed space 22 adjacent the first end 38. A plurality of containers 10 may be stacked while culturing.

The container 10 may be placed on the first end 38 for the purpose of extracting the cell culture medium C for exchange, or cell collection. A pipette, or other extraction device, may be introduced to extract the cell culture medium C from near the first end 38. This allows for complete removal. A pipette may be introduced into the enclosed space 22 between the top 14 and the adjacent rack 20 into the space adjacent the first end 38 for extraction. Under negative pressure, cell culture medium C can be drawn through flow aperture 32b to the space adjacent top 14 for withdrawal therefrom. Alternatively, the cell culture medium C can be poured out through the opening 18. Pancreatin, or other digestive agents, may be used to release the cells for harvesting.

Claims (13)

1. A container for culturing cells, comprising:

a base including a floor, an upwardly extending wall at least partially bounding the floor of the base;

a top including a base, a downwardly extending wall at least partially bounding the base of the top;

a tubular neck extending from the container defining an opening therein; and

one or more shelves, each shelf comprising a base, an upwardly extending wall at least partially bounding the base of the shelf, each shelf comprising at least two flow apertures,

wherein the upwardly extending wall of the first shelf is contiguous with the downwardly extending wall of the top, the first shelf being located between the bottom and the top,

wherein the bottom, the top and the one or more shelves together define an enclosed space for culturing cells accessible through the opening in the tubular neck, the bottom, the top and portions of the one or more shelves defining portions of the outer surface of the container,

wherein each shelf comprises at least one constraining wall disposed along an edge of at least one of the at least two apertures such that the at least one of the at least two apertures is disposed between the upwardly extending wall and the at least one constraining wall of the shelf, an

Wherein the substrate of each shelf comprises at least one of at least two flow apertures formed therein.

2. The container of claim 1, comprising a second shelf positioned between the bottom and the first shelf, the upwardly extending wall of the bottom abutting the second shelf.

3. The container of claim 2, wherein the container has a first end defining a first space between the top and a first shelf extending a predetermined length from the first end, and a second space substantially equal to the first space between the bottom and a second shelf extending a predetermined length from the first end.

4. The container of claim 3, wherein the base of the top includes a first portion and a second portion, the first portion being spaced a first distance from the base of the first shelf, the second portion being spaced a second distance from the base of the first shelf, the second distance being less than the first distance.

5. The container of claim 4, wherein the second portion extends a predetermined length from the first end of the container.

6. The container of claim 4, wherein the first portion and the second portion are parallel.

7. The container of claim 4, wherein the base of the top includes a third portion extending between and abutting the first portion and the second portion.

8. The container of claim 7, wherein the third portion is generally flat and is disposed at an angle relative to a plane defined by the second portion, the angle being in the range of 10-90 degrees.

9. The container of claim 8 wherein the included angle is in the range of 10 to 30 degrees.

10. The container of claim 8 wherein the included angle is 20 degrees.

11. A container for culturing cells, comprising:

a base including a floor, an upwardly extending wall at least partially bounding the floor of the base;

a top including a base, a downwardly extending wall at least partially bounding the base of the top;

a tubular neck extending from the container defining an opening therein; and

one or more shelves, each shelf comprising a base, an upwardly extending wall at least partially bounding the base of the shelf, each shelf comprising at least two flow apertures,

wherein each shelf comprises at least one constraining wall disposed along an edge of at least one of the at least two apertures such that the at least one of the at least two apertures is disposed between the upwardly extending wall and the at least one constraining wall of the shelf,

wherein the bottom, top and one or more shelves together define an enclosed space for culturing cells, the bottom, top and portions of the one or more shelves define portions of an outer surface of the container, and

wherein the substrate of each shelf comprises at least one of at least two flow apertures formed therein.

12. A container for culturing cells, comprising:

a base including a floor, an upwardly extending wall at least partially bounding the floor of the base;

a top including a base, a downwardly extending wall at least partially bounding the base of the top;

a tubular neck extending from the container defining an opening therein; and the number of the first and second groups,

one or more shelves, each shelf comprising a base, an upwardly extending wall at least partially bounding the base of the shelf, each shelf comprising at least two flow apertures,

wherein the upwardly extending wall of the first shelf is contiguous with the downwardly extending wall of the top, the first shelf being located between the bottom and the top,

wherein the base of each shelf has at least one of at least two flow apertures formed therein,

wherein each shelf comprises at least one constraining wall disposed along an edge of at least one of the at least two apertures such that the at least one of the at least two apertures is disposed between the upwardly extending wall and the at least one constraining wall of the shelf, an

The top base has a first portion spaced a first distance from the base of the first shelf, and the top base has a second portion spaced a second distance from the base of the first shelf, the first distance being greater than the second distance.

13. A container for culturing cells, comprising:

a base including a floor, an upwardly extending wall at least partially bounding the floor of the base;

a top including a base, a downwardly extending wall at least partially bounding the base of the top; and

one or more shelves, each shelf comprising a base, an upwardly extending wall at least partially bounding the base of the shelf, each shelf comprising at least two flow apertures,

wherein the upwardly extending wall of the first shelf is contiguous with the downwardly extending wall of the top, the first shelf being located between the bottom and the top,

wherein the base of each shelf has at least one of at least two flow apertures formed therein,

wherein each shelf comprises at least one constraining wall disposed along an edge of at least one of the at least two apertures such that the at least one of the at least two apertures is disposed between the upwardly extending wall and the at least one constraining wall of the shelf, and

the bottom, top and one or more shelves together define an enclosed space for culturing cells, and the bottom, top and portions of the one or more shelves define portions of the outer surface of the container that are accessible through the opening in the tubular neck into the enclosed space.