Abstract:
The present invention discloses a vessel for culturing cells which includes: a bottom, a top, a tubular neck, and, one or more shelves. The first shelf adjoins the top with the first shelf being located intermediate the bottom and the top. The bottom, the top and the one or more shelves collectively define an enclosed volume for culturing cells with the enclosed volume being accessible by the opening in the tubular neck. Advantageously, this vessel provides high volume cell culture in a manner that increases efficiency and reduces the cost of culturing cells.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application is a continuation of U.S. application Ser. No. 12/840,758, filed on Jul. 21, 2010, now U.S. Pat. No. 8,778,669, the entire contents of this application being incorporated by reference herein. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to laboratory vessels. More particularly, the present invention relates to multilayer tissue culture vessels. 
       BACKGROUND OF THE INVENTION 
       [0003]    Cells, such as eukaryotic cells, are cultured for a variety of purposes, including basic research and high-throughput screening. However, culturing cells under sterile conditions is both laborious and expensive. Thus, there is a need for more efficient and cost-effective laboratory vessels for culturing cells. 
       SUMMARY OF THE INVENTION 
       [0004]    The present invention discloses a vessel for culturing cells which includes: a bottom including a base with an upwardly extending wall at least partially bounding the base of the bottom; a top including a base with a downwardly extending wall at least partially bounding the base of the top; a tubular neck with an opening defined therein; and, one or more shelves, wherein, each shelf includes a base with an upwardly extending wall at least partially bounding the base of the shelf. The upwardly extending wall of a first shelf adjoins the downwardly extending wall of the top with the first shelf being located intermediate the bottom and the top. The base of each of the shelves having at least one aperture formed therein. The bottom, the top and the one or more shelves collectively define an enclosed volume for culturing cells. The tubular neck extends from the vessel with the enclosed volume being accessible by the opening in the tubular neck. Advantageously, this vessel provides high volume cell culture in a manner that increases efficiency and reduces the cost of culturing cells. 
         [0005]    The vessels of the present invention increase the total culture area per surface area of the footprint of a culture vessel. The vessels also increase the percent recovery of cells per surface area of the footprint of a culture vessel. Thus, the vessels provide means for efficient high volume cell culture. Such vessels may be used in a manual and/or automated fashion. Exemplary embodiments include, but are not limited to, vessels which maintain the general footprint of a standard BD Falcon T-175 flask, and are thus compatible with automated cell culture systems, such as The Automation Partnership&#39;s SelecT™ and CompacT™ automated cell culture systems. The vessels, however, may be expanded or reduced in comparison to the height of a standard flask, such as a BD Falcon T-175 flask. In particular, the stacking arrangement of shelves within the vessel permits the height to be varied such that the surface area for culturing cells can be expanded. 
         [0006]    In addition, the vessel&#39;s design minimizes the number of manipulations required to fill and remove media thereby increasing the efficiency of culturing cells and decreasing the chance for contamination of the vessel with each movement. Preferably, the vessels include an opening sufficiently large for a pipette to access the back wall of the vessel thereby promoting good laboratory practices for good cell culture technique. Exemplary embodiments may include, but are not limited to, vessels wherein the opening is sufficiently large for a 10 mL pipette or even for a 50 mL pipette to access the back wall. 
         [0007]    The vessels&#39; design also decreases the amount of media as well as cells “hung up” along the walls and corners of the vessel&#39;s interior and allows for efficient removal of media and cells. The vessels&#39; design also distributes liquid (e.g., media, phosphate buffered saline (PBS), trypsin) efficiently to the different layers of cells thereby decreasing the amount of liquid required, promoting more even distribution of liquid and promoting more even cell distribution during seeding. This provides more consistent distribution of nutrients, nutrient consumption rate, cell growth rate and dissociation rate at the time of cell harvest thereby fostering more uniform cell growth and/or differentiation as well as a healthier population of cells overall. Such vessels also provide a great cost savings with respect to the amount of media and dissociation agent (e.g., trypsin) required for cell growth and dissociation of cells, respectively. In short, the vessels of the present invention decrease the amount of labor and expense associated with culturing cells. 
         [0008]    These and other features of the invention will be better understood through a study of the following detailed description and accompanying drawings. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0009]      FIG. 1  is a perspective view of a vessel formed in accordance with the subject invention. 
           [0010]      FIG. 2  is a cross-sectional view taken along line  2 - 2  of  FIG. 1 . 
           [0011]      FIG. 3  is a top plan view of the vessel. 
           [0012]      FIG. 4  is a cross-sectional view taken along line  4 - 4  of  FIG. 3 . 
           [0013]      FIG. 5  is a side view of the vessel. 
           [0014]      FIG. 6  is a perspective view of a bottom useable with the subject invention. 
           [0015]      FIG. 7  is a cross-sectional view taken along line  7 - 7  of  FIG. 6 . 
           [0016]      FIG. 8  is a perspective view of a top useable with the subject invention. 
           [0017]      FIG. 9  is a cross-sectional view taken along line  9 - 9  of  FIG. 8 . 
           [0018]      FIG. 10  is a cross-sectional view taken along line  10 - 10  of  FIG. 8 . 
           [0019]      FIG. 11  is a perspective view of a shelf useable with the subject invention. 
           [0020]      FIG. 12  is a cross-sectional view taken along line  12 - 12  of  FIG. 11 . 
           [0021]      FIG. 13  is a perspective view of a vessel formed in accordance with the subject invention. 
           [0022]      FIG. 14  is a cross-sectional view taken along line  14 - 14  of  FIG. 13 . 
           [0023]      FIG. 15  is a perspective view of a shelf useable with the subject invention. 
           [0024]      FIG. 16  is a schematic demonstrating equilibration of cell media in a vessel formed in accordance with the subject invention. 
           [0025]      FIG. 17  is a schematic demonstrating pipette access in a vessel formed in accordance with the subject invention. 
           [0026]      FIG. 18  is a schematic demonstrating layered arrangement of cell media in a vessel formed in accordance with the subject invention. 
           [0027]      FIG. 19  is a top plan view of a shelf useable with the subject invention. 
           [0028]      FIG. 20  is a cross-sectional view taken along line  20 - 20  of  FIG. 19 . 
           [0029]      FIGS. 21 and 22  are enlarged views of Sections  21  and  22 , respectively, of  FIG. 20 . 
           [0030]      FIGS. 23 and 24  depict different containment wall configurations. 
           [0031]      FIGS. 25-27  depict caps useable with the subject invention. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0032]    With reference to the Figures, a vessel  10  for cell culturing is depicted. The vessel  10  may be utilized with various cell cultures, as will be appreciated by those skilled in the art. The vessel  10  generally includes a bottom  12 , a top  14 , a tubular neck  16 , having an opening  18  defined therein, and one or more shelves  20  located intermediate the bottom  12  and the top  14 . The bottom  12 , the top  14  and the shelves  20  collectively define an enclosed volume  22  for culturing cells. The tubular neck  16  extends from the vessel  10 , with the enclosed volume  22  being accessible by the opening  18  in the tubular neck  16 . 
         [0033]    The bottom  12  is generally tray-shaped with a base  24  from which upwardly extends a wall  26  which at least partially bounds the base  24 . Preferably, the wall  26  fully bounds the base  24  perimetrically. 
         [0034]    Depending on the overall desired size of the vessel  10 , one or more of the shelves  20  may be utilized. The greater the quantity of the shelves  20 , the greater the cell culturing capacity of the vessel  10 . Due to limits in gas flow and distribution, excessive size may not be desired. Embodiments utilizing two or four of the shelves  20  are envisioned. However, other quantities may be utilized. Each of the shelves  20  is generally tray-shaped having a base  28  with a wall  30  extending upwardly therefrom to at least partially bound the base  28 . Preferably, the wall  30  fully perimetrically bounds the base  28  for each of the shelves  20 . In addition, at least one aperture  32  is formed through the base  28  of each of the shelves  20 . Preferably, each of the shelves  20  includes at least two apertures  32 , a gas-transmission aperture  32   a  and a flow aperture  32   b.  As discussed further below, the gas-flow apertures  32   a  permit gas transmission through the shelves  20  so as to permit gas flow throughout the vessel  10 . The flow apertures  32   b  may be provided for equilibrating cell media disposed into the vessel  10  so as to distribute the cell media between the various support layers of the vessel  10  as defined by the bottom  12  and the shelves  20 . Although it is preferred to utilize a two-aperture arrangement for each of the shelves  20 , a single aperture  32  may be provided for each of the shelves  20  which may perform both functions of gas transmission and equilibration. Further, more than two apertures  32  may be utilized for one or both of the functions of gas transmission and equilibration. It is preferred that the gas-transmission aperture  32   a  and the flow aperture  32   b  of a respective shelf  20  be spaced apart. 
         [0035]    The top  14  has a generally inverted tray-shape with a base  34  and a wall  36  which extends downwardly therefrom to at least partially bound the base  34 . Preferably, the tubular neck  16  is formed integrally with the top  14  so as to extend therefrom. Alternatively, an opening may be formed in the top  14 , with the tubular neck  16  being formed as a separate component which is secured to the top  14  using any known technique, such as fusion or adhesive. It is further possible to form an opening which extends from the top  14  and into one or more of the shelves  20  to which the tubular neck  16  is attached. The opening  18  in the tubular neck  16  may be elliptical (including being circular) or semi-circular and may be formed with a constant or variable profile along the length of the tubular neck  16  (e.g., a portion of the opening  18  may be elliptical and a portion may be semi-circular along the length of the tubular neck  16 ). 
         [0036]    The bottom  12 , the top  14  and one or more of the shelves  20  are arranged in a stacked fashion so as to collectively form the vessel  10 . Preferably, the components are formed of a thermoplastic material which is compatible with the cells of interest. Polystyrene may be utilized. As will be appreciated by those skilled in the art, other materials may be utilized. The components may be formed clear, tinted (e.g., blue tint) or colored (e.g., amber). In addition, various portions of the bottom  12 , the top  14 , and the shelves  20  may be modified or treated so as to enhance certain conditions. For example, one or more biological agents may be applied to one or more portions of the bottom  12 , the top  14  and/or the shelves  20 , including, but not limited to, an extracellular matrix, or components thereof, such as laminin, fibronectin, and collagen, in any combination. In addition, or alternatively, synthetic agents may be applied. The surfaces may also be pre-treated, such as with tissue-culture treatment or plasma polymerization. As will be appreciated by those skilled in the art, these various treatments or modifications may be used in various combinations and utilized depending on intended objectives. 
         [0037]    The bottom  12 , the top  14  and the shelves  20  are adjoined to form the vessel  10 . In particular, the wall  36  of the top  14  is adjoined to the wall  30  of the shelf  20  located adjacent to the top  14 . All of the shelves  20  are located intermediate the bottom  12  and the top  14 . Adjacent shelves  20  are arranged in stacked fashion and adjoined with the wall  30  of the lower stacked shelf  20  being adjoined to the higher stacked adjacent shelf  20 . The bottom  12  is adjoined to the shelf  20  adjacent to the bottom  12 , which shall be the lowest stacked shelf  20  if a plurality of the shelves  20  is utilized, with the wall  26  of the bottom  12  being adjoined to the adjacent shelf  20 . Any known technique, such as fusion (e.g., by ultrasonic welding), adhesion, and/or mechanical interconnections (e.g., interlocking tongue and groove) may be utilized in any combination, so as to provide liquid-tight seals at the interfaces thereof. Sealing members, such as gaskets, may be interposed between adjacent components, such as between the top  14  and the adjacent shelf  20 . The gasket material may be chosen so as to be gas permeable, but liquid impermeable. In addition, one or more vented openings  37  may be formed in the bottom  12 , the top  14  and/or one or more of the shelves  20 . A gas permeable/liquid impermeable membrane  39  may be provided to extend across one or more of the vented openings  37 . 
         [0038]    The bottom  12 , the top  14  and the shelves  20  may be sized with various shapes to provide different surface areas for cell culturing. Accordingly, the vessel  10  may be formed with different dimensions and different shapes, such as a flask shape, as shown in the figures. Alternatively, the vessel  10  may have a rectangular shape, or be formed of other polygonal or other shapes. Preferably, the vessel  10  includes a first end  38  formed to support the vessel  10  in an upright position where the shelves  20  are aligned upwardly. This is a preferred state for loading cell media to achieve equilibration. More preferably, the first end  38  is located at an opposite location on the vessel  10  from the opening  18  of the tubular neck  16 . The first end  38  may be a flat surface or a locus of points which collectively define a resting surface. 
         [0039]    The bottom  12 , the top  14  and the shelves  20  form the vessel  10 . The walls  26 ,  30 ,  36  of the bottom  12 , the top  14  and the shelves  20 , respectively, and the bases  24 ,  28  of the bottom  12  and the top  14 , respectively, define portions of the exterior surface of the vessel  10 . 
         [0040]    To achieve equilibration, it is preferred that the flow apertures  32   b  be located in proximity to the first end  38 . In this manner, as shown in  FIG. 16 , with cell media C being disposed in the vessel  10 , and with the vessel  10  resting on the first end  38 , the cell media C may pass through the flow apertures  32   b  and achieve divided volumes (V 1 , V 2 , V 3 . . . ) in an equilibrated state between the bottom  12 , the shelves  20  and the top  14 . From the equilibrated state, the vessel  10  is caused to be placed horizontally resting on the bottom  12 , as shown in  FIG. 18 . The divided amounts of the cell media C are thus caused to spread across the supporting underlayer, which may be defined by the bottom  12  or the shelves  20 . 
         [0041]    Preferably, the cell media C is caused to equilibrate into equal volumes between the bottom  12 , the shelves  20  and the top  14 . Equal spacing, such as spacing z, between the bases  24 ,  28  and  34  of the bottom  12 , the top  14  and the shelves  20 , respectively, can cause equal volumes to be entrapped between the layers, particularly adjacent to the first end  38  of the vessel  10 . 
         [0042]    As shown in  FIGS. 13-14 , the base  34  of the top  14  may be formed generally planar throughout. To permit good pipette access, the base  34  of the top  14  may have portions thereof spaced from the base  28  of the adjacent shelf  20  greater than the spacing z found between the bases  28  of the shelves  20  or between the base  24  of the bottom  12  and the base  28  of the adjacent shelf  20 , as shown in  FIG. 14 . This increased spacing (spacing q) enhances pipette access through the entire length of the vessel  10  into contact with the first end  38 , as shown in  FIG. 17 . Thus, post cell culturing, and with the vessel  10  resting on the first end  38 , efficient recovery of cell media and cells may be conducted utilizing the pipette access. Increased spacing, however, between the base  34  of the top  14  and the base  28  of the adjacent shelf  20  may result in an enlarged volume being entrapped between the top  14  and the adjacent shelf  20  as compared to the volumes entrapped between the shelves  20  and the volume entrapped between the bottom  12  and the adjacent shelf  20 . 
         [0043]    Preferably, the base  34  of the top  14  is formed with first, second and third portions,  42 ,  44 ,  46 , respectively, which reduce the volume entrapped between the base  34  of the top  14  and the base  28  of the adjacent shelf  20  in proximity to the first end  38 . In particular, with reference to  FIG. 18 , the first portion  42  is located a distance x from the base  28  of the adjacent shelf  20 , the second portion  44  is located a distance y from the base  28  of the adjacent shelf  20 , with the distance x being greater than the distance y. The second portion  44  extends from the first end  38  so as to partially enclose the volume adjacent to the first end  38 . Preferably, the distance y is equal to the spacing z set between each adjacent pairs of the shelves  20  and between the bottom  12  and the adjacent shelf  20 . It is further preferred that the second portion  44  have a predetermined length L extending from the first end  38  which is equal to or greater than the length necessary to accommodate the target equilibrated volume. In this manner, as shown in  FIG. 16 , with the cell media C being equilibrated, the second portion  44  extends coextensively with or beyond the height of the cell media C (as equilibrated as volumes V 1 , V 2 , V 3 . . . ). It is preferred to have the height of the cell media C be below the third portion  46  in the equilibrated state. Height of the cell media C above the second portion  44  and into contact with the third portion  46  may result in uneven 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 , which includes access to the volume adjacent to the second portion  44  in proximity to the first end  38 , with the pipette passing through the greater height area adjacent to the first portion  42 . This arrangement also permits for equal volumes to be equilibrated between the various layers. The first and second portions  42 ,  44  may be each formed flat and may be arranged in parallel although other configurations (e.g., arcuate) are useable. 
         [0044]    The third portion  46  of the base  34  extends between and joins the first and second portions  42 ,  44 . Preferably, the third portion  46  is formed flat, but may be formed with other shapes, such as with an arcuate profile. Advantageously, the third portion  46  may define a suitable surface for printing or for bearing other indicia, such as bar coding. With respect to a plane defined by the second portion  44 , the third portion  46  is disposed at an angle α in the range of about 10-90 degrees, more preferably in the range of about 10-30 degrees, and more preferably at the angle α of about 20 degrees. The third portion  46 , as being disposed angularly relative to the second portion  44 , provides a tapered surface which directs the cell media C towards the first end  38  with the vessel  10  resting on the first end  38 . Although an angle α of 90 degrees is possible, it is preferred that the angle α be less than 90 degrees so as to prevent any cell media or cells being trapped at the intersection of the first and third portions  42 ,  46 . 
         [0045]    With the use of the second portion  44 , as shown in  FIG. 1 , a recess  48  may be defined in the vessel  10 . The wall  36  of the top  14  may partially bound the recess  48 . The recess  48  provides a handle function in exposing portions of the wall  36  in permitting gripping access thereto for a user. 
         [0046]    As shown in  FIG. 2 , it is preferred that the gas-transmission apertures  32   a  be in linear registration so as to define a gas flow channel  40  inside the enclosed volume  22 . Preferably, the gas flow channel  40  extends into proximity to the opening  18  defined in the tubular neck  16 . The gas flow channel  40 , by extending through the shelves  20 , permits gas flow to reach various portions of the enclosed volume  22 . 
         [0047]    As discussed above, and shown in  FIG. 18 , in a cell culturing use state, the vessel  10  is disposed to rest on the bottom  12 . In this position, the cell media C, due to its liquid nature, is disposed across the face of the base  24 ,  28  of the corresponding bottom  12  or shelf  20 . With respect to the bottom  12 , the wall  26  provides fluid containment in maintaining the cell media C atop the base  24 . With respect to the shelves  20 , to provide containment in preventing the cell media C from passing through any of the apertures  32  ( 32   a,    32   b ), a containment wall  50  may be disposed along the edge of each of the apertures  32  to act as a levee. Preferably, the height of the walls  30  of the shelves  20  is greater than the height of the containment walls  50 . In this manner, an open space can be maintained above each layer of the cell media C between the shelves  20 . 
         [0048]    The bottom  12  and the shelves  20  may be configured to handle different volumes of the cell media C, although it is preferred that within the same vessel  10 , the same volume is accommodated for each layer. The base  24  and the wall  26  of the bottom  12  define the volume for receiving the cell media C on the bottom  12 . The base  28 , the wall  30 , and the containment wall(s)  50  of each of the shelves  20  define the volume for receiving the cell media C thereupon. The bottom  12  and the shelves  20  may be configured to each accommodate a volume of the cell media C in the range of 4-50 mL. The surface area of the bottom  12  may be greater than the surface area of each of the shelves  20 , as the bottom  12  has none of the apertures  32  formed therein; as such, the height of the cell media C layer on the bottom  12  may have a slightly lower height than the height of the cell media C layers on the shelves  20 . The spacing z is set to take into consideration the amount of volume of the cell media C for each layer to ensure sufficient head-space above the layer of the cell media C for proper gas distribution. 
         [0049]    Concerns exist over the cell media C being wicked by the containment walls  50  into the corresponding apertures  32 . It is preferred that the containment walls  50  be bowed along an arcuate longitudinal axis, as shown in  FIGS. 11, 15 and 19 . The bowed shape acts against capillary attraction and provides an anti-wicking effect to the containment walls  50 . The apertures  32  ( 32   a,    32   b ) may be formed with arcuate shapes with the containment walls  50  being disposed along edges thereof. In addition, as shown in  FIGS. 11, 15 and 19 , the apertures  32  may have various configurations. In particular, the gas-transmission apertures of  32   a  may be formed to extend across a portion of a side edge  52  of the shelves  20  ( FIGS. 11 and 15 ) or be formed to extend along the full length of the side edge  52  of the shelves  20  ( FIG. 19 ). 
         [0050]    The containment walls  50  may be provided with other anti-wicking features such as being angularly disposed relative to the base  28  of the respective shelf  20  ( FIG. 21 ); being provided with arcuate cross-sections ( FIG. 23 ); and/or, include non-smooth surfaces  53  facing away from the corresponding aperture  32 . The non-smooth surface  53 , as shown in  FIGS. 20-24 , may include various surface interruptions or protrusions, such as bumps, dimples, roughened areas, striations, and the like. The anti-wicking features are intended to disrupt capillary attraction. In addition, or alternatively, the containment walls  50  may be made or prepared to have hydrophobic portions to repel the cell media C. For example, free edges  54 , at which the containment walls  50  terminate, may be prepared to be hydrophobic. Other portions of the containment walls  50  may be likewise treated. To further enhance this effect, portions of the bases  28  of the shelves  20  may be formed hydrophilic so as to enhance the retentiveness of the cell media C on the bases  28  of the shelves  20 . These various anti-wicking features may be used in various combinations. 
         [0051]    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 different angular orientations relative to the vessel  10 . As shown in  FIG. 2 , the tubular neck  16  extends along a longitudinal axis which is disposed at an angle β relative to a plane defined by the base  28  of the uppermost shelf  20 . The angle β may be in the range of about 0 degrees-90 degrees. At 0 degrees, the tubular neck  16  protrudes from a side of the top  14 , as shown in  FIG. 2 . At 90 degrees, the tubular neck  16  has a vertical orientation and protrudes from the base  34  of the top  14 , as shown in  FIG. 25 . With this arrangement, pipette access may be provided through the gas flow channel  40  to the volume located adjacent to the base  24  of the bottom  12  for cell media or cell removal. The angle β may be alternatively an acute angle. 
         [0052]    It is preferred that the vessel  10  be formed to be stackable with other vessels. To achieve such stacking, it is preferred that the top  14  define an upper resting surface which is parallel to a resting surface defined by the bottom  12 . In this manner, two or more of the vessels  10  may be stacked with the bottom  12  of the upper-stacked vessel being supported by the top  14  of the lower-stacked vessel  10 . To enhance stability, a bead  56  may be defined on the exterior surface of the bottom  12 . Correspondingly, the wall  36  of the top  14  may be formed to slightly protrude from the base  34 . The wall  36  may be formed to nestingly receive the bead  56  to provide lateral stability in a stacked configuration. It is noted that the wall  36  of the top  14 , particularly by protruding from the base  34 , may be configured to primarily bear the weight of the upper-stacked vessel  10 . Instability issues due to the existence of the recess  48  may thus be avoided. 
         [0053]    The bases  28  of the shelves  20  are preferably formed to be flat. Alternatively, the bases  28  may be formed with a rippled, wavy, or other configuration to increase surface area. With any configuration, it is preferred that the layers of the cell media C supported by the shelves  20  be disposed to be parallel with the vessel  10  resting on the bottom  12 . The base  24  of the bottom  12  may be likewise configured to be rippled, wavy or otherwise formed to have increased surface area. With any configuration, it is preferred that the layer of the cell media C supported by the bottom  12  be parallel to the other layers of the cell media C. 
         [0054]    The vessel  10  may be provided as an assembly with a corresponding cap  58  formed to mount onto the tubular neck  16  so as to seal the opening  18 . Any known arrangement for permitting mounting, such as a friction-fit, interference-fit, threaded or bayonet mounting, may be utilized. The cap  58  may be formed solid so as to be non-vented. Alternatively, the cap  58  may be provided with one or more vents  60  which may include gas permeable/liquid impermeable membrane  61 . Further, the cap  58  may include one or more adjustable valves  62  which permit selective aseptic connection between the enclosed volume  22  and an external fixture. Further, the cap  58  may include one or more tube connections  64  to permit direct and continuous communication with one or more supply tubes T (e.g., gas supply tubes). 
         [0055]    In use, the vessel  10  is set upon the first end  38  and a sufficient amount of the cell media C is introduced into the enclosed volume  22  via the opening  18  to provide target volume amounts for the bottom  12  and each of the shelves  20 . A pipette may be used to introduce the cell media C with the pipette being inserted through the opening  18  and into the enclosed volume  22  in between the top  14  and the adjacent shelf  20 . The introduced cell media C collects in the volume adjacent to the first end  38  and equilibrates between the flow apertures  32   b  to provide divided volumes of the cell media C corresponding to the bottom  12  and each of the shelves  20 . Once equilibrated, the vessel  10  is adjusted to rest on the bottom  12  with the cell media C dispersing across the bottom  12  and each of the shelves  20 . The cap  58  may be mounted to the tubular neck  16  to seal the opening  18 . Thereafter, the vessel  10  may be placed in an incubator. For transportation, it is preferred that the vessel  10  be held upright with the cell media C being accumulated in the enclosed volume  22  adjacent to the first end  38 . A plurality of the vessels  10  may be stacked during incubation. 
         [0056]    To extract the cell media C for exchange or cell harvesting purposes, the vessel  10  may be set upon the first end  38 . A pipette, or other extraction device, may be introduced to extract the cell media C from adjacent the first end  38 . This permits thorough removal. The pipette may be introduced into the enclosed volume  22  in between the top  14  and the adjacent shelf  20  to access the volume adjacent to the first end  38  for extraction. Under negative pressure, the cell media C may be caused to be drawn through the flow apertures  32   b  to the volume adjacent the top  14  for extraction therefrom. Alternatively, the cell media C may be caused to be poured through the opening  18 . Trypsin, or other disassociation agents, may be utilized to release cells for harvest.