Abstract:
A flask for the culturing of cells is disclosed. The cell culture chamber is defined by top and bottom walls connected by side and end walls, one end wall shaped such that media can drain to a bottommost spot thereby allowing for the complete removal of media via a vertically oriented pipette inserted into the interior of the flask body through an open neck.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Ser. No. 61/731,842 filed on Nov. 30, 2012, the entire content of which is hereby incorporated by reference. 
     
    
     TECHNICAL FIELD 
       [0002]    The present disclosure relates generally to the cellular biological field and, in particular, to a cell cultivating flask. 
       BACKGROUND 
       [0003]    In vitro culturing of cells provides material necessary for research in pharmacology, physiology, and toxicology. The environmental conditions created for cultured cells should resemble as closely as possible the conditions experienced by the cells in vivo. One example of a suitable medium for culturing cells is a common laboratory flask such as demonstrated in U.S. Pat. No. 4,770,854 to Lyman. The cells attach to and grow on the bottom wall of the flask, immersed in a suitable sustaining media. The flask is kept in an incubator to maintain it at the proper temperature and atmosphere. 
         [0004]    Desirably, flasks are stacked together in an incubator and a number of cultures are simultaneously grown. Small variations in the growth medium, temperature, and cell variability have a pronounced effect on the progress of the cultures. Consequently, repeated microscopic visual inspections are needed to monitor the growth of the cells. As such, cell culture flasks are typically constructed of optically clear material that will allow such visual inspection. 
         [0005]    With the advent of cell-based high throughput applications, fully automated cell culture systems have been the subject of serious development work (see e.g. A Review of Cell Culture Automation, M. E. Kempner, R. A. Felder, JALA Volume 7, No. 2, April/May 2002, pp. 56-62.) These automated systems employ traditional cell culture vessels (i.e. common flasks, roller bottles, and cell culture dishes). These systems invariably require articulated arms to uncap flasks and manipulate them much like the manual operator. During such automated manipulation, it is often required to remove all media within a cell culture flask. Conventional flasks require angled manipulation of a pipette through the neck in order to fully remove all media. This limits the size of the pipette that may be employed. 
         [0006]    There is a need for a cell culture flask having a rigid structure that is capable of use with any number of conventional pipette sizes while being suitable for use in the performance of high throughput assay applications that commonly employ robotic manipulation. There is also a need for a cell culture flask that can be fully emptied with a pipette from a single position. 
       SUMMARY 
       [0007]    According to an illustrative embodiment of the present disclosure, a flask for the efficient culturing of cells is disclosed. The illustrative flask includes a unitary body including a bottom wall defining a cell growth area and a top wall, connected by side walls and end walls. For the addition and removal of media, the flask is equipped with a wide neck defining an opening or aperture allowing access to the body of the flask itself. A sloped shoulder region is included which tapers toward the neck and enables pouring. The end wall opposite the neck is configured in such a way as to enable media pooling at a point directly opposed to the neck opening. This allows the removal of media from a single point opposite the neck opening when the flask is oriented with the neck opening facing upward. In addition, the flask of the present disclosure is shaped and configured to enable robotic access to the flask interior without requiring cumbersome robotic arm manipulation. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0008]    The disclosure is best understood from the following detailed description when read with the accompanying drawing figures. It is emphasized that the various features are not necessarily drawn to scale. In fact, the dimensions maybe arbitrarily increased or decreased for clarity of discussion. 
           [0009]      FIG. 1  is a perspective view according to one embodiment. 
           [0010]      FIG. 2  is a bottom view according to one embodiment. 
           [0011]      FIG. 3  is a side view according to one embodiment. 
           [0012]      FIG. 4  is a view of the use of a pipette with an illustrative embodiment. 
       
    
    
     DETAILED DESCRIPTION 
       [0013]    In the following detailed description, for purposes of explanation and not limitation, exemplary embodiments disclosing specific details are set forth in order to provide a thorough understanding of the present disclosure. However, it will be apparent to one having ordinary skill in the art that the present disclosure may be practiced in other embodiments that depart from the specific details disclosed herein. In other instances, detailed descriptions of well-known devices and methods may be omitted so as not to obscure the description. 
         [0014]    The laboratory flask shown in the drawing includes a body  10 , a neck  12  and a threaded region  14  to receive a removable coverall screwcap. In this specification and when referring to top, bottom, sides, etc., the flask will be generally described in an orientation where the neck portion is in an approximate horizontal position and the bottom wall  16  facing the observer in  FIG. 1  is in contact with a flat surface such as a laboratory bench. However, it is of course understood that the flask is not limited in any way to that particular position. 
         [0015]    The body  10  has a bottom wall  16  and a top wall  18  that generally lie in parallel planes, and they are connected together by side walls  20  and  22 , and first and second end walls  24  and  26 . The neck  12  is integrally formed with the central section  28  of the second end wall  26 . The second end wall  26  also includes diverging sloping portions  30  and  32  which extend from the ends of the central portion  28  to the side walls  20  and  22 , respectively. The top wall  18  is essentially flat throughout its full extent. The bottom wall  16  throughout its major rectangular and arched portion  34  is flat and parallel to the top wall  18  while the remaining portion  36  of the bottom wall defines an inclined ramp from the neck  12  to horizontal, arched portion  34  of the bottom wall. The ramp  36  is disposed in an angle of approximately 15-30° with the horizontal while the margins of the ramp defined by the end wall portions  30  and  32  diverge from one another in a sloped arching fashion. 
         [0016]    In  FIGS. 1-3 , the rectangular arching portion  34  of the bottom wall  16  is shown to carry a downwardly extending bead  42  about its periphery, which functions as one part of a stacking facility provided in the flask to enable identical flasks to be stacked compactly and positively with one another. The other part of the stacking facility is in the form of an upwardly extending flange  44  formed about the edge of the top wall  18 . Because the plan dimensions of the top wall  18  slightly exceed the corresponding dimensions of the bottom wall  16 , when one flask is stacked upon another, the bead  42  on the bottom wall just fits within the flange  44  on the top wall. There is an interruption in the flange  44  to prevent a vacuum when stacked or placed on a wet surface and in no way affects the stacking facility. 
         [0017]    A sloping feature along the innermost portion of the first end wall  24  enables complete drainage and removal of media when the flask is arranged in position in which the neck  12  faces upward. In the embodiment disclosed and in the orientation illustrated in  FIGS. 1 ,  2  and  4 , the first end wall  24  is comprised of two opposed portions sloping downward at an angle of between 5-25° angle and together intersecting at an obtuse angle of less than 180 degrees, and in one embodiment between 150 and 175°. The focal point of the angle forms the bottommost point for liquid containment within the flask body in this orientation. As shown by  FIG. 3 , this focal point may take the form of a line  25 . In such a position, media will pool along the line at the bottom most portion  25  of the sloped end wall, which is located in direct vertical alignment with the center of the screw cap neck. For example, in  FIG. 4 , a pipette  11  is shown entering the flask through the neck  12  and engaging the lowermost point  25  in the sloped inner portion of first end wall  24 . In such a position, the neck opening can accommodate the largest possible size pipette and still be capable of draining the contents of the flask in its entirety. In other embodiments, there is both a slope in the x-axis as well as the y-axis of the first end wall thereby creating a single bottommost focal point in the end wall within which all remaining liquid within the flask body will pool. 
         [0018]    The neck portion may be straight or canted. The diameter of the opening defined by the neck may be any size. In one embodiment, the opening ranges from approximately 25-35 mm. A generally accepted standard size for cell culture flasks is approximately 30 mm diameter fitting to a 33 mm cap. 
         [0019]    Attached to the first end wall  24  are two feet  46 ,  48  that are shaped to accommodate any slope in the first end wall and will allow the flask to stand upright as demonstrated in the orientation displayed in  FIGS. 1 ,  2  and  4 . 
         [0020]    The flask is injection molded in two parts from a clear plastic material such as a polysterene. One part of the flask includes the bottom wall  16 , side walls  20  and  22 , end walls  24  and  26 , ramp  36  and neck  12 . The other part comprises the top wall  18 , a short skirt  60  that fits over the top edges of the side and end walls  20 ,  22 ,  24  and  26 , and a generally semi-circular collar  40  that surrounds the upper half of neck  12  as is shown in  FIGS. 1 and 2 . The collar  40  assists in positioning the top wall and skirt on the bottom part of the container when the two are cemented or otherwise secured together in sealed relationship. The collar  70  also serves to strengthen the connection between the neck  12  and the second end wall  26 . It will be appreciated that a slight draft is provided in the side walls  20  and  22  and end walls  24  and  26  to facilitate removal of the lower part of the container from the mold during manufacture. This in turn results in the slightly larger surface for top wall  18  so as to provide a firm seat for the bottom wall of another flask when one is stacked upon another. The parts are held together and are adhesive bonded along the seam, ultrasonically welded, or scan welded. Preferably, scan welding equipment is utilized in a partially or fully automated assembly system. The two parts are properly aligned while a scan weld is made along the outer periphery of the joint. For flasks made with polystyrene, the thickness is preferably greater than 0.5 mm and more preferably greater than 1 mm. This thickness ensures that the flask bottom wall be perfectly flat, which in use provides a durable surface that will readily attach a uniform cell layer. Although not required, for optical clarity, it is advantageous to maintain a thickness of no greater than 2 mm. 
         [0021]    Advantageously and in order to enhance cell attachment and growth, the surface of the bottom wall is treated to make it hydrophilic. Treatment may be accomplished by any number of methods known in the art which include plasma discharge, corona discharge, gas plasma discharge, ion bombardment, ionizing radiation, and high intensity UV light. Although cell attachment is typically targeted for only one surface (the inner potion of the bottom wall), other parts of the flask may be treated so as to enable cell growth on all surfaces of the flask interior. 
         [0022]    The configuration of the flask provides several advantages. The fluid collection area enables a serological pipette to access the flask through the opening and since the low spot in the first end wall is located directly opposite the opening in the neck, no manipulation of the pipette is required to fully empty the flask of media. Pipette size will range with flask size, but generally, the following pipette sizes may be used with the relative associated flasks:
       25 cm2 with pipettes 10 ml, 5 ml, 2ml, 1 ml   75 cm2 with pipettes 50 ml, 25 ml, 10 ml, 5 ml, 2 ml, 1 ml   150 cm2 with pipettes 50 ml, 25 ml, 10 ml, 5 ml, 2 ml, 1 ml   175 cm2 with pipettes 100 ml, 50 ml, 25 ml, 10 ml, 5 ml, 2 ml, 1 ml   225 cm2 with pipettes 100 ml 50 ml, 25 ml,10 ml, 5 ml, 2 ml, 1 ml       
 
         [0028]    Such an arrangement also has benefits in automated cell growth procedures since conventional robots are more capable of a vertical pipette insertion and less prone or capable of angular pipette manipulation. 
         [0029]    Finally, a cap (not shown) is provided, in one embodiment having a septum that is integral with the cap top. This will allow a cannula, tip or needle to access the contents of the flask without the need for unscrewing. The septum is leak proof, puncturable and capable of resealing once the needle, tip or cannula is removed from the flask, even after multiple punctures. 
         [0030]    In use, the flask of the current disclosure is employed according to accepted cell growth methods. Cells are introduced to the flask though the threaded neck. Along with the cells, media is introduced such that the cells are immersed in the media. The flask is arranged such that the cell containing media covers the cell growth surface of the bottom wall. It is important not to completely fill the flask so as to allow for proper oxygenation of the media and cells. The flask is then placed within an incubator and maybe stacked together with similar flasks such that a number of cell cultures are simultaneously grown. The flask is situated such that the bottom wall assumes a horizontal position that will allow it to be completely covered by media. Cell growth is monitored from time to time by microscopic inspection through the bottom wall. During the cell growth process, it may become necessary to extract the exhausted media and insert fresh media. As previously described, media replacement may be achieved through insertion of a pipette, for example, through the opening in the neck. Once the cells are ready for harvesting, a chemical additive such as trypsin is added to the flask through the opening in the neck. The trypsin has the effect of releasing the cells from the flask walls. The cells are then harvested from the flask. 
         [0031]    Being thus described, it would be obvious that the same may be varied in many ways by one of ordinary skill in the art having had the benefit of the present disclosure. Such variations are not regarded as a departure from the spirit and scope of the disclosure, and such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims and their legal equivalents.