Abstract:
An apparatus to facilitate precise and efficient evaluation of biomaterials using direct contact cell culture techniques. The apparatus positions the biomaterial and creates the potential to form a fluid-tight seal between the biomaterial and the apparatus, at which point the biomaterial is exposed to cells and/or media. An assay method based on the apparatus is claimed.

Description:
RELATED APPLICATIONS  
       [0001]    This application claims the benefit of U.S. Provisional Patent Application No. 60/294,536 filed May 30, 2001 and which is incorporated herein by reference. 
     
    
     
       BACKGROUND OF THE INVENTION  
         [0002]    1. Field of the Invention  
           [0003]    This invention is directed towards a laboratory device that facilitates studies using cell culture techniques to assay biomaterials. Specifically this invention is a device that facilitates control of the exposure of animal cells and/or media to biomaterials and the measure of the main and interaction effects of the cells, tissue and media on the biomaterials. By way of example, but not as a limitation, the device can be used to evaluate biomaterial toxicity or drug release from films. The laboratory device also facilitates the recovery of biomaterials, cells, tissues, and/or the cell-material interface following controlled experiments.  
           [0004]    2. Background  
           [0005]    Technology related to the continued development of medical devices for humans comprises two fundamental areas of research and development: design and fabrication of said devices and development of minimally toxic, biologically compatible materials (biomaterials) to be used in the manufacture of said medical devices. Safety and health considerations require that the potential of toxic effects of biomaterials that are otherwise suitable for medical devices must be fully evaluated, and performance considerations require that the material maintain its function in an in vivo environment. Devices to facilitate cell culture and study are known in the art as shown and claimed in U.S. Pat. Nos. 5,578,492 and 5,139,951, which are hereby incorporated by reference in their entirety.  
           [0006]    Direct contact cell culture is employed to evaluate biomaterial reactions and interaction of cells with a biomaterial. Evaluation includes toxicity, drug delivery, or material degradation analysis. Such studies require a laboratory apparatus that supports cellular growth, allows cell cultures to be exposed to known amounts of biomaterials, and to be handled for study purposes which includes observation of cells, sampling materials and media, changing media, and moving samples into and out of controlled environment facilities while protecting samples from contamination. Additionally, such evaluation apparatuses must provide a container which provides surfaces to support cellular growth.  
           [0007]    Details of the preparation of media and methods of culture of cells are well known and comprehended by those skilled in the art. Specific environmental conditions including factors such as minimizing contamination of cultures and maintaining controlled temperature, humidity, and light conditions are common to all studies although specific conditions of light, temperature, and humidity may vary with the material to be tested. Nonetheless, the specific conditions are well known to those skilled in the art or are otherwise readily available without the need for excessive experimentation. See for example, R. I. Freshney, “Culture of Animal Cells”, 2 nd  ed., Wiley/Liss, 1994, N.Y., N.Y., which is hereby incorporated by reference in its entirety.  
           [0008]    With current technology, biomaterials may float or otherwise move during the study making precise observations more difficult. To minimize these issues, materials are commonly glued or weighted, which introduces additional complications. Additionally, current technology necessitates mechanical collection using a spatula or similar instrument to recover the cells from bioassay apparatus. Commonly, this results in damage to the cells thereby reducing the value of the cells for further analysis. These and related difficulties limit aspects of the accuracy and dependability of biomaterial assays. Accordingly, there remains room for variation and improvement in the art.  
         SUMMARY OF THE INVENTION  
         [0009]    It is an object of the present invention to provide a biomaterial assay apparatus and process which provides a stable, controlled surface for cell growth and study. Further the apparatus and process may expose the cells to only a single surface of the biomaterial. The fixed positioning of the biomaterial being evaluated minimizes damages to cell material and resultant experimental error. In addition, the apparatus is readily manufactured using injection-molding techniques as would be recognized by one skilled in the art.  
           [0010]    This and other objects of the invention are accomplished by a well-plate insert comprising a support platform and at least one cylinder that traverses and is connected to the platform. A portion of the cylinder extends below the platform and fits into a well of a multi-well plate. The distal end of the extended portion of the cylinder contacts the floor of the well and is capable of forming a fluid-tight seal with a biomaterial placed on the floor. The well-plate is positioned in a frame connected to the platform of the sleeve insert. The connection can be adjusted to increase a compressible force between the interface of the cylinder and biomaterial, thereby creating the potential for a fluid-tight seal between the biomaterial and cylinder and simultaneously preventing excessive movement of the biomaterial to be assayed. In this configuration, only a specified portion of the biomaterial is exposed to cell growth, and cells are protected from damage.  
           [0011]    Further, the invention includes a process for the assay of biomaterial, for using the growth of animal cells on the biomaterial as a bio-indicator of toxicity of the biomaterial. The process requires providing a container suitable for cell culture and placing a substantially flat sample of biomaterial on the floor of the container, followed by inserting a hollow, open-ended cylinder into the container with the distal end of the cylinder over and contacting the biomaterial throughout its full circumference. These steps are followed by applying compressible pressure on the cylinder thereby allowing a fluid-tight seal between the cylinder and biomaterial, followed by introducing animal cells and appropriate, supporting growth media to the cylinder and contacting the biomaterial with the cells and media, and next culturing the cells, followed by assaying the cells, and finally recovering the sample of biomaterial for additional study, assays, and observations. 
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0012]    [0012]FIG. 1 illustrates the well plate insert.  
         [0013]    [0013]FIG. 2 provides a cross-section view of a well plate and its relationship to the biomaterial and cylinder.  
         [0014]    [0014]FIG. 3 illustrates the base that supports the well plate and is connected to the sleeve insert.  
         [0015]    [0015]FIG. 4 provides a cross-section diagram of a well plate positioned in and supported by the base.  
         [0016]    [0016]FIG. 5 is a cross-section illustration of the platform connected to the base and the relationship of the well-plate insert, platform, cylinders, and connectors  
         [0017]    [0017]FIG. 6A illustrates adaptation of the cylinder to facilitate sealing biomaterial disks with a compressible O-ring positioned in the distal end of the cylinder.  
         [0018]    [0018]FIG. 6B illustrates an alternative adaptation to sealing biomaterials of different thicknesses by fabricating a portion of the cylinder with a compressible material, such as rubber.  
         [0019]    [0019]FIG. 7A illustrates a modified platform to accommodate a moveable cylinder.  
         [0020]    [0020]FIG. 7B provides illustrates modifications of a cylinder to permit unidirectional movement in a modified platform.  
         [0021]    [0021]FIG. 7C illustrates interlocking surface of the platform and cylinder.  
         [0022]    [0022]FIG. 8 illustrates adaptation of bioassay apparatus to biomaterials of different thicknesses.  
         [0023]    [0023]FIG. 9 illustrates the four major elements of a bioassay apparatus.  
         [0024]    [0024]FIG. 10 provides an angular top view of the nested bioassay apparatus.  
         [0025]    [0025]FIG. 11 provides a front view of the nested bioassay apparatus. 
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0026]    Detailed Description of the Figures  
         [0027]    [0027]FIG. 1 illustrates a well plate insert  1  with a platform  2  with a bottom surface  3 , a top surface  4 , a cylinder  5 , with an open longitudinal core traversing the platform  2  from the top surface  4  to the bottom surface  3 , and the cylinder  5  having a lower portion  7  extending below the platform  2 , a distal end  9 , a proximal end  8  and an outside diameter  6 . Apertures  13  are positioned for connectors  11  to physically connect the platform to the base  30 . The cylinder  5  traverses the platform  2  and is either molded as part of the platform  2  or secured to an aperture traversing the platform  2  (aperture not illustrated).  
         [0028]    [0028]FIG. 2 describes the spatial relationship of the lower portion of the cylinder  7 , the well-plate  21  with well  23  having a sample of biomaterial  25  positioned on the floor  24  of well  23 . The outer diameter  6  of the lower portion of the cylinder  7  is less than the diameter of the well  26  such that the the lower portion of the cylinder  7  can be inserted into the well  23  with the distal end of the cylinder  9  contacting the biomaterial  25  and creating the potential to form a fluid-tight seal at the point of contact  27 .  
         [0029]    [0029]FIG. 3 describes a rectangular base  30  capable of supporting a multi-well plate (as illustrated in FIG. 2) and of being connected to the platform  2  of the well plate insert  1  by means of threaded connectors  11 . The base  30  comprises a back piece  31 , a front piece  32 , and side pieces  33 . A ledge  34  is created by a groove on the interior of the front, side, and back pieces. The width  35  and length  36  of the ledge are determined by the corresponding dimensions of the well plate to be supported. Threaded apertures  10  are defined by the edge of the base, and positioned to align precisely with corresponding apertures  13  in the platform  2  to receive threaded connectors  11 .  
         [0030]    [0030]FIG. 4 illustrates a cross-section of the base  30  with a well-plate  21 . Well-plate  21 , including wells  23 , is illustrated here positioned on ledge  34  formed on sidewalls  33  of base  30 . Threaded apertures  10  are positioned to correspond to and align with apertures  13  of the platform  2 .  
         [0031]    [0031]FIG. 5 illustrates in cross-section the spatial and functional relation of the components. Well plate  21  is positioned on ledge  34  of side wall  33  of base  30 . Cylinder  5  is connected to platform  2  with lower portion of cylinder  7  extending below bottom surface of platform  3 . Biomaterial  25  is positioned on floor of well  24 . Distal end  9  of cylinder  5  is inserted in well  23  and contacts biomaterial  25 . Apertures  13  in platform  2  and threaded apertures  10  in base  30  align such that connectors  11  physically connect platform  2  and base  30 . Tightening connectors  11  creates the potential of a fluid-tight seal at the distal end of the cylinder  9 , between the biomaterial  25  and cylinder  5  by bringing well plate insert  1  relatively closer to base  30  thereby producing a compressive force on the interface  27  of the distal end  9  of the cylinder  5  and the biomaterial  25 .  
         [0032]    [0032]FIG. 6A illustrates a longitudinal cross section of the cylinder  5  adapted to position and hold a compressible gasket or O-ring  61  on the distal end of the cylinder  9 . A groove  63  to receive the O-ring  61  is formed in the distal end face of the cylinder  64 . The O-ring  61  fits into the groove  63  with approximately one-half of its thickness  65  exposed to form a seal with the biomaterial  25 . This creates the potential to form a fluid-tight seal between the O-ring  61  and biomaterial  25  when compressed as a result of the compressive connection joining platform  2  and base  30 .  
         [0033]    [0033]FIG. 6B illustrates the position of a compressible material as a segment of the lower portion of the cylinder  7 . Cylinder  5  traverses platform  2 , and lower portion of cylinder  7  extends below the bottom surface of the platform  3 . Any portion of the length  67  of the lower portion of cylinder  7  starting at point  66  of the lower portion of the cylinder  7  and extending towards the distal end of the cylinder  9  may be fabricated from a compressible material such as, but not limited to rubber. This portion  67  serves essentially the same function as the previously described function of O-ring  61 .  
         [0034]    [0034]FIG. 7A describes a modification of the platform  2  in which an opening  70  with a diameter  71  traverses the platform  2  from its top surface  4  through its bottom surface  3 . Opening  70  is defined by a wall  72  with horizontal, uniformly spaced ridges  73  formed on the surface of the wall  72 . One skilled in the art would recognize that, alternatively, the ridges  73  may be formed and characterized as threads.  
         [0035]    [0035]FIG. 7B describes modifications of cylinder  5  that permits only unidirectional movement of cylinder  5  through opening  70  in platform  2 . Uniformly spaced, horizontally parallel ridges  75  are formed over at least a portion of the outer surface  76  of the cylinder  5 . The ridges  75  are spaced and shaped to permit cylinder  5  to be inserted at the top surface  4  of platform  2  and to move downward. The configuration prevents opposite movement. One skilled in the art would recognize that, alternatively, the ridges  75  may be formed and characterized as threads that circumscribe the outer surface of the cylinder  76 . The threads are adapted to receive threads formed on the surface of wall  72 . In this configuration, the cylinder may be moved upward or downward by reversing the rotation of the cylinder as it is inserted in opening  70 .  
         [0036]    [0036]FIG. 7C details how relative movement of the cylinder  5  through opening  70  is restricted. When cylinder  5  is inserted in opening  70 , the flat surface  77  of ridge  75  formed on cylinder  5  contacts the corresponding flat surface  78  of ridge  73  formed on wall  72  of opening  70  in platform  2 . Opposing flat surfaces resist upward pressure, arrow  81 , of the cylinder  5  in relation to platform  2 . Corresponding beveled surfaces on the cylinder  79  and beveled surfaces on the platform  80  will allow downward movement of cylinder  5  through opening  70  in platform  2 . Thus, when platform is physically linked to base, and cylinder is inserted into a well, downward pressure relative to platform on cylinder can create a fluid-tight seal to be maintained between the distal end of the cylinder and biomaterial positioned on the floor of the well.  
         [0037]    [0037]FIG. 8 illustrates cylinders  5 A and  5 B traversing corresponding openings  70 A and  70 B in platform  2 . Distal ends  9 A and  9 B of corresponding cylinders are inserted into corresponding wells  23 A and  23 B of multi-well plate  21 . Platform  2  is connected to base  30  by connectors  11 . Samples of biomaterial  25 A and  25 B of different thicknesses are positioned in corresponding wells  23 A and  23 B. Interlocking ridges  73  and  75  formed on the adjacent, opposing surfaces of cylinders  5 A and  5 B and corresponding wall of opening  72 A and  72 B allow cylinders to be pressed downward so that contact is made with biomaterial samples. Biomaterial sample  25 B for illustrative purposes is thicker than biomaterial sample  25 A.  
         [0038]    [0038]FIG. 9 illustrates the four basic elements of a bioassay apparatus  100 . When assembled the units are stacked in a nested configuration. Base unit  30  serves as a rectangular frame capable of supporting multi-well cell plate  21 . By way of illustration, but not limitation, multi-well cell plate  21  comprises six wells  23 . The well plate insert comprises one or more cylinders  5  that traverse a platform  2  and are structurally attached to the platform. Edges of the platform further define a plurality of apertures  13 . Lid unit  90  rests on and covers the proximal ends  8  of the cylinders  5 .  
         [0039]    [0039]FIG. 10 describes and illustrates the relationship of the elements of a bioassay apparatus  100  from the perspective of an angular top. Multi-well plate  21  is nested into base  30 . Distal ends (illustrated as  9  in FIG. 1) of plurality of cylinders  5  are inserted into wells  23  of well plate  21 . Connectors  11  are inserted through apertures  13  and are threaded into threaded apertures  10  and tightening connectors  11  creates a compressive force at point of contact  27  of cylinder  5  and biomaterial  25 . Lid unit  90  fits over the proximal ends of cylinders (illustrated as  8  in FIG. 1) and fits nest fashion on platform  2 .  
         [0040]    [0040]FIG. 11 provides a face on view of bioassay apparatus  100 . Multi-well plate  21  is positioned on ledge  34  formed by groove in base  30 . Well plate insert  1  is positioned above multi-well plate  21  with cylinders  5  inserted into wells  23 . Connectors  11  are fully tightened producing a compressive force at interface of distal end of cylinder and biomaterial positioned on floor of well  23 .  
       EXAMPLE I  
       [0041]    As seen in reference to FIG. 9, the major elements of a bioassay apparatus  100  are the base  30 , a multi-well plate  21  with a plurality of wells  23 , a well plate insert  1  comprising a plurality of open ended, hollow cylinders  5  attached to a platform  2 , and a lid  90 . Details of these elements and their spatial and functional relationships are described in the following example and discussion of certain figures.  
         [0042]    As seen in reference to FIG. 3, a base  30  is provided which may be in the form of a rectangular frame. An outer margin of the frame can define a plurality of threaded apertures  10 . An upper surface of the base defines a ledge  34  formed by a notch or groove which further defines a receiving surface for a conventional multi-well plate  21  (as illustrated in FIG. 2).  
         [0043]    As seen in reference again to FIG. 9, multi-well plate  21  may be provided by a conventional six-well plate as are commercially available from, for example Fisher Scientific, Pittsburgh, Pa. 15275. While the illustrated embodiment provides for a six-well plate, the number, size, and spacing of the individual wells can vary. The ledge  34  on the interior of the base  30  is adapted for nesting with the lower rim of the multi-well plate  21 .  
         [0044]    As seen by FIG. 5 which represents the detail of only one of a plurality of wells  23  and associated elements of the assay apparatus, open-ended, hollow cylinder  5  traverses the platform  2  of the well plate insert  1 . Cylinder  5  is formed as part of, or attached to platform  2 . A lower portion  7  of cylinder  5  extends below the bottom surface of the platform  2 . The proximal end  8  (as illustrated in FIG. 1) of the cylinder  5  extends above the top surface of the platform  2 . Thus each of the plurality of cylinders  5  corresponds to one well  23  of the plurality of wells in a multi-well plate  21 , and the cylinder  5  allows access via the proximal end  8  of the cylinder  5  through the platform  2  to the distal end  9  of the cylinder  5 . The bottom edges of the multi-well plate  21  are nested within the groove of the corresponding edge of the base resting on and supported by the ledge  34 . A sample of biomaterial  25  is positioned on the floor of a well. The distal end  9  of the cylinder  5  is inserted into the well  23  and contacts the biomaterial  25 . Both the well-plate insert  1  and the base  30  further define a plurality of threaded apertures  10  which are vertically aligned when cylinders  5  are inserted into corresponding wells  23  in the multi-well plate  21  positioned on the base  30 , and the bioassay apparatus  100  (as illustrated in FIG. 9) is in a stacked configuration. Threaded connectors  11  inserted through the apertures  13  and  10  connect the well plate insert  1  and base  30  and provide a means of exerting a compressive force between these elements by tightening the connectors  11 . It is to be noted that FIG. 5 represents and illustrates only a single cylinder-well association in a cross-section view from the front of a bioassay apparatus  100 . Reference to FIG. 9 illustrates a configuration with six wells  23 , by means of example, not limitation.  
         [0045]    As seen in reference to FIG. 9, a lid  90  is provided having an upper surface and a lower surface. A lower surface of the lid  90  is surrounded by a protruding flange which extends around the perimeter of the lid  90 . As seen in further reference to FIG. 10, the lid has a similar size and shape to the platform  2 , which is adapted to engage the lid  90 . The inner surface of the lid  90  defines a plurality of circular ridges which correspond to the proximal ends  8  of each cylinder  5 . For purposes of this invention, it has been found that a conventional lid  90  of commercially available multi-well plates  21  may be used. Fisher Scientific, Pittsburgh, Pa. 15275.  
         [0046]    As best seen in reference to FIGS. 10 and 11, the assembled bioassay apparatus  100  uses the base  30  to engage a lower surface of a multi-well plate  21 . Next, the well-plate insert  1  (as illustrated in FIG. 5) is positioned over the multi-well plate  21 . As seen in the referenced figures, for each well  23  within the multi-well plate  21  a corresponding cylinder  5  can be provided and appropriately spaced so as to align each cylinder  5  with a corresponding well  23 .  
         [0047]    When so aligned, the apertures defined on the edges of the platform  2  (as illustrated in FIG. 5) and base  30  are aligned so as to receive a threaded connector  11  such as a bolt or screw. In this manner, the threaded connectors  11  can be used to apply a compressive force between the lower ends  7  of the cylinder  5  and the corresponding bottom portion of the multi-well plate  21 . The lid  90  may then be placed over the top surface  4  of the platform  2 , the lower surface of the lid  90  being in contact with at least the proximal end  8  of each cylinder  5 , which extends above the upper surface of the platform  2 .  
         [0048]    As best seen in reference to FIG. 5, the bioassay apparatus  100  can be used to test the compatibility of various biomaterials  25  as they are placed in contact with a test medium, which may contain living cells. For instance, a sheet of biomaterial  25  may be provided in which circular portions of a biomaterial  25  are cut and sized so as to be placed on the floor  24  of each well  23  of the multi-well plate  21 . Thereafter, when the well-place insert  1  is brought into engagement with the multi-well plate  21 , the engaging cylinder walls are placed in contact with the biomaterial  25 . As seen in reference to FIG. 6A, a lower edge of each cylinder wall can support a corresponding “O” ring  61  or similar flexible gasket-like material. When the gasket material of the lower sleeve wall is brought in contact with the biomaterial, a seal, which may be fluid tight, results. The use of the threaded connectors  11  helps maintain the necessary compressive force between the cylinder  5  and the biomaterial  25  which may provide and maintain a fluid-tight seal. While threaded connectors are illustrated in the preferred embodiment, it is recognized that there are alternative means of supplying a suitable compressive force between the cylinder  5  and the biomaterial  25 . For instance, spring-loaded clips could be used to secure the margins of the platform  2  to the base  30 . Likewise, clamps or other tensioning devices may be used to supply the necessary compressive force.  
         [0049]    For instance, by selecting the use of dense materials such as glass or dense plastics, the weight of the well-plate insert  1  could be sufficient to provide a necessary compressive force.  
         [0050]    As is readily appreciated by one having ordinary skill in the art, the amount of compressive force that needs to be supplied would vary depending upon the presence of a gasket or other sealing material. Additionally, some biomaterials may have sufficient physical properties that a seal can be formed without the necessity of a separate gasket. In addition, it is recognized that depending upon the texture and surface features of the biomaterial being assayed, a rough or textured material may require a more specialized gasket and/or increased compressive forces to bring about an effective seal. In specific cases a seal is not necessary or will not be possible. In these cases the insert will simply position the material.  
         [0051]    Once a seal has been established, a test medium, for example a population of cells and growth media may be introduced through the upper opening defining each cylinder and brought into contact with the biomaterial. In this manner, the biomaterial is maintained in intimate contact with the growth media and resident population of cells. The biomaterial is firmly held in place by the compressive forces of the cylinder walls. Accordingly, the biomaterial is immobilized which eliminates cell damage attributed to movement of the biomaterial.  
         [0052]    The above described embodiment is preferred in that it makes use of conventional and readily available multi-well assay plates. However, the process of carrying out the biomaterial assay can employ a variety of different apparatuses. For instance, a base unit may be provided in which a flat sheet of biomaterial is placed. A hollow cylinder-like structure may thereafter be brought into contact with the biomaterial so as to bring about a fluid-tight seal between the biomaterial and the engaging cylinder surface. An upper opening in the cylinder can provide an entry way for the addition of a cell culture and growth media. In this arrangement, a conventional assay plate is not needed in that the hollow cylinder is used to define an enclosure relative to the biomaterial which can contain the cells and media.  
         [0053]    The entire device may be made of any materials that tolerate sterilization. In a preferred embodiment, the material is polystyrene. The invention anticipates a variety of materials including, but not limited to, appropriate polymers, glass, and metals.  
       EXAMPLE II  
       [0054]    As seen by reference to FIG. 8, illustrating a well plate  21  with two wells  23 , the current invention may be adapted for the study of biomaterials  25  of significantly different thickness. The number of wells  23  is for illustration purposes and not as a limitation. As seen by reference to FIG. 7A, a cylinder  5  adapted with closely spaced ridges, teeth, or serrations  75  in horizontally parallel arrangement along part of its exterior surface. Reference to FIG. 7B illustrates corresponding structures. The serrations  75  circumscribing the walls  72  define an aperture  70  in the platform  2 . Reference to FIG. 7C illustrates how a cylinder  5  inserted into the aperture  70  moves downward, but the shape and structure of the serrations  75  on the opposing surfaces of the aperture  70  and cylinder  5  for a locking interface that allows downward movement and restricts movement upward. The seal is created by tightening the connectors. Any previously described modification to the cylinder to enhance sealing may be incorporated into the cylinders employed in this example. This example requires the use of individual lids for each cylinder. Common types of commercially available laboratory petri dish lids have been found to be suitable. Fisher Scientific, Pittsburgh, Pa. 15275.  
         [0055]    One of average skill in the art would recognize that the threads on the opposing faces of the cylinder and wall of the aperture in the platform could replace the serrations. In this configuration, the cylinder could be screwed into the aperture and depth adjusted in either an upward or downward direction. All other aspects of the invention remain as described, discussed and illustrated. In this configuration, the cylinder represents the male unit and the aperture the female unit. Regardless of the configuration, as can be inferred by reference to FIG. 7C, the sensitivity of the adjustment is a function of the space between serrations or the number of threads per centimeter of length of the cylinder or aperture.  
       EXAMPLE III  
       [0056]    As seen in reference to FIG. 6B, a segment of the lower portion  7  of the cylinder  5  can be fabricated with a compressible material such as rubber. This adaptation serves the same function as the interlocking ridges and moveable cylinders, which is to accommodate biomaterials of different thicknesses. Thus, the modification described in FIG. 6B is appropriate for uses described in Examples I and II.