Patent Publication Number: US-6218178-B1

Title: Loading station assembly

Description:
RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/084,728, filed May 8, 1998 entitled “Loading Post Assembly”. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to an assembly positionable beneath a flexible cell culture substrate membrane that supports the membrane and allows the membrane to stretch across the assembly equally in all directions when vacuum is applied at the periphery of the membrane thereby imparting equibiaxial strain to the growth surface of the membrane and to a cell culture apparatus incorporating the assembly. Alternatively, other geometries of the assembly can be employed that yield uniaxial or gradient strain in the membrane. 
     2. Prior Art 
     Elastomeric substrates have been used as surfaces for culturing cells in vitro. These substrates are usually treated to make them hydrophilic and are preferably bonded with matrix proteins. This development arose from the desire to flex cell cultures in vitro instead of culturing cells in a static environment such as exists in conventional cell culture devices. In particular, when cell cultures are grown on membranes which are flexed, the cells are strained and stressed which simulates a dynamic in vivo environment. Stressing of cells causes dramatic morphologic changes and biomechanical responses in the cells which are both long term and short term. Cell culture devices using an elastomeric substrate or a flexible cell culture membrane are flexed or stretched to induce mechanical stress and strain on the cells adhering thereto. 
     Such flexible cell culture membranes have been incorporated in devices by the Applicant including the single well and multi-well cell culture plates and devices disclosed in U.S. Pat. No. 4,789,601 which is incorporated herein by reference. The wells of this cell culture plate have a floor made from a flexible membrane which is treated to enable cells to adhere and grow thereon. Typically, negative pressure, from a vacuum, is applied to the underside of the cell culture plate to deform downwardly the flexible membranes in the wells of the culture plate. 
     An improved device is the subject of Applicant&#39;s copending U.S. patent application Ser. No. 09/201,570, filed Nov. 30, 1998 entitled “Culture Plate for Applying Mechanical Load to Cell Cultures”, which is incorporated herein by reference. The device disclosed in the &#39;570 application is similar to that of the device in the &#39;601 patent in that it includes a multi-well culture plate having flexible cell culture membranes as the floors of the wells which may be subjected to negative pressure to downwardly flex the membrane. However, the membrane in the device in the &#39;570 application is fixed between a base and a body so that either positive or negative pressure may be applied thereto causing upward or downward flexing of the membrane. The pressure differential applied to either of these devices may be released and reapplied to repeatedly apply stress and strain to the cells, thereby mimicking the mechanical load experienced by cells in mechanically active tissues such as the heart, lungs, skeletal muscles, bone, ligament, tendon, cartilage and the like. In this manner, the biological or biochemical responses of cells subjected to a flexed environment may be tested and studied. 
     However, certain cells normally experience equibiaxial strain in the plane of the cell. By equibiaxial it is meant that the strain is applied equally along both the x-axis and y-axis of the plane of the cell. In some applications of membrane stretching induced by either positive or negative pressure where the membrane is not supported, the cell culture membrane becomes arcuately shaped upon application of a differential pressure to the device. Such arcuate flexing renders difficult an analysis of the biologic responses of the cells since the strain varies in both the circumferential and radial directions. The impact of uniaxial strain in the plane of a cell (a strain in one of the x-axis or y-axis) is likewise important and difficult to analyze. 
     Accordingly, a need remains for a device for use with a cell culture plate with flexible cell culture membrane wells which provides for well-defined strain to be imposed on cells cultured thereon, wherein the strain is applied equibiaxially, uniaxially or in other defined strain field directions. 
     SUMMARY OF THE INVENTION 
     This need is met by the loading station assembly of the present invention. The loading station assembly is designed to allow equibiaxial, uniaxial or other directional stretching of a flexible cell culture membrane. In equibiaxial stretching the membrane is maintained in a generally planar configuration while strain is applied equally in both the radial and circumferential directions or in one direction only. The assembly includes a planar member and at least one post extending from a surface of the planar member. An upper surface of each post is adapted to support the flexible cell culture membrane. The planar member defines at least one aperture and each post is removably received within one of the apertures such that when one of the posts is removed, the membrane is not restricted from flexing downwardly at the position of the absent post. Each post includes a body receivable within one of the apertures and a flange having the upper surface, wherein the flange is positionable against the planar member surface. 
     The upper surface may be configured to allow equibiaxial, uniaxial or other directional stretching of a membrane. For equibiaxial stretching, the upper surface is preferably circular in shape. For uniaxially stretching, the upper surface preferably has a length which is longer than its width. The upper surface may be formed in a horizontal plane or in a plurality of planes. The planar member also defines a passageway adapted to allow fluid to flow through from one side of the planar member to an opposite side of the planar member such that a pressure differential may be applied to the underside of the membrane. 
     The present invention further includes a cell culture plate assembly having a cell culture plate defining at least one opening and a flexible cell culture membrane covering the opening. A loading station assembly is positioned adjacent the cell culture plate. The loading station assembly includes a planar member and at least one post extending from a surface of the planar member. An upper surface of each post supports a portion of the flexible membrane covering the opening. The planar member defines at least one aperture and the post is removably received within one of the apertures such that when one of the posts is removed, the cell culture membrane covering an opening is not restricted from flexing downwardly. Each post includes a body receivable within one of the apertures and a flange including the upper surface. The flange is positionable against the planar member. The flange upper surface may be formed in a horizontal plane or in a plurality of planes. 
     Preferably, each of the flexible member and the loading station assembly is made from a transparent material. In one embodiment of the invention, the post upper surface supports all of the flexible membrane covering the opening. The upper surface may be configured to allow equibiaxial, uniaxial or other directional stretching of the membrane. For equibiaxial stretching, the upper surface of the flange is preferably circular in shape. For uniaxially stretching, the upper surface of the flange preferably has a dimension which is approximately equal to a dimension of the membrane. More preferably, the opening in the cell culture plate is circular and the length of the upper surface is approximately equal to the diameter of the opening and the width of the upper surface is less than the diameter of the opening. 
     The planar member also defines a passageway adapted to allow fluid to flow from one side of the planar member to an opposite side of the planar member. 
     A complete understanding of the invention will be obtained from the following description when taken in accordance with the accompanying drawing figures wherein like reference characters identify like parts throughout. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 is a plan view of a loading station assembly with a loading post and a planar member made in accordance with the present invention; 
     FIG. 2 is a side elevation view of the loading post depicted in FIG. 1; 
     FIG. 3 is a plan view of the planar member depicted in FIG. 1; 
     FIG. 4 is a cross sectional view of the loading station assembly shown in FIG. 1 taken along line  4 — 4 ; 
     FIG. 5 is an exploded perspective view of the loading station assembly shown in FIG. 1 in use with a cell culture plate with a membrane, a gasket and a cell culture base plate; 
     FIG. 6 is a perspective view of the cell culture plate shown in FIG. 5; 
     FIG. 7 is a cross sectional view of the cell culture plate shown in FIG. 6 taken along line  7 — 7  in relationship with the loading station assembly; 
     FIG. 8 shows the loading station assembly with cell culture plate shown in FIG. 7 upon application of negative pressure to the underside of the membrane; 
     FIG. 9 is a side elevation view of a second embodiment of the loading post; 
     FIG. 10 is a top view of the loading post show: 
     in FIG. 9; 
     FIG. 11 is an end elevation view of the loading post shown in FIG. 9; 
     FIG. 12 is a side elevation view of a third embodiment of the loading post; 
     FIG. 13 is a top view of the loading post shown in FIG. 12; and 
     FIG. 14 is an end elevation view of the loading post shown in FIG.  12 . 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     For purposes of the description hereinafter, the terms “upper”, “lower”, “right”, “left”, “vertical”, “horizontal”, “top”, “bottom” and derivatives thereof shall relate to the invention as it is oriented in the drawing figures. However, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. It is also to be understood that the specific devices and processes illustrated in the attached drawings, and described in the following specification, are simply exemplary embodiments of the invention. Hence, specific dimensions and other physical characteristics related to the embodiments disclosed herein are not to be considered as limiting. 
     The loading station assembly of the present invention is designed for use with a multi-well cell culture plate wherein the floors of the wells are formed from a flexible material. Suitable multi-well cell culture plates are those disclosed in U.S. Pat. No. 4,789,601 and U.S. patent application Ser. No. 09/201,570. For convenience, the loading station assembly is described hereinafter as used with the devices disclosed in the &#39;570 application, and this is only meant to be exemplary. It should be understood that the loading station assembly may be used with other multi-well cell culture plates having flexible-bottom wells which are not specifically described herein. It should be understood that the loading station assembly also may be used with single well cell culture plates or devices having flexible-bottom wells. 
     As shown in FIGS. 1-4, one embodiment of the loading station assembly  2  of the present invention includes a planar member  10  having a plurality of loading posts  12  extending therefrom. The planar member  10  defines a plurality of spaced apart apertures  14 . The loading posts  12  each include a column  16  which is releasably fitted into one of the apertures  14 . A head or flange  18  integral with the column  16  has a lower surface  20  which seats onto an upper surface  22  of the planar member  10 . An upper surface  24  of the flange  18  is adapted to contact a flexible cell culture membrane as described in detail below and preferably is chamfered as indicated at reference numeral  26 . 
     Preferably, the planar member  10  defines six apertures  14  which each receive a column  16 . The number of apertures  14  may vary and is selected to match the number of wells in a multi-well cell culture plate having flexible-bottom wells. 
     The planar member  10  also defines a central opening  28  which is adapted to allow fluid flow from one side of the planar member  10  to an opposite side of the planar member  10 . By the term fluid it is meant a gas (such as air) or a liquid. In particular, the space above the upper surface  22  of the planar member  10  may be evacuated through the central opening  28  when negative pressure is applied from below to the underside of the planar member  10  as described in further detail below. 
     As shown by example in FIGS. 5-8, the loading station assembly  2  is sized and configured for use with a multi-well culture plate  50  which is fitted into a gasket  60  receivable with a cell culture base plate  70 . FIGS. 7 and 8 show the assembled relationship between the loading station assembly  2  and the cell culture plate  50 . The culture plate  50  includes a base  51 , a body  52  and a flexible membrane  53  sandwiched therebetween. The body  52  includes a plurality of well walls  54  which define wells  55 . Floors  56  of the wells  55  are formed from the flexible membrane  53 . The loading station assembly  2  is sized and configured to be positioned in an underlying relationship to the cell culture plate  50  such that the upper surfaces  24  of the flanges  18  directly support and contact an underside of the flexible cell culture membrane  53  in the location of each well  55 . The entire underside of the floors  56  of the wells  55  are not covered by the flanges  18  but instead, as shown in FIG. 7, only a portion of the flexible membrane  53  is supported by the loading posts  12  beneath each well  55 . Preferably, the upper surface  24  of each flange  18  is centered on the underside of each floor  56 . Although the cell culture plate  50  is depicted as having circular shaped wells  55  and the upper surfaces  24  of the flanges  18  are depicted as being circular, the invention is not limited to these forms. Other geometrical configurations for all these elements may be employed. 
     The planar member  10  is adapted to abut against an underside of the base  51  of the cell culture plate  50 . When installed with the gasket  60  and cell culture base plate  70 , the planar member  10  engages with the underside of the base  51 . As an example, the cell culture base plate  70  includes recessed portions  71 , each being adapted to receive the gasket  60 , the loading station assembly  2  and the cell culture plate  50 . A bottom of each recessed portion  71  defines an opening  72 . The openings  72  communicate via a passageway (not shown) defined in the base plate  70  with ports  73  and  74 . Ports  73  and  74  are adapted to be connected to a vacuum or other source of negative pressure. 
     In operation, when negative pressure is applied to the ports  73  or  74 , a vacuum is drawn through the passageway and openings  72  to the underside of the planar member  10  and through the central opening  28 . The volume of space between the upper surface  22  of planar member  10  and the underside of the well floors  56  is at least partially evacuated. The flexible membrane  53  of each floor  56  is pulled in the direction of the planar member  10  as shown schematically in FIG.  8 . Because the upper surfaces  24  of the flanges  18  have smaller surface areas than the surface areas of the well floors  56 , only a periphery of the flexible membrane  53  within the wells  55  is pulled downwardly. In so doing, the membrane  53  is stretched in a plane across the upper surfaces  24  of the flanges  18  resulting in an equibiaxial strain on the flexible membrane  53 . Cells which are adhered to the flexible membrane  53  likewise experience equibiaxial strain. The vacuum may be applied once, intermittently, regularly or in a variety of frequencies to induce equibiaxial strain on the cells over time. The chamfered portion  26  of the flange  18  provides a guide against which the flexible membrane  53  may slide as the flexible membrane  53  is pulled toward the planar member  10  thereby minimizing wear on the flexible membrane  53 . 
     When the flexible membrane  53  is made from a transparent silicone material such a material produced by the Bisco Company under Product No. HT 6240, the loading station assembly is preferably formed from a transparent plastic material. In this manner, light is able to pass through the loading station assembly  2  and flexible membrane  53  so that cells adhering to the flexible membrane  53  are clearly visible. Additionally, such an arrangement also permits the use of a microscope therewith to view the cells while an equibiaxial strain or other strain is applied thereto since the cells remain in the same plane throughout the stretching paradigm. 
     Various strains or stresses on cells adhering to the flexible membrane  53  may be induced by employing loading posts having alternative geometrical shapes on their upper surfaces. For example, the plane of the upper 
     surface may be slanted, the flange of the loading post may be frustoconical or frustopyramidal in shape or the upper surface may have single or multiple peaks. Such various geometrical shapes of the loading post flange or upper surface of the flange induce different strains and/or stresses upon the cells adhered to the flexible membrane. 
     One such alternative loading post  112  is shown in FIGS. 9-11. The loading post  112  includes a generally rectangularly shaped flange  118  having a pair of slanted upper surfaces  124  terminating in a pair of ridges  126  and defining a groove  130  therebetween and a pair of vertical sides  128 . In use with a cell culture plate having wells with bottoms formed from a flexible membrane, the membrane is stretched across the groove  130  and along the slanted upper surfaces  124 . Ends  132  of the ridges  126  preferably are rounded to reduce wear on the membrane. Cells adhering to the portion of the membrane deformed over the slanted upper surfaces  124  experience strain as well as some shear stress due to movement of the cell culture fluid thereover. 
     Another alternative loading post  212 , shown in FIGS. 12-14, allows for uniaxial stretching of the membrane to induce uniaxial strain on cells attached thereto. The loading post  212  has an upper surface  224  with a length which is sized and configured to completely support the underside of the membrane  53  in one direction only. Ends  225  of the upper surface  224  are rounded to conform with the circular shape of the underside of the floor  56  of the well  55 . The distance between the ends  225  is approximately equal to the diameter of the opening of the well  55 , and the length of the upper surface  224  between the ends  225  is greater than the width of the upper surface  224  between sides  227 . In use, the membrane  53  is pulled downwardly under vacuum across the sides  227  of the upper surface  224 . The membrane  53  is not pulled downwardly over the ends  225  but is stretched generally only in the direction of the arrow A shown in FIG. 13 so that uniaxial strain in the direction of arrow A is experienced by cells attached to the membrane  53 . The width of the upper surface  224  can be varied according to the particular stretching paradigm desired. The loading post  212  may be rotated within the aperture  14  to induce uniaxial strain in various directions. For example, the loading post  212  may be used in uniaxially stretching a membrane  53  with cells attached thereto in a first direction. The loading post  212  may then be rotated by a preselected amount, e.g., 90°, and used in uniaxially stretching the membrane  53  again. The cells will have experienced strain in a pair of perpendicular directions. 
     The loading posts  12 ,  112  and  212  of the loading station assembly are interchangeable such that the loading posts positioned on the planar member  10  may have the same or different upper surface profiles. In addition, one or more of the apertures  14  may be vacant such that the membrane positioned thereover is permitted to flex arcuately when negative pressure is applied thereto. Alternatively, the surface area of the upper surface  24  of the flange  18  may be the same as the surface area of the well floor  56  such that negative pressure applied to the loading post  12  does not impact the flexible membrane  53  in the corresponding well  55  and the flexible membrane  53  is stationary. The cells adhering to the stationary flexible membrane  53  may be compared to cells which are actively strained. In such a manner, varying stresses and strains may be applied to cells cultured in different wells of the same cell culture plate by altering the configuration or presence of the upper surface of the loading post. The various geometric shapes of the loading posts may be used so that one membrane of one well in the culture plate is deformed over one type of loading post and then a different loading post is used for the same well. This effect can be achieved by rotating the loading posts from one position to another on the planar member. 
     In an alternative embodiment (not shown), the height of the loading post may be adjustable. For example, the loading post flange may be formed in two parts. An upper part includes an upper portion having an upper horizontal surface and a lower portion having a lower surface angled at about 45° to vertical. The angled lower surface mates with a lower part having a complementary angled upper surface. The upper and lower parts are positioned adjacent to each other along their complementary angled surfaces. The position of the upper part relative to the lower part determines the overall height of this loading post. 
     In another embodiment of the loading station assembly (not shown), an adjustable pin such as a screw or the like is received within the aperture  14  and is upwardly and downwardly moveable. The pin extends into the aperture and is positioned adjacent the loading post column. By moving the pin up or down, the post column is likewise upwardly or downwardly positionable. 
     Alternatively, a central plunger may be received within a central area of the column and is upwardly and downwardly extendable through the aperture in the loading post upper surface. By moving the plunger to various positions, the geometrical profile of the loading post against which the membrane bears may be altered. Likewise, a sleeve may surround the loading post and is upwardly and downwardly movable to change the profile of the loading post. 
     It will be readily appreciated by those skilled in the art that modifications may be made to the invention without departing from the concepts disclosed in the foregoing description. Such modifications are to be considered as included within the following claims unless the claims, by their language, expressly state otherwise. Accordingly, the particular embodiments described in detail herein are illustrative only and are not limiting to the scope of the invention which is to be given the full breadth of the appended claims and any and all equivalents thereof.