Abstract:
A device and method for analyzing cells includes a housing with a chamber, a barrier supported by a frame disposed within the chamber, and a plate arranged at a bottom surface of the housing interior of the chamber. The plate is adapted to receive and sustain cells and the barrier separates the plate into at least two contiguous separate areas. In some embodiments, a thin rubber strip is arranged at the bottom edge of the barrier, which facilitates control of the area in which each cell type is grown, the size of the gap between the cells, and helps prevents over growth of the two cell types on to each other.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to a device and method for growing and analyzing cells, e.g., in at least two areas on a cell culture separated by a removable barrier. 
       BACKGROUND INFORMATION 
       [0002]    It is conventional to conduct studies of the interrelationships between two or more cell types grown in the same culture dish or between two regions of the same cell type that have been differentially treated, e.g., by over expression of a gene in one region only. Pijnappels et al. describe physically disrupting an existing monolayer of one cell type and then adding a second layer of another cell type over the disrupted area. Pijnappels et al., “Progressive Increase in Conduction Velocity Across Human Mesenchymal Stem Cells Is Mediated by Electrical Coupling,”  Cardiovasc Research  (2006), doi:10.1016/j.cardiores.2006.07.016. 
         [0003]    There is believed to be a need for device and method for studying the interrelationships between two or more cell types grown in the same culture dish or between two regions of the same cell type that have been differentially treated without physically disrupting the cells. 
       SUMMARY 
       [0004]    An example embodiment of the present invention includes a housing with a chamber, a barrier disposed within the chamber, and a rubber seal adhered to a bottom surface of the housing where it contacts the plate. The plate is adapted to receive and sustain cells and the barrier separates the plate into at least two separate areas. 
         [0005]    A rubber strip may be placed between a bottom edge of the barrier and the plate. The rubber strip may be adhered to the bottom edge of the barrier. The rubber strip allows for the area in which each cell type is grown and the size of the gap between the cells to be controlled and over growth of the two cell types on to each other avoided. 
         [0006]    The plate may include a multichannel electrode array (MEA), for example, an MEA produced by Multi Channel Systems, in Reutlingen, Germany. 
         [0007]    The device may include a frame disposable within the chamber. The frame may include a slot adapted to accommodate the barrier. 
         [0008]    The device may include locking members adapted to frictionally engage and secure the frame within the housing. 
         [0009]    The locking members may be adapted to apply a downward force on the frame so as to create a seal between the frame and the plate. The downward force may be sufficient to prevent cell growth across the barrier but not high enough to leave an indentation on the plate. 
         [0010]    The device may include locking members adapted to frictionally engage and secure the barrier within the housing. 
         [0011]    The locking members may include rubber members adapted to be wedged between the housing and the frame. 
         [0012]    The locking members may include screws. 
         [0013]    The locking members may include flat set screws that pass through the housing at an angle and press against the frame forcing it downward against the plate. 
         [0014]    An exemplary device for use with a plate adapted to receive and sustain cells according to the present invention includes a removable barrier and housing with a chamber. The removable barrier may be disposed within the chamber, adapted to seal against the plate, and separate the plate into at least two contiguous separate areas. 
         [0015]    An exemplary method of the present invention includes: 
         [0000]    a) placing a barrier on a plate so as to divide the plate into at least two separate areas; b) forming cells on the plate on opposite sides of the barrier; c) removing the barrier; and d) detecting at least one parameter of the cells after removing the barrier. 
         [0016]    The at least one parameter may include an electrical activity of the cells. 
         [0017]    The barrier may be forced against the plate at a predetermined pressure, which, for example, may be sufficient to prevent cell growth across the barrier but not so high as to leave an indentation on the plate. 
         [0018]    A study may be conducted of the interrelationships between two or more cell types grown in the same culture dish or between two regions of the same cell type that have been differentially treated without physically disrupting the cells being analyzed. 
         [0019]    The time course interaction as the cell types grow together may be monitored. 
         [0020]    One cell type may be monitored while an adjacent cell type, on an opposite side of the barrier prior to its removal, is selectively exposed to a source or agent, which may have an effect on the cells, e.g., drugs. 
         [0021]    The conduction across and between multiple cell types may be measured, for example, using an MEA. 
         [0022]    The device may be used as a test system for experiments intended for the development and improvement of AV node bypass. 
         [0023]    Example embodiments of the present invention are described in more detail below with reference to the appended Figures. The foregoing description and examples have been set forth merely as illustrative and are not intended as being limiting. Each of the disclosed aspects and embodiments may be considered individually or in combination with other aspects, embodiments, and variations thereof. The steps of the methods described herein are not confined to any particular order of performance. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0024]      FIG. 1  is a top view of a device according to an exemplary embodiment of the present invention. 
           [0025]      FIG. 2  illustrates the device of  FIG. 1  with the locks, frame and barrier removed and placed to the side of the device housing. 
           [0026]      FIG. 3  is a perspective view of the device illustrated in  FIG. 1  with the frame and barrier removed. 
           [0027]      FIG. 4  is a perspective view of the divider and a magnified view of the rubber strip on the bottom edge of the divider. 
           [0028]      FIG. 5  is a perspective view of the frame illustrated in  FIGS. 1 and 2 . 
           [0029]      FIG. 6  is a grid including plots of the action potential of cardiomyocyte cells at multiple locations on an MEA plate 24 hours after removal of the barrier. 
           [0030]      FIG. 7  is the grid of  FIG. 6  but showing action potential plots of cardiomyocyte cells 72 hours after removal of the barrier. 
           [0031]      FIG. 8  is a perspective view of an exemplary embodiment of the housing including screws. 
           [0032]      FIG. 9  is a photograph of a device according to an exemplary embodiment of the present invention. 
           [0033]      FIG. 10  is a perspective view of an exemplary embodiment of the present invention not including a housing. 
       
    
    
     DETAILED DESCRIPTION 
       [0034]    As illustrated in the top view of  FIG. 1 , an exemplary embodiment the present invention, generally designated  10 , is designed to facilitate studies of the interrelationships between two or more cell types grown in the same culture dish, i.e., on plate  12 , or between two regions of the same cell type that have been differentially treated, e.g., by over expression of a gene in one region only. Monolayers of the studied cells are separated by a thin barrier  14  at the time of initial plating. The barrier is subsequently removed leaving in place a gap on the plate  12  between cells on the plate that is suitable to allow opposing groups of cells to grow across it and make physical contact with each other. 
         [0035]    The device  10  may be used to test the response of one cell type to pharmacologic agents administered only to the adjacent cell type, since the separation of the two cell types allows the use of a perfusion arrangement that restricts drug exposure to only a portion of the total area, even after the barrier  14  is removed. The device  10  may also be used for modeling of specific arrhythmia mechanisms, e.g., reentry. Such modeling can be used to facilitate an understanding of the likelihood that various interventions, including pharmaceuticals, will suppress or induce arrhythmias. 
         [0036]    A frame  16  is placed inside a housing  18 , which is placed over plate  12 . The housing  18  has recesses  20 , which accommodate locks  22  used to secure frame  16  to housing  18 . Locks  22 ,  22 ′ include an elongate member  24 ,  24 ′ capped on opposite ends with a high friction member  26 ,  26 ′, for example, made from rubber. Locks  22 , which may be adjustable in length, are sized to frictionally engage housing  18  to mechanically fix frame  16  in place in the housing  18 . Housing  18  also includes opposing rack members  28 ,  30  which include slots  32 ,  33  for receiving one or more of the barriers  14  at various positions along the length of the housing  18 . The use of more than one barrier  14  allows for the study of more than two kinds of cells. Locks  34 ,  34 ′ also made from a high friction material, such as rubber, are used to secure barrier  14  in place within slots  32  by frictionally engaging opposing members  28 ,  30  and side edges of barrier  14 . The frame  16 , housing  18 , and rack members  28 ,  30  may be made from conventional materials, for example, from a plastic such as polystyrene or polycarbonate. All parts of the device can be easily sterilized, e.g., using alcohol. 
         [0037]      FIG. 2  illustrates the device  10  in a disassembled state with the locks  22 ,  22 ′,  34 ,  34 ′ and frame  16  placed to the side of the housing  18 . As illustrated in  FIG. 5 , the frame  16  includes a slot  36  to accommodate the barrier  14  and is mounted to the plate  12  using a layer  19  of glue or sealing material, such as a 2 mm thick layer of Sylgard-184, for example, adhered to the frame  16  but not the plate  12 . The frame  16  restricts the area of the growing cells and prevents leakage of media and cells around the side edges of the barrier  14 . Any space between the barrier  14  and the frame  16  in slot  36  may be filled, for example, with a glue such as Sylgard-184. Frame  16  also allows for easy management of barrier  14  which, given its thin structure, may be difficult to hold and manipulate without damage. 
         [0038]    Housing  18  may be removably fixed to a separate support member, i.e., a table, or may be removably fixed to the plate  12  over a portion not used for cell growth. The housing  18  is fixed so that it can be used for leverage to create a downward force on the frame  16  to create a seal between the frame  16  and the plate  12 . The downward force may be created, for example, using locks  22 ,  22 ′ or using screws. Screws may be passed in an upward angled direction through a bottom of the housing into the frame effectively pulling the frame downwardly toward the plate  12 . Alternatively, flat tipped set screws may be passed through holes in the housing  18  so as to press against the frame  16  and bear down against it at a predetermined pressure so as to create a seal between the frame  16  and the plate  12 . A frame  16 ′ including set screws  46  is illustrated in  FIG. 8 . 
         [0039]    Plate  12  may be any substrate suitable for cell culture and may include a multichannel electrode array (MEA) system (Multi Channel Systems, Reutlingen, Germany). The use of an MEA allows for the measurement of conduction across and between multiple cell types. Alternatively, conduction across and between the cell types may be measured using voltage sensitive dyes or other methods for recording electrical events. 
         [0040]    Barrier  14  may be made from a glass sheet, e.g., a commercial 9×18 mm coverslip, for example, having thickness of 100 or 120 micrometers, or any other suitable rigid and thin material. Barrier  14  provides a seal on the surface of plate  12  to assure that the cell lines are separated. Barrier  14  may include a thin strip of softer material  38 , such as rubber, to assure that it does not scratch or otherwise alter the surface of the plate  12 . The thin strip of softer material  38  may be formed by covering a Teflon plate with a thin layer of sil-poxi-glue (silicone rubber adhesive, Smooth-On Inc., Easton, Pa.) and vertically mounting a bottom edge of the barrier  14  (the one which sits on the plate  12 ) on the thin layer of the sil-poxi-glue. After allowing the rubber to harden, e.g., for 15 to 20 minutes, the barrier  14  is removed from the Teflon plate by cutting the glue along both sides of the barrier  14  edge. This procedure may be repeated to increase the thickness of the rubber strip, for example, up to a thickness of 100 micrometers. The rubber strip adheres tightly to the barrier  14  thus allowing the barrier to be reused multiple times. 
         [0041]      FIG. 9  is a photograph of device  10  over an MEA shown without locking members  34 ,  34 ′. 
         [0042]    The device  10  may be used as follows. Cardiomyocyte cells are plated and cultured on an MEA plate  12  inside the housing  18  with the glass barrier  14  in place. The MEA is used to record the electrical activity of the cells, e.g., from sixty points in the culture area, for example, three days after the cells are plated and the barrier  14  is removed. Within, e.g., 24 hours after removing the barrier  14 , the two areas of cells on opposite sides of the barrier  14  may have different rate and timing of action potentials. Within, e.g., 72 hours after the barrier  14  is removed, both cell areas may have synchronized action potentials, which means that they develop physical and functional contacts.  FIG. 6  is a grid including multiple plots of the action potentials of the cardiomyocyte cells occupied by the cells 24 hours after removing the barrier  14  at 60 different locations along the plate  12 , and  FIG. 7  is similar grid including multiple plots of the action potentials 72 hours after removing the barrier  14 . Each box or cell of the grid in  FIGS. 6 and 7  (except for the corners) includes a plot of electrical activity, i.e., extracellular potential (y-axis), over time (x-axis). The grids include plots for each of the 60 monitored cell areas on the plate&#39;s multichannel electrode array. No electrical activity is observed in  FIG. 6  in the column labeled  41  since this column represents the location at which the barrier  14  is placed, indicating that cells do not bridge the gap after twenty four hours. 
         [0043]    In an exemplary embodiment of the device  10 ′ illustrated in  FIG. 10 , the frame  16 ′ may be connected or adhered to the plate  12 ′ without a housing, using, for example, screws, or as illustrated, using an adhesive  19 ′, such as Sylgard-184. The frame  16 ′ may be made, for example, from plastic. A spring  42 , for example, made from metal or fiberglass, is supported on a post  44  and used to bear pressure against the barrier  14 ′ so as to press it against the frame  16 ′ and the plate  12 ′ below. Alternatively, the frame  16 ′ may be made from Sylgard-184, which is set, for example, in a mold using a removable insert to form the slit for the barrier  14 ′ and the center space used for cell growth.