Abstract:
An electric field stimulation (EFS) device is provided for stimulating a plurality of cultured cells. The EFS device includes a transparent substrate, an insulator plate secured adjacent to the transparent substrate and having at least one well formed therethrough for containing the plurality of cultured cells, a surface of the transparent substrate defining a floor of the well, a first transparent electrode disposed on the surface of the transparent substrate for covering at least a portion of the floor, and a second electrode in electrical communication with the first transparent electrode. A voltage is selectively induced across the first transparent electrode and the second electrode for stimulating the plurality of cultured cells.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
         [0001]    This application claims the benefit of U.S. Provisional Application No. 60/299,913, filed Jun. 22, 2001.  
         FIELD OF THE INVENTION  
         [0002]    The present invention relates to screening candidate compounds and more particularly to electric field stimulation of cells for high throughput screening of candidate compounds.  
         BACKGROUND OF THE INVENTION  
         [0003]    Organic cells include cell membranes which isolate the cell interior from the surrounding environment. These membranes include channels, through which a cell is able to communicate with its environment. These channels are made up of single molecules, or complexes of molecules, and selectively enable the passage of ions therethrough. Over 50% of the pharmaceuticals produced today affect the opening and closing of the ion channels. Therefore, it is important for pharmaceutical researchers to be able to observe the affect of pharmaceuticals on the ion channels.  
           [0004]    Ion channels respond to a variety of stimuli including an applied bias across the cellular membrane. Therefore, to understand the operation of ion channels researchers often stimulate cells with an applied voltage. To achieve this, earlier devices included metal micro-electrodes patterned on a glass substrate to stimulate cells and record the subsequent electrical response. These configurations used gold (Au), titanium/platinum (Ti/Pt), or indium tin oxide (ITO) micro-electrodes, that were capped with black platinum (Pt) to reduce electrode impedance and facilitate recording by increasing the signal-to-noise ratio. These devices were used to study the complex signaling between neurons. In particular, these devices were optimized for monitoring one or a few cells, since the primary interest was in understanding the growth, adaptation and signaling between neural networks. These devices, however, were not wholly transparent, and were not optimized for uniform stimulation of multiple cells.  
           [0005]    Other devices enable high throughput screening (HTS) of pharmaceuticals and other compounds targeting ion channels. These devices include standard well-plate configurations, which include a modified substrate to electrically stimulate cells. A fluorescent, voltage-sensitive fluid is used to monitor the resulting change in transmembrane potential. These devices are designed to enable light to shine through the substrate, as the optical monitoring equipment of the HTS machines is located below the device.  
           [0006]    Three different device configurations for monitoring cell reactions have been previously employed. The first, the dipper electrode configuration, includes two or more electrodes dipped into the top of the wells, and between which a voltage is applied, horizontally across the well. The second is similar to the first, but includes satellite electrodes in an attempt to make the electric field more uniform. Both the first and the second electrode configurations do not modify the bottom surface of the well, and thus, a clear line of sight through the floor of the well is maintained. The third includes electrodes plated within the well, either to the wall of the well, or to a perimeter of the floor of the well. The electrodes in the third configuration are arranged so as to leave a clear line of sight through the floor of the well.  
           [0007]    Each of the above described configurations retain specific disadvantages. These disadvantages include (1) requiring cleaning of the electrodes between use, (2) occluding the well openings due to one or more electrodes, and (3) indirectly stimulating the monitored cells, resulting in an inefficient, non-uniform electrical field.  
           [0008]    Therefore, it is desirable in the industry to provide an improved HTS device for observing the affect of pharmaceuticals and other compounds on ion channels of organic cells. The device should enable direct, substantially uniform stimulation of the cells, and enable unobstructed optical monitoring thereof. The device should also preferably be configured to enable the passage of light from beneath the device to enable operation with standard HTS machines. Furthermore, the device should minimize the number of electrodes inserted into the well from above.  
         SUMMARY OF THE INVENTION  
         [0009]    Accordingly, the present invention provides an electric field stimulation (EFS) device for stimulating a plurality of organic cells contained therein. The EFS device includes a transparent substrate, an insulator plate secured adjacent to the transparent substrate and having at least one well formed therethrough for containing the plurality of cultured cells, a surface of the transparent substrate defining a floor of the well, a first transparent electrode disposed on the surface of the transparent substrate for covering at least a portion of the floor, and a second electrode in electrical communication with the first transparent electrode. A voltage is selectively induced across the first transparent electrode and the second electrode for stimulating the plurality of cultured cells.  
           [0010]    In accordance with one preferred embodiment, the second electrode is selectively positionable into the top of the well. The EFS device applies a voltage potential vertically, across the cells. In this manner, a substantially uniform voltage is applied, for stimulating the cells. The transparent electrode attached to the floor of the wells enables the passage of light therethrough for improved observation of the cell reactions. In this manner, existing HTS machines retain an unobstructed view of the cells.  
           [0011]    In accordance with an alternative preferred embodiment, the second electrode is also transparent and disposed on the surface of the transparent substrate for covering at least a portion of the floor. Preferably, the first transparent electrode and the second electrode include respective inter-digitated fingers. The EFS device applies a voltage potential between the electrodes resulting in electric field lines substantially horizontal to the well floor. In this manner, a substantially uniform voltage is applied, for stimulating the cells. The first and second transparent electrodes attached to the floor of the wells enable the passage of light therethrough for improved observation of the cell reactions.  
           [0012]    Further areas of applicability of the present invention will become apparent from the detailed description provided hereinafter. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. 
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0013]    The present invention will become more fully understood from the detailed description and the accompanying drawings, wherein:  
         [0014]    [0014]FIG. 1 is a top view of an EFS device in accordance with the principles of the present invention;  
         [0015]    [0015]FIG. 2 is a cross-sectional view of the EFS device of FIG. 1, along line  2 - 2 ;  
         [0016]    [0016]FIG. 3 is an expanded, cross-sectional view of a portion of the EFS device, as shown in FIG. 2, detailing operation thereof; and  
         [0017]    [0017]FIG. 4 is a detailed view of a single well illustrating an alternative electrode configuration.  
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0018]    The following description of the preferred embodiments is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses.  
         [0019]    With particular reference to FIG. 1, an electric field stimulation (EFS) device  10  is shown. The EFS device  10  includes a plurality of wells  12  formed therein for retaining organic cells, as described in further detail below. EFS device  10  includes ninety-six (96) wells  12  formed therein with center-to-center spacing and diameters corresponding to industry standards for micro-well plates. Further, per industry standard, the wells  12  are arranged in eight (8) rows by twelve (12) columns. It is anticipated, however, that although the number, size and configuration of the wells  12  is provided per industry standard, each may be varied in accordance with particular design requirements. More specifically, the EFS device can be formed to include 384 or 1536 wells  12 . The EFS device  10  is designed for operation with industry standard, high-throughput screening (HTS) machines, such as a fluorometric imaging plate reader (FLIPR®).  
         [0020]    [0020]FIG. 2 is a cross-sectional view of the EFS device  10  along line  2 - 2  of FIG. 1. The EFS device  10  includes a substantially transparent substrate  14 , preferably manufactured from glass or some other transparent material. The transparent substrate  14  includes a surface  16 , to which a transparent electrode  18  is attached. In one embodiment, the transparent electrode completely covers the surface  16  of the transparent substrate  14 . As described in further detail herein, other preferred configurations for the transparent electrode  18  are anticipated. By completely covering the transparent substrate, a common electrode is provided for each of the wells  12 , as described in further detail herein.  
         [0021]    In accordance with a preferred embodiment, the transparent electrode  18  comprises indium tin oxide (ITO). However, it will appreciated that the transparent electrode  18  may be made from any other electrically conductive, transparent material known in the art. An insulator plate  20 , having apertures  22  formed therethrough is then bonded to the transparent substrate  14  and transparent electrode  18  combination with a bio-compatible adhesive. In this manner, a circumferential wall  24  of each aperture  22  defines a wall of each well  12 , and the surface  16  defines a floor of each well  12 . The insulator plate  20  is preferably opaque to prevent light cross-talk between wells  12 .  
         [0022]    The transparent electrode  18  electrically communicates with a terminal  30  of an external power source  32  that is selectively attachable to the transparent electrode  18 . A movable robotic arm  34  includes a series of electrodes  36  extending downward therefrom, which are constructed from an opaque, bio-compatible material. The number of electrodes  36  generally corresponds with the number of wells  12  in a row or the number of wells  12  in a column. The electrodes  36  electrically communicate with another terminal  38  of the external power source  32 , and are dipped into the wells  12  for selective inducement of a voltage potential within the wells  12 . More specifically, the voltage potential is selectively induced across the electrode  36  and the transparent electrode  18 , vertically within each well  12 . In this manner, a substantially uniform voltage is applied through the well  12  for more effective stimulation of the cells. It will be appreciated that, although transparent electrode  18  is shown connected to the negative terminal of the power supply  32 , and electrode  36  is shown connected to the positive terminal of the power supply  32 , the opposite polarity connection can be employed. Further, both AC and DC voltages can be applied.  
         [0023]    The robotic arm  34  is a modified version of a standard HTS robotic arm. In particular, standard robotic arms include fluid dispensing conduits for simultaneously dispensing liquid into each well of a column or a row. However, the robotic arm of the present invention includes electrodes  36  for each well  12  of a row (i.e. 8 electrodes), as well as, one fluid dispensing conduit  40  for each well  12  of a row (i.e. 8 fluid dispensing conduits). In this manner, the robotic arm  34  is able to dispense fluid into each of the wells  12  and induce the voltage potential in each well  12 , along a particular row.  
         [0024]    As described above, the transparent electrode  18  electrically communicates with the power source  32  via a lead  42 . Alternatively, however, the transparent electrode  18  may electrically communicate with the power source  32  through an electrode  36 . More particularly, the eighth electrode  36  in the row may electrically communicate with the negative terminal  30  of the power source  32 , and be longer than the other electrodes  36 . As the robotic arm  34  dips the electrodes  36  into the wells  12 , the eighth electrode  36  contacts the floor (i.e. transparent electrode  18 ) of the eighth well  12 , to establish an electrical connection. In such a case, there are no cells cultured in the eighth well  12 . In this manner, a separate lead from the EFS device is not required, thereby simplifying the EFS device and usage thereof.  
         [0025]    In operation, a growth enhancing coating  50 , of a type known in the art, is preferably applied to the floor of the wells  12 . It will be appreciated, however, that the coating  50  may not be required. Organic cells  52  are selectively placed within the wells  12 , using a variety of methods known in the art. These cells  52  may be cultured within the wells  12  for a predetermined period of time. After the cells  52  have cultured for a predetermined period of time, a voltage sensitive, fluorescent fluid  54  is dispensed from the fluid dispensers  40  into the wells  12 . The fluid  54  interacts with the cells  52  for facilitating observation.  
         [0026]    A voltage potential is applied across the electrode  36  and the transparent electrode  18  for stimulating the cells  52 . In response to the applied voltage, a transmembrane potential of the cell changes. The fluorescent fluid  54  reacts to this change, itself changing fluorescence. In this manner, cellular reaction to the applied voltage can be observed. In particular, the effect of pharmaceuticals on the cell membrane ion channels can be determined. Pharmaceuticals, or other compounds may be added to observe their effect on the cells  52 .  
         [0027]    With particular reference to FIG. 4, an alternative preferred embodiment of the EFS device, indicated as  10 ′, will be described in detail. The EFS device  10 ′ includes a patterned, transparent electrode  18 ′. The transparent electrode covers a portion of the floor of a well  12 ′ (shown in phantom). More particularly, the transparent electrode  18 ′ includes a negative electrode portion  18   a′  and a positive electrode portion  18   b′  having inter-digitated fingers  60 ,  62 , respectively. The electrode portions  18   a′ , 18   b′  include respective leads  64 , 66  for interconnection with the power source  32 . Although only a single well  12 ′ is shown including the patterned transparent electrode  18 ′, it is anticipated that all of the wells  12  may include such a patterned, transparent electrode, interconnected to form a circuit.  
         [0028]    In operation, the configuration of FIG. 4, removes the need for an external electrode to be dipped into the well  12 ′. Instead, the electric field is induced between the inter-digitated fingers  60 ,  62 , horizontally across the floor of the well  12 ′. The inter-digitated fingers  60 ,  62  are formed of such a width and have such a spacing between the electrodes so that a cell  52  typical contacts two or more electrodes  18   a′ , 18   b′  (one positive and one negative). This results in very efficient stimulation of the cells. Different widths of electrodes  18   a′ , 18   b′  and spacing therebetween can be implemented for varying cell types having different average cell diameters.  
         [0029]    The EFS device  10  applies a voltage potential vertically across the cells  52 . In this manner, cells  52  in the cell layer are substantially uniformly stimulated. Alternatively, the EFS device  10 ′ applies a voltage between two electrodes on the floor of the well  12 , creating an electric field which is substantially horizontal with respect to the floor. In this manner, cells  52  in the cell layer are also substantially uniformly stimulated. In both configurations, the cells  52  are in direct contact with one or more electrode. Further, the transparent electrodes  18 ,  18 ′ covering the floor of the wells  12 , enables the passage of light therethrough for observation of the cell reactions. Therefore, existing HTS machines retain an unobstructed view of the stimulated cells  52 .  
         [0030]    The description of the invention is merely exemplary in nature and, thus, variations that do not depart from the gist of the invention are intended to be within the scope of the invention. Such variations are not to be regarded as a departure from the spirit and scope of the invention.