Cell potential measurement apparatus having a plurality of microelectrodes

Abstract

A cell potential measurement apparatus, which uses a planar electrode enabling a multi-point simultaneous measurement of potential change arising from cell activities, is provided which can conduct measurements accurately and efficiently as well as can improve convenience of arranging measurement results. According to the configuration of the cell potential measurement apparatus of this invention, it includes an integrated cell holding instrument, which includes a planar electrode provided with a plurality of microelectrodes arranged in a matrix form on the surface of a substrate, a cell holding part for placing cells thereon, drawer patterns from the microelectrodes, and electric contact points for outside connections; an optical observation means for optical observations of cells; a stimulation signal supply means to be connected to the cell holding instrument for providing electric stimulation to the cells; and a signal processing means to be connected to the cell holding instrument for processing an output signal arising from electric physiological activities of the cells. It is preferable that a cell culturing means is also provided for maintaining a culture atmosphere of the cells placed on the integrated cell holding instrument.

What is claimed is:  
     
       1. A cell potential measurement apparatus for measurement of electrical physiological characteristics of cells, comprising: 
       (A) an integrated cell holding instrument provided with a plurality of microelectrodes arranged in a matrix form on the surface of a glass plate, said microelectrodes each having an electrode area of  4 × 10   2    μm   2    to  4 × 10     4    μm   2   ,  conductive patterns connected to the microelectrodes, electric contact points which are connected to edge parts of these conductive patterns, an insulation coating covering the surface of said conductive patterns, said microelectrodes being in electrical connection to a cell holding part which is constructed so as to contain cells and arranged in an area including said plurality of microelectrodes, and an electric connection means for providing an electric signal to said microelectrodes and for leading out an electric signal from said microelectrodes, said electric connection means including a half-split holder which has contacts touching said electric contact points and fixes said glass plate by holding the plate at the top and bottom of the plate;  
       (B) a stimulation signal supply means to be connected to the electric connection means of said integrated cell holding instrument for providing electric stimulation to said cells; and  
       (C) an optical observation means for observing the cells optically, and  
       ( D )    a signal processing means to be connected to the electric connection means of said integrated cell holding instrument for processing an output signal arising from electric physiological activities of said cells.  
     
     
       2. The cell potential measurement apparatus as in  claim 1 , further comprising an optical observation means for observing the cells optically. 
     
     
       3. The cell potential measurement apparatus as in  claim 1 , further comprising a cell culturing means for maintaining an environment for culturing cells which are placed on said integrated cell holding instrument. 
     
     
       4. The cell potential measurement apparatus as in  claim 3 , wherein the cell culturing means comprises a temperature adjustment means for maintaining a constant temperature, a means for circulating a culture solution, and a means for supplying a mixed gas of air and carbon dioxide. 
     
     
       5. The cell potential measurement apparatus as in  claim 1 , wherein said plurality of microelectrodes comprise 64 electrodes arranged in 8 columns and 8 rows. 
     
     
       6. The cell potential measurement apparatus as in  claim 1 , wherein said microelectrodes each have an electrode area of 4×10 2  μm 2  to 4×10 4  μm 2 . 
     
     
       7. The cell potential measurement apparatus as in  claim 1 , wherein said electric connection means fixes said half-split holder, and said apparatus further comprises a printed circuit board having an outside connection pattern which is connected to the contacts of said holder via a connector. 
     
     
       8. The cell potential measurement apparatus as in  claim 1 , wherein contact resistance of said electric contact points with said contacts and contact resistance of said contacts with said connector are both less than 30 m ohm. 
     
     
       9. The cell potential measurement apparatus as in  claim 1 , wherein said optical observation means comprises an optical microscope, and an image pick-up device and an image display device connected to the optical microscope. 
     
     
       10. The cell potential measurement apparatus as in  claim 9 , wherein said optical observation means further comprises an image storage device. 
     
     
       11. The cell potential measurement apparatus as in  claim 1 , wherein said stimulation signal supply means comprises a pulse signal generator. 
     
     
       12. The cell potential measurement apparatus as in  claim 1 , wherein said signal processing means comprises a multi-channel amplifier which amplifies a detection signal arising from cell activities and a multi-channel display device which displays an amplified signal waveform in real-time. 
     
     
       13. The cell potential measurement apparatus as in  claim 1 , further comprising a computer which outputs said stimulation signal via a D/A converter and receives and processes an output signal arising from electric physiological activities of said cells via an A/D converter, said computer controlling said optical observation means and said cell culturing means. 
     
     
       14. A method for the continuous, simultaneous measurement of electrical physiological characteristics of at least one neural sample, comprising the steps of: 
           1 . )  placing said at least one neural sample on a microelectrode array of a measuring device, which device comprises:    
       ( A )  an integrated neural sample holding instrument provided with said microelectrode array having plurality of microelectrodes arranged in a matrix form, conductive pathways connected to said microelectrodes, a neural sample holding part which is constructed to contain at least one said neural sample and an area including said plurality of microelectrodes, and said conductive pathways for providing electric stimulation signals to said microelectrodes and for leading out a responsive electric signal from said microelectrodes;    
       ( B )  a stimulation signal supply connectable to each said conductive pathways for providing electric stimulation to said neural sample; and    
       ( C )  a signal processor connectable to all of said conductive pathways of said integrated neural sample holding instrument suitable for processing said signals arising from electric physiological activities of said at least one neural sample and reflecting said signals as said complex waveforms,    
           2 . )  electrically stimulating at least one of said neural samples to cause an electrical response stimulus, and    
           3 . )  continuously simultaneously detecting said electrical response stimulus output signals arising from electric physiological activities of said neural sample and processing said signal to reflect said electrical, physiological responsive, complex waveform.   
     
     
       15. The method of  claim 14  further comprising the step of maintaining an environment for culturing said neural sample on said integrated neural sample holding instrument. 
     
     
       16. The method of  claim 15  further comprising the step of maintaining a constant temperature, circulating a solution, and supplying a gas supply to said neural sample holding part. 
     
     
       17. The method of  claim 14  wherein said plurality of microelectrodes comprise  64  electrodes arranged in eight columns and eight rows. 
     
     
       18. The method of  claim 14  wherein said microelectrodes each have an electrode area of  4 × 10   2    μm   2    to  4 × 10     4    μm   2 . 
     
     
       19. The method of  claim 14  wherein said electrical stimulation comprises a pulse signal. 
     
     
       20. The method of  claim 14  wherein said at least one neural sample comprises a section of a nerve organ. 
     
     
       21. The method of  claim 14  wherein said at least one neural sample comprises a nerve cell. 
     
     
       22. A method for the measurement of electrical, physiological response, complex waveforms in at least one neural samples, comprising the steps of 
           1 . )  placing at least one neural sample on a microelectrode array of a measuring device, which device comprises:    
       ( A )  an integrated, neural sample holding instrument provided with i. )  said microelectrode array comprising a plurality of microelectrodes arranged in a matrix form, ii. )  conductive pathways connected to the microelectrodes, and said conductive pathways for providing electric stimulation signals to said microelectrodes and for leading out an electric signal from said microelectrodes and iii. )  a neural sample holding part which is constructed to contain said at least one neural sample and include said plurality of microelectrodes,    
       ( B )  a signal processor connectable to said conductive pathways of said integrated neural sample holding instrument suitable for processing said signals arising from electric physiological activities of said at least one neural sample and reflecting said signals as said complex waveforms,    
           2 . )  stimulating more than one of said neural samples to cause an electrical response stimulus, and    
           3 . )  continuously, simultaneously detecting and processing said electrical response stimulus to reflect said complex waveforms.   
     
     
       23. The method of  claim 22  wherein the stimulation is an electrical signal from a stimulation signal supply connectable to all of said conductive pathways to said neural sample through said microelectrodes. 
     
     
       24. The method of  claim 22  further comprising the step of maintaining an environment for culturing said neural sample on said integrated neural sample holding instrument. 
     
     
       25. The method of  claim 22  further comprising the step of maintaining a constant temperature, circulating a solution, and supplying a gas supply to said neural sample holding part. 
     
     
       26. The method of  claim 22  wherein said plurality of microelectrodes comprise  64  electrodes arranged in eight columns and eight rows. 
     
     
       27. The method of  claim 22  wherein said microelectrodes each have an electrode area of  4 × 10   2    μm   2    to  4 × 10     4    μm   2 . 
     
     
       28. The method of  claim 23  wherein said electrical stimulation comprises a pulse signal. 
     
     
       29. The method of  claim 22  wherein said said at least neural sample comprises a section of a nerve organ. 
     
     
       30. The method of  claim 22  wherein said said at least neural sample comprises a nerve cell.