Cell fusion chamber, cell fusion device, and method for cell fusion using the same

Abstract

The present invention relates to a cell fusion chamber in which two types of cells having different diameters are fused, the cell fusion chamber including: a cell fusion region in which cell fusion is carried out; a pair of electrodes formed by a conductor and disposed opposite to each other in the cell fusion region; and a partition wall having at least one fine pore; near the fine pore, a cell fusion device including a cell fusion container containing a cell fusion region; a pair of electrodes; a spacer; and an insulator disposed between the spacer and one of the electrodes and having at least one fine pore; and an electronic power supply which applies an alternating voltage and a voltage pulsed direct current to the electrodes, and a cell fusion method using the same.

1 . A cell fusion chamber in which two types of cells having different diameters are fused, the cell fusion chamber comprising: 
 a cell fusion region in which cell fusion is carried out;    a pair of electrodes formed by a conductor and disposed opposite to each other in the cell fusion region; and    a partition wall disposed between the pair of electrodes to divide the cell fusion region into two compartments, the partition wall having at least one fine pore penetrating through the partition wall in a direction of the pair of electrodes, and the fine pore having a diameter no larger than that of the cell having a larger diameter than the other cell and no smaller than that of the cell having a smaller diameter than the other cell.    
     
     
         2 . A cell fusion chamber according to  claim 1 , further comprising: 
 an alternating-current power supply which applies an alternating voltage to the electrodes;    a direct current pulsed power supply which applies a voltage pulsed direct current to the electrodes; and    a switching device which connects the electrodes with the alternating-current power supply or the direct current pulsed power supply.    
     
     
         3 . A cell fusion chamber according to  claim 1 , further comprising: 
 a device which flows a suspension containing the cells into the cell fusion region.    
     
     
         4 . A cell fusion method using the cell fusion chamber of  claim 3 , comprising: 
 separately introducing two types of cells having different diameters into each compartments of the cell fusion region divided by the partition wall;    drawing the cells to the fine pore of the partition wall by applying an alternating voltage from the alternating-current power supply to the electrodes;    switching a connection with the electrodes from the alternating-current power supply to the direct current pulsed power supply using the switching device after drawing the cells;    fusing the cells by applying a voltage pulsed direct current from the direct current pulsed power supply to the electrodes; and    transferring the fused cell in a direction from the compartment into which the cell having a smaller diameter is introduced to the compartment into which the cell having a larger diameter is introduced.    
     
     
         5 . A cell fusion device comprising: 
 a cell fusion container comprising: 
 a cell fusion region in which cell fusion is carried out;  
 a pair of electrodes formed by a conductor and disposed opposite to each other in the cell fusion region;  
 a tabular spacer disposed between the pair of electrodes; and  
 a tabular insulator disposed between the spacer and one of the electrodes and having at least one fine pore penetrating through the insulator in a direction of the pair of electrodes; and  
   an electronic power supply which applies an alternating voltage or a voltage pulsed direct current to the pair of electrodes,    wherein the insulator is disposed on a cell fusion region side-surface of one of the electrodes.    
     
     
         6 . A cell fusion device according to  claim 5 , wherein the electronic power supply comprises: 
 an alternating-current power supply which applies an alternating voltage to the pair of electrodes;    a direct current pulsed power supply which applies a voltage pulsed direct current to the pair of electrodes; and    a switching device which connects the electrodes with the alternating-current power supply or the direct current pulsed power supply.    
     
     
         7 . A cell fusion device according to  claim 5 , wherein the fine pore has a shape which traps a single cell.  
     
     
         8 . A cell fusion device according to  claim 5 , wherein the electronic power supply applies to the electrodes an alternating voltage having a waveform fixing a single cell in each fine pore.  
     
     
         9 . A cell fusion device according to  claim 5 , wherein the electronic power supply applies to the electrodes an alternating voltage having a waveform periodically repeating charge and discharge of cells.  
     
     
         10 . A cell fusion device according to  claim 5 , wherein the electronic power supply applies to the electrodes an alternating voltage having a waveform including per half cycle thereof at least one plateau in which a predetermined voltage at a level other than 0 is maintained for a predetermined time.  
     
     
         11 . A cell fusion device according to  claim 5 , wherein the electronic power supply applies to the electrodes an alternating voltage having a waveform selected from the group consisting of a rectangular wave waveform, a trapezoidal wave waveform, and a waveform combining a rectangular wave with a trapezoidal wave.  
     
     
         12 . A cell fusion device according to  claim 5 , wherein the electronic power supply applies to the electrodes an alternating voltage having a waveform including a plateau in which a predetermined voltage at a level other than 0 is maintained for a predetermined time which is no shorter than a time constant calculated by multiplying a cellular capacitance by a resistance of a cell suspension containing the cells.  
     
     
         13 . A cell fusion device according to  claim 5 , wherein a diameter of a maximum circle inscribed in a planar shape of the fine pore is no less than equal to but less than twice as large as that of a cell to be fixed in the fine pore and a depth of the fine pore is no larger than the diameter of the cell to be fixed in the fine pore.  
     
     
         14 . A cell fusion device according to  claim 5 , wherein the insulator has plural fine pores in a face thereof, each fine pore being formed evenly spaced apart from adjacent fine pores.  
     
     
         15 . A cell fusion device according to  claim 5 , wherein the plural fine pores are formed in a face of the insulator in an array state.  
     
     
         16 . A cell fusion device according to  claim 14 , wherein a distance between the adjacent fine pores is no less than 0.5 times but less than 6 times as large as a diameter of a cell to be fixed in the respective fine pores.  
     
     
         17 . A cell fusion device according to  claim 5 , wherein the spacer comprises: 
 a through-hole forming the cell fusion region.    
     
     
         18 . A cell fusion device according to  claim 5 , wherein the spacer comprises: 
 an introducing channel through which a cell is introduced; and    an exhausting channel through which the cell is exhausted.    
     
     
         19 . A cell fusion method using the cell fusion device of any one of  claims 5  to  18 , comprising: 
 introducing a first cell into the cell fusion region;    fixing the introduced first cell in the fine pore by applying an alternating voltage to the electrodes;    introducing a second cell into the cell fusion region after fixing the first cell;    bringing the introduced second cell in contact with the first cell at the fine pore by applying the alternating voltage to the electrodes; and    fusing the first and second cells making contact by applying a voltage pulsed direct current.    
     
     
         20 . A cell fusion method according to  claim 19 , wherein the alternating voltage is an alternating voltage having a waveform described in any one of  claims 8  to  12 .  
     
     
         21 . A cell fusion method according to  claim 19 , wherein the first cell and the second cell are fused at a farthest position of the fine pore from the bottom of the fine pore.  
     
     
         22 . A cell fusion method according to  claim 19 , wherein the first cell has a diameter smaller than that of the second cell.