Electroporation apparatus for control of temperature during the process

Abstract

An electroporation method and apparatus generating and applying an electric field according to a user-specified pulsing and temperature profile scheme. The apparatus includes a cuvette holder with a Peltier device forming part of the electrode structures that form part of the holder. Advantageously, one such pulse includes a low voltage pulse of a first duration, immediately followed by a high voltage of a second duration, immediately followed by a low voltage of a third duration. The low voltage electroporation field accumulates molecules at the surface of a cell, the appropriately high voltage field creates an opening in the cell, and the final low voltage field moves the molecule into the cell. The molecules may be DNA, portions of DNA, chemical agents, the receiving cells may be eggs, platelets, human cells, red blood cells, mammalian cells, plant protoplasts, plant pollen, liposomes, bacteria, fungi, yeast, sperm, or other suitable cells. The molecules are placed in close proximity to the cells, either in the interstitial space in tissue surrounding the cells or in a fluid medium containing the cells.

We claim:  
     
         1 . An electroporation apparatus, the apparatus comprising: 
 receptacle means for providing a) an interface to a voltage of specified output pulsing, and b) temperature control of materials undergoing electroporation disposed within;    at least two spaced apart electrodes, at least one of the electrodes contains a Peltier device;    at least one opening to provide transfer of the materials;    a source of energy for energizing the at least two electrodes; and    a controller to receive user specification of an integrated output electroporation pulsing event with a corresponding temperature profile superimposed therewith, the controller outputs gating signals to the source of energy to generate the specified output pulsing and required polarity and magnitude to the Peltier device at the at least two electrodes, whereby the at least two electrodes enable temperature control and electroporation.    
     
     
         2 . The apparatus of  claim 1 , the controller including a microprocessor and the user specification of the electroporation pulsing event and required temperature of the materials undergoing electroporation by superposition of the gating signals to power supplies for electroporation and temperature control.  
     
     
         3 . The apparatus of  claim 1 , further comprising a user interface coupled to the controller for inputting the temperature profile.  
     
     
         4 . The apparatus of  claim 1 , the electrodes comprising plate electrodes.  
     
     
         5 . The apparatus of  claim 1 , further comprising: 
 a gap sensor to sense a distance between the electrodes and provide a representative electronic gap distance output signal.    
     
     
         6 . The apparatus of  claim 1 , wherein the controller and the energy source for generating the user specified electroporation pulse shape comprising: 
 a first power supply to provide a first output voltage;    a second power supply to provide a second output voltage, the first and second power supplies are separate sources of electrical energy;    a transformer having electromagnetically coupled primary and secondary windings, the secondary winding being interposed between a pair of terminals;    a first switch coupled to the first power supply and the primary winding and being responsive to a first gating signal to apply the first output voltage from the first power supply to the primary winding;    a second switch coupled to the second power supply and the secondary winding and responsive to a second gating signal to apply the second output voltage from the second power supply to the secondary winding; and    a controller to receive user specification of an output pulse shape and provide the first and second gating signals to generate the specified output pulse shape at the terminals.    
     
     
         7 . The apparatus of  claim 5 , the controller further being programmed to perform steps comprising: 
 receiving an input specifying a desired electric field;    receiving an input specifying an electrode gap distance; and    calculating a first voltage to provide the specified electric field across the specified electrode gap distance.    
     
     
         8 . The apparatus of  claim 6 , further comprising: 
 a pair of electrodes electrically connected to predetermined contacts of the transformer;    a gap sensor to sense an electrode gap distance between the electrodes and provide a representative electrode gap distance output signal;    wherein the controller is further programmed to perform steps comprising:    receiving an input of a desired electric field;    receiving the electrode gap distance output signal; and    computing the first voltage to provide the input desired electric field across the electrodes.    
     
     
         9 . The apparatus of  claim 1 , wherein the controller and the energy source for generating the user specified temperature state during the electroporation pulsing event includes means for controlling polarity of electrical energy at the at least two electrodes from the controller that controls heat flow using the Peltier device.  
     
     
         10 . The apparatus of  claim 6 , wherein the controller and the energy source includes means for controlling polarity and power magnitude to the at least two electrodes thereby controlling direction of heat transfer from the Peltier device, the controller includes a temperature sensor for closed loop control of the Peltier device.  
     
     
         11 . The apparatus of  claim 1 , wherein the receptacle means comprises a cuvette with at least one integral electrode contact surface attached to external surfaces of the cuvette, the at least one cuvette electrode is configured to slidably interface with and maintain positioning in a holder device, the holder device includes the at least one Peltier device in a complementary electrode structure that interfaces with at least one of the electrodes forming part of the cuvette, the holder has terminals with means for connection to the controller.  
     
     
         12 . The apparatus of  claim 11 , wherein the cuvette is a non-flow type containing device.  
     
     
         13 . The apparatus of  claim 11 , wherein the cuvette is a flow through containing device.  
     
     
         14 . A receptacle device and complementary holder device for electroporation in kit form, the kit comprising: 
 the receptacle device has at least two electrode structures for providing an interface to a specified output voltage pulsation waveform and heat transfer control for materials contained within the device;    at least one Peltier device forming a junction with at least one of the electrode structures; and    means for providing positional stability for the receptacle device and connecting the at least two electrode structures to a connectable external controller.    
     
     
         15 . The kit of  claim 14 , the electrodes comprising plate electrodes.  
     
     
         16 . The kit of  claim 14 , further comprising: 
 a gap sensor to sense a distance between the electrodes and provide a representative electronic gap distance output signal.    
     
     
         17 . The kit of  claim 14 , wherein the receptacle device provides for liquid flow through the receptacle device that includes: 
 the receptacle device including an elongated flow through chamber having an inlet and an outlet at opposite ends thereof; and    a pair of elongated spaced apart parallel internal electrodes disposed in and extending along opposite sides of the chamber between the inlet and the outlet for fluid to flow between.    
     
     
         18 . The kit of  claim 17 , wherein the receptacle device comprises a generally elongated nonconductive bar member having a rectangular cross section and an elongated through slot intermediate the ends thereof, 
 the electrodes are elongated flat conductive members sealingly applied to opposite sides of the bar member closing the slot and defining the elongated flow through chamber.    
     
     
         19 . The kit of  claim 18 , wherein the inlet and the outlet are formed in one of the electrodes and communicate with opposite ends of the slot.  
     
     
         20 . The kit of  claim 19 , wherein the receptacle device further comprises a generally U-shaped housing formed of a non-conducting material and having a pair of parallel side walls with an opening therebetween for removably receiving the flow through chamber.  
     
     
         21 . The kit of  claim 20 , wherein the U-shaped housing includes slots in one side thereof for accommodating tubing connected to the inlet and the outlet.  
     
     
         22 . The kit of  claim 17 , wherein the electrodes extend substantially the full length of the flow through chamber.  
     
     
         23 . The kit of  claim 22 , wherein the flow through chamber comprises an elongated nonconductive tubular member; 
 a header on one end of the tubular member defining one of the inlet and the outlet; and    a pair of elongated conductive bars extending along opposite sides of the tubular member defining the electrodes.    
     
     
         24 . The kit of  claim 17 , wherein the flow through chamber comprises an elongated conductive tubular member defining one of the electrodes, and a pair of headers on the ends of the tubular member defining the inlet and the outlet; and 
 a conductive rod extending coaxially of the tubular member defining the other of the conductors.    
     
     
         25 . The kit of  claim 17 , wherein the flow through chamber substantially rectangular in cross section.  
     
     
         26 . The kit of  claim 25 , wherein the flow through chamber comprises an elongated nonconductive tubular member; 
 a header on one end of the tubular member defining one the inlet and the outlet; and    a pair of elongated conductive bars extending along opposite sides of the tubular member defining the electrodes.    
     
     
         27 . A method for regulating temperature of an electroporation pulse apparatus that includes a receptacle means for containing and controlling temperature of a material contained within, the receptacle means has an electrode structure that includes a Peltier device; 
 receiving user input specifying an output pulse pattern of at least one output pulse for effectuating electroporation of the material, the user input specifying a duration for each pulse wherein the pulse pattern defines an event;    receiving a second user input specifying an output pulse pattern for controlling temperature of the material during the event.    
     
     
         28 . The method of  claim 27 , the material comprising cells removed from a living being.  
     
     
         29 . A method of electroporation using a cuvette holder comprising: 
 positioning a pair of electrodes containing a Peltier device relative to a region of cells;    applying at least one voltage pulse to the electrodes of specified polarity and    delivering a predetermined implant agent to the region of cells at a specified temperature;    moving molecules of the implant agent toward the cells by applying at least one pulse at a    second voltage to the electrodes for a second predetermined time;    creating pores in a plurality of the cells; and    moving molecules of the implant agent into a plurality of the pores while controlling temperature.    
     
     
         30 . The method of  claim 29 , the second predetermined magnitude of voltage providing a resultant electric field at the electrodes in the range of 300-3000 V/cm.  
     
     
         31 . The method of  claim 29 , further comprising the steps of computing the second predetermined magnitude of voltage by multiplying a desired electric field by measurement of a gap existing between the electrodes.  
     
     
         32 . An article of manufacture comprising a data storage medium tangibly embodying a program of machine-readable instructions executable by a digital processing apparatus to perform method steps for generating an electroporation pulse pattern in an electroporation pulse apparatus that includes a electrode structure containing Peltier material, the method comprising: 
 receiving user input specifying an output pulse pattern of one or more output pulses, the user input specifying a of an integrated output electroporation pulsing event with a corresponding temperature profile; and    providing gating signals to generate the specified output pulsing at the electrode structure.    
     
     
         33 . The article of manufacture of  claim 32  wherein the data storage medium tangibly embodying a program of machine-readable instructions executable by a digital processing apparatus to perform method steps for electroporation and temperature control, the method further comprising: 
 (a) applying an electric field of a first predetermined magnitude to a region of cells for a first predetermined duration;  
 (b) increasing the electric field to a second predetermined magnitude greater than the first predetermined magnitude; and  
 (c) reducing the electric field to a third predetermined magnitude less than the second predetermined magnitude.  
 
     
     
         34 . An apparatus for programmable control of an electroporation device, comprising: 
 a receptacle for containing a material undergoing electroporation, the receptacle includes at least two electrodes for effectuating the electroporation process of the material when disposed in the receptacle;    programmable means for controlling a excitation of a pulsing scheme at the electrodes for controlling the magnitude and polarity of the electric field of a Peltier means connected to the electrodes for regulating the temperature of the material within the receptacle and the electroporation process.    
     
     
         35 . The apparatus as recited in  claim 34 , wherein the programmable controlling means comprises: 
 a control board electrically connected to each electrode for controlling magnitude, polarity and duration of electrical energy supplied to each the electrode.    
     
     
         36 . The apparatus as recited in  claim 34 , wherein the Peltier means comprises: 
 within each electrode having an internal and external surface, the internal surface configured for containing the material whereby the internal surface is a thermally conductive sheet;    a Peltier device having an internal and external surface, whereby the internal surface of the cell is attached to the external surface of each the plate; and    a control board electrically connected to each cell for inputting electrical input to the cell which, in response to the electrical input, the cell transfers heat to and from the material.    
     
     
         37 . The apparatus as recited in  claim 34  wherein the control board is a microprocessor and memory-based controller with programmable pulse generator.  
     
     
         38 . An apparatus for programmable control of an electroporation process comprising: 
 means for containing a material undergoing electroporation;    programmable means for controlling a magnitude of an electric field acting upon the material;    a controllable Peltier device for controlling heat flow to the material; and    control means for controlling the programmable means, the Peltier device, independent of one another.