Cell handling, electroporation and electrofusion in microfluidic systems

Abstract

Method and systems provide improved cell handling in microfluidic systems and devices using lateral cell trapping and methods of fabrication of the same that allow for selective low voltage electroporation and electrofusion.

1 . A method of selectively introducing substances of interest into cells comprising:
 placing one or more cells into an integrated microfluidic chip providing controllable cell trapping; optical characterizations; and simple cell loading for multiple single cell analysis;   locally electroporating a trapped cell with a focused electric field.   
     
     
         2 . The method of  claim 1  further wherein:
 said integrated chip comprises a PDMS microfluidic device with individual lateral cell trapping sites. 
 said field is generated using low applied voltages. e.g., about ˜0.76V. 
 said field is generated by electrodes that are located at a distance from said cell, said distance sufficient to reduce the potential of adverse products from electrode reactions. 
 
     
     
         3 . A method of fabricating an integrated device for cell electroporation and/or electrofusion comprising:
 preparing a mold by making height patterns defining narrow patch channels using deep etching;   adding patterns for wide connection regions;   introducing a settable material into the mold and curing;   detaching the set material from the mole;   placing holes for connection of tubes;   connecting tubes to reservoirs, via said holes, to load cells and/or electrolyte solutions and to apply suction to patch channel.   
     
     
         4 . The method of  claim 3  further wherein:
 said mold is constructed from silicon. 
 
     
     
         5 . The method of  claim 3  further wherein:
 said mold is constructed from one or more of: a ceramic; a metal; metal alloy; a hardenable polymer. 
 
     
     
         6 . The method of  claim 3  further wherein:
 said moldable material comprises polydimethylsiloxane (PDMS) and a curing agent. 
 
     
     
         7 . A Low-Voltage Single Cell Electroporation Array for Intracellular Compound Delivery device comprising:
 a substrate;   a main reservoir able to hold cells in a fluidic medium;   at least one lateral opening in a side of said main reservoir;   at least one trapping channel operatively connected to said at least one lateral opening; and   at least two electrical connections, one to said main reservoir and one to said trapping channel, such that an electric field can be focused where a cell contacts said lateral opening.   such that a cell in said main reservoir can be selectively immobilized at said lateral opening by negative pressure in said trapping channel.   
     
     
         8 . The device according to  claim 7  further wherein:
 said substrate is a three dimensional structure comprising a length, a width and a thickness, said thickness being a smallest dimension; and 
 said side of said main reservoir is roughly parallel to said thickness. 
 
     
     
         9 . The device according to  claim 7  further wherein:
 said at least two electrical connections can also be used measuring electrical characteristics between said main reservoir and said trapping channel. 
 
     
     
         10 . The device according to  claim 7  further wherein:
 said lateral opening has effective dimensions of less than about 3 microns by 3 microns. 
 
     
     
         11 . The device according to  claim 7  further comprising:
 at least three lateral openings in said main channel, said lateral openings spaced less than 40 microns apart. 
 
     
     
         12 . The device according to  claim 11  further wherein:
 said lateral openings are electrically connected to operate as independent electroporation locations. 
 
     
     
         13 . The device according to  claim 12  further wherein:
 patch lateral openings are in a horizontal plane with multiplexed electroporation sites having a distance between sites of between one hundred μm and one thousand μm. 
 
     
     
         14 . The device according to  claim 7  further comprising:
 microfluidic features to move substances to appropriate positions of said device. 
 
     
     
         15 . A multiple cell electroporation/electrofusion device comprising:
 a substrate;   a main reservoir able to hold cells in a fluidic medium running parallel to the largest dimensions of said substrate;   a plurality of lateral openings in sides of said main reservoir, at least some of said openings operatively connected to a plurality of trapping channels;   a microfluidic input for introducing cells in a fluid to said main reservoir;   one or more microfluidic trapping connections for applying negative pressure to said lateral openings;   such that cells in said main reservoir can be selectively immobilized at said lateral openings;   one or more electrical connections for applying an electric field to a cell at one of said openings, said field focused where a cell membrane contacts said opening.   
     
     
         16 . The device according to  claim 15  further wherein:
 said substrate is formed of an elastomer; 
 said lateral openings have a cross section less than about 3 microns by 3 microns; and 
 said lateral openings are operatively connected to trapping channels with cross sections less than about 3 microns by 3 microns. 
 
     
     
         17 . A multiple low voltage cell electroporation device comprising:
 a substrate;   means for holding cells in fluid suspension in a main channel, said means running parallel to the largest dimensions of said substrate;   lateral cell trapping means formed in said substrate and operatively connected to said means for holding cells in fluid suspension;   means for applying negative pressure to said lateral cell trapping means in order to selectively immobilize cells at said lateral trapping means;   means for applying an electric field between said means for holding cells and said lateral trapping means. such that said field is focused at an area of an immobilized cell.   
     
     
         18 . The device according to  claim 17  further comprising:
 means for measuring electrical properties between said means for holding cells and said lateral trapping means.