Dynamic mixing and electroporation chamber and system

Abstract

An electroporation apparatus and its novel chamber with inlet ports for mixing cells and exogenous material. The inlet ports are oriented in nonparallel to each other immediately adjacent at the same top comer of the first wall of the chamber. The mixing chamber comprises successive wall sections, two curved walls at its bottom; the first curved comer is on the same side of the chamber where the liquids enter the chamber, and directs the liquids to the second curved comer at the opposing side of the chamber, which in turn further redirects the mixing to the first curved corner. The direction of the liquid flow mixture change direction at least twice into the mixing chamber.

What is claimed is: 
     
         1 . A method of electroporation comprising:
 introducing a first liquid stream comprising biological cells through one of at least two non-parallel adjacent inlet ports into a closed electroporation chamber having a wall comprising two curved wall sections, two electrodes, a voltage generator, and at least one gas venting port;   introducing a second liquid stream comprising exogenous material through another one of said at least two non-parallel adjacent inlet ports;   initiating mixing of said first and said second liquid streams as soon as they make contact near the said non-parallel adjacent inlet ports;   further mixing of said first and said second liquid streams by redirecting the said first and said second liquid streams at least twice, along said curved wall sections of the electroporation chamber;   venting the electroporation chamber as it get filled with said liquid streams through the gas venting port;   applying a pulsed voltage from the voltage generator to the electroporation chamber; and   removing mixed liquid stream contents from the electroporation chamber through an outlet port on the electroporation chamber.   
     
     
         2 . The method of  claim 1 , wherein the liquid streams are introduced into the chamber perpendicular to each other. 
     
     
         3 . The method of  claim 1 , wherein the liquid streams are directed to flow along the walls as soon as they are introduced into the chamber. 
     
     
         4 . The method of  claim 1 , further comprising maintaining a homogenous suspension of the biological cells before they are introduced into the electroporation chamber. 
     
     
         5 . The method of  claim 1 , wherein mixing the liquid streams within the electroporation is complete within 0.25 seconds to 1 minute. 
     
     
         6 . The method of  claim 1 , further comprising filtering the air displaced through the gas venting port. 
     
     
         7 . The method of  claim 1 , wherein the exogenous material is any substance that is to be delivered into the biological cells. 
     
     
         8 . The method of  claim 1 , wherein the substances are selected from one or more of a combination of proteins, polypeptides, nucleic acids, polynucleotides, expression vectors, viral RNA vectors, mRNA, polymers, carbohydrates, or other pharmaceutical molecules. 
     
     
         9 . The method of  claim 1 , wherein the liquid streams comprise low conductivity media having conductivity lower than 8 milliseimens/cm. 
     
     
         10 . The method of  claim 1 , wherein pulse is applied through an agile pulse protocol. 
     
     
         11 . The method of  claim 10 , wherein the voltage is less than 5000 volts/cm. 
     
     
         12 . The method of  claim 1 , wherein the electric fields are substantially uniform through out treatment volume of the electroporation chamber. 
     
     
         13 . The method of  claim 1 , wherein the method is scalable from 0.5 ml to at least 20 ml. 
     
     
         14 . The method of  claim 13 , wherein scaling does not affect consistency of electroporation efficiency through at least 20 repeated electroporation cycles. 
     
     
         15 . The method of  claim 1 , wherein cell density of the biological cells can be any cell density. 
     
     
         16 . The method of  claim 1 , further comprising:
 storing the biological cells for electroporation in a first container in a liquid in communication with the one of said at least two inlet ports;   storing the exogenous material for electroporation in a second container in a liquid in communication with the second one of said at least two inlet ports; and   receiving the mixed liquid stream contents from the electroporation chamber in a third container in communication with the outlet port.   
     
     
         17 . The method of  claim 16 , further comprising regulating the flow and quantities of said biological cells and said exogenous materials from said first and said second containers, as well as said outlet through automated means. 
     
     
         18 . The method of  claim 16 , wherein liquid streams up to 500 times the volume of the electroporation chamber is sequentially processed. 
     
     
         19 . The method of  claim 18 , wherein sequential processing comprises:
 moving a unit volume of the biological cells and the exogenous material into the chamber;   mixing them in the chamber and venting gas as the chamber is filled;   applying a voltage for electroporating the cells;   moving the electroporated cells out of the chamber;   moving another unit volume of the biological cells and the exogenous material into the chamber; and   repeating the process until there are no more biological cells or exogenous material or both left.   
     
     
         20 . The method of  claim 19 , wherein the electroporation system automatically shuts down when there are no more biological cells or exogenous material or both left.