DEVICES, SYSTEMS, AND KITS FOR ELECTROPORATION AND METHODS OF USE THEREOF

• US 20200131500A1
• Filed: 10/28/2019
• Published: 04/30/2020
• Est. Priority Date: 10/26/2018
• Status: Active Application

• Current Assignee: Kytopen Corp., Massachusetts Institute of Technology
• Original Assignee: Kytopen Corp.
• Inventors: GARCIA, Paulo Andres, SIDO, Jessica, HEMPHILL, James, MCCORMACK, Rameech, BRALOWER, Harrison
• Application Number:
  US16/666,302
• Publication Number:
  US 20200131500A1
• Time in Patent Office: Days
• CPC Class Codes: A61N 1/327 for enhancing the absorption properties of tissue, e.g. by electroporation
• CPC Class Codes: C12M 1/42 Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12N 13/00 Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

Devices, systems, and kits for cell electroporation are provided. A device includes a first electrode, a second electrode, and an electroporation zone therebetween where an electrical potential difference applied to the first and second electrodes generates an electric field in the electroporation zone sufficient to electroporate at least a subset of the cells in the flow path. Methods of introducing a composition into at least a portion of a plurality of cells using the devices, systems, and kits of the invention are also provided.

Claims

1. A device for electroporating a plurality of cells suspended in a liquid, the device comprising:

(a) a first electrode comprising a first inlet, a first outlet, and a first lumen comprising a minimum cross-sectional dimension;

(b) a second electrode comprising a second inlet, a second outlet, and a second lumen comprising a minimum cross-sectional dimension; and

(c) an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone has a length of between 0.5 mm and 50 mm and comprises a minimum cross-sectional dimension between 0.1 mm and 50 mm, wherein the electroporation zone has a substantially uniform cross-sectional area;

wherein the ratio of the minimum cross-sectional dimension of each of the first and second lumen to the minimum cross-sectional dimension of the electroporation zone is independently between 1;

10 and 10;

1, and wherein the first outlet, the electroporation zone, and the second inlet are in fluidic communication.

2. The device of claim 1, wherein the length of the electroporation zone is between 0.5 mm and 25 mm.

3. The device of claim 1, wherein the minimum cross-sectional dimension of the electroporation zone is between 0.1 mm and 10 mm.

4. The device of claim 1, wherein the ratio of the minimum cross-sectional dimension of either of the first or second electrode to the minimum cross-sectional dimension of the electroporation zone is between 1:

5 and 5;

1.

5. The device of claim 1, further comprising a first reservoir in fluidic communication with the first lumen and/or a second reservoir in fluidic communication with the second lumen.

6. A method of introducing a composition into a plurality of cells suspended in a flowing liquid, the method comprising:

(a) providing a device comprising;

(i) a first electrode comprising a first outlet, a first inlet, and a first lumen comprising a minimum cross-sectional dimension;

(ii) a second electrode comprising a second outlet, a second inlet, and a second lumen comprising a minimum cross-sectional dimension; and

(iii) an electroporation zone disposed between the first outlet and the second inlet, wherein the electroporation zone has a length of between 0.5 mm and 50 mm and comprises a minimum cross-sectional dimension between 0.1 mm and 50 mm, and wherein the electroporation zone has a substantially uniform cross-sectional area;

wherein the ratio of the minimum cross-sectional dimension of each of the first and second lumen to the minimum cross-sectional dimension of the electroporation zone is between 1;

10 and 10;

1, and wherein the first outlet, the electroporation zone, and the second inlet are in fluidic communication;

(b) applying an electrical potential difference between the first and second electrodes, thereby producing an electric field in the electroporation zone; and

(c) passing the plurality of cells and the composition in the flowing liquid through the electroporation zone, thereby enhancing permeability of the plurality of cells and introducing the composition into the plurality of cells.

7. The method of claim 6, wherein the electric field is between 100 V/cm and 1,000 V/cm in the electroporation zone.

8. The method of claim 6, wherein the plurality of the cells is in a separate liquid than the composition before step (c).

9. The method of claim 6, wherein the liquid delivered from a fluid delivery source from the first lumen to the electroporation zone has a flow rate between 1 mL/min and 100 mL/min, wherein the fluid delivery source is configured to deliver the liquid and/or the plurality of cells in suspension through the first lumen to the second outlet.

10. The method of claim 6, wherein the plurality of cells suspended in the liquid has a residence time in the electroporation zone between 0.5 ms and 50 ms.

11. The method of claim 6, wherein the electric field is produced by voltage pulses.

12. The method of claim 11, wherein the voltage pulses energize the first electrode at a particular applied voltage and the second electrode at a particular applied voltage, thereby applying the electrical potential difference between the first and second electrodes, wherein the voltage pulses each have an amplitude between −

3 kV and 3 kV.

13. The method of claim 11, wherein the voltage pulses each have a duration of between 0.01 ms and 1,000 ms.

14. The method of claim 11, wherein the voltage pulses are applied to the first and second electrodes at a frequency between 1 Hz and 50,000 Hz.

15. The method of claim 11, wherein the voltage pulses comprise a waveform selected from the group consisting of DC, square, pulse, bipolar, sine, ramp, asymmetric bipolar, or arbitrary.

16. The method of claim 11, wherein the voltage pulses comprise a duty cycle between 0.001% and 100%.

17. The method of claim 11, wherein the liquid has a conductivity of between 0.001 mS/cm and 500 mS/cm.

18. The method of claim 7, wherein the composition comprises at least one compound selected from the group consisting of a therapeutic agent, a vitamin, a nanoparticle, a charged molecule, an uncharged molecule, DNA, RNA, a CRISPR-Cas system, a peptide, a protein, a virus, a polymer, a ribonucleoprotein, a polysaccharide, an engineered nuclease, a transcription activatory-like effector nuclease, a zinc-finger nuclease, a homing nuclease, a meganuclease, a megaTAL, an enzyme, and a transposon.

19. The method of claim 7, wherein the plurality of cells suspended in the liquid is selected from a group consisting of eukaryotic cells, prokaryotic cells, plant cells, mammalian cells, animal cells, red blood cells, human cells, primary cells, cell lines, cells in suspension, adherent cells, stem cells, blood cells, Chinese hamster ovary cells, immune cells, human embryonic kidney cells, unstimulated cells, stimulated cells, activated cells, induced pluripotent stem cells, primary human induced pluripotent stem cells, embryonic stem cells, mesenchymal stem cells, hematopoietic stem cells, and synthetic cells.

20. The method of claim 19, wherein the immune cells comprise peripheral blood mononuclear cells, adaptive immune cells, innate immune cells, antigen presenting cells, monocytes, T-cells, B-cells, dendritic cells, macrophages, neutrophils, natural killer cells, Jurkat cells, primary human T-cells, THP-1 cells, primary human macrophages, primary human monocytes, primary human natural killer cells, unstimulated cells, activated cells, or stimulated cells.