Modification of cells by introduction of exogenous material

• US 10,443,074 B2
• Filed: 09/28/2018
• Issued: 10/15/2019
• Est. Priority Date: 09/30/2017
• Status: Active Grant

• Current Assignee: Inscripta, Inc.
• Original Assignee: Inscripta, Inc.
• Inventors: Bernate, Jorge, Masquelier, Don
• Primary Examiner(s): Bowers, Nathan A
• Application Number:
  US16/147,353
• Publication Number:
  US 20190100774A1
• Time in Patent Office: 382 Days
• CPC Class Codes: C12M 23/44 Multiple separable units; Modules
• CPC Class Codes: C12M 35/02 Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
• CPC Class Codes: C12M 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation

Abstract

The present disclosure provides methods and devices for rapid and efficient modification of a variety of cell types, including mammalian cells, plant cells, archaea, yeasts, and bacteria, by novel methods of introducing exogenous materials, e.g. nucleic acids.

Claims

1. A method of electroporating cells comprising:

providing a flow-through electroporation (FTEP) device comprising a top surface and a bottom surface, wherein the flow-through electroporation device comprises an inlet coupled to an inlet reservoir and an inlet channel for receiving a fluid comprising electrocompetent cells and exogenous material into the FTEP device, wherein the inlet reservoir originates from the top surface of the FTEP device;

an outlet coupled to an outlet reservoir and an outlet channel for removing a fluid comprising transformed cells and exogenous material from the FTEP device, wherein the outlet reservoir originates from the top surface of the FTEP device;

a flow channel intersecting and positioned between the inlet channel and the outlet channel, wherein the flow channel decreases in width to a dimension no smaller than at least 2×

diameter of the cells being electroporated to form a constriction of the flow channel between the inlet channel and the outlet channel; and

a first electrode and a second electrode each positioned in an electrode channel, wherein the electrode channels originate from the top surface of the FTEP device, wherein the first electrode is positioned between the inlet channel and the constriction, wherein the second electrode is positioned between the constriction and the outlet channel, and wherein the electrodes are in fluid and electrical communication with fluid in the flow channel but not directly in the path of the cells traveling through the flow channel;

pulling the cells and exogenous material into the inlet reservoir, inlet, inlet channel, flow channel, and past the two electrodes;

providing electrical pulses to the cells as the cells are pulled through the flow channel past the electrodes producing electroporated cells;

collecting the electroporated cells in the outlet reservoir;

detecting an increase in air flow indicative of fluid being drained from the inlet reservoir;

reversing pressure to push the electroporated cells through the outlet reservoir, outlet, outlet channel, flow channel and past the two electrodes;

providing electrical pulses to the cells as the cells are pushed through the flow channel past the electrodes producing twice-electroporated cells; and

collecting the twice-electroporated cells in the inlet reservoir.

2. The method of electroporating cells of claim 1, wherein the flow channel decreases in width to between 10 μ

m and 5 mm.

3. The method of electroporating cells of claim 2, wherein the flow channel decreases in width to between 50 μ

m and 2 mm.

4. The method of electroporating cells of claim 1, wherein the flow channel decreases in width to 3 mm to 7 mm.

5. The method of electroporating cells of claim 1, wherein the electrodes are configured to deliver a voltage of 1-25 Kv/cm.

6. The method of electroporating cells of claim 5, wherein the electrodes are configured to deliver a voltage of 5-20 Kv/cm.

7. The method of electroporating cells of claim 6, wherein the electrodes are configured to deliver a voltage of 10-20 Kv/cm.

8. The method of electroporating cells of claim 1, wherein the flow rate of the FTEP device is between 0.1 mL to 5 mL per minute.

9. The method of electroporating cells of claim 8, wherein the flow rate of the FTEP device is between 0.5 mL to 3 mL per minute.

10. The method of electroporating cells of claim 1, further comprising the steps of

detecting an increase in air flow indicative of fluid being drained from the outlet reservoir;

reversing the pressure to pull the twice-electroporated cells through the inlet reservoir, inlet, inlet channel, flow channel and past the two electrodes;

providing electrical pulses to the twice-electroporated cells in the fluid as the twice-electroporated cells are pulled through the flow channel past the electrodes producing thrice-electroporated cells; and

collecting the thrice-electroporated cells in the outlet reservoir.

11. The method of electroporating cells of claim 10, wherein the electrodes are between 0.5 mm to 10 mm apart.

12. The method of electroporating cells of claim 11, wherein the electrodes are between 3 mm to 7 mm apart.

13. The method of electroporating cells of claim 10, wherein the electrodes are between 10 μ

m to 5 mm apart.

14. The method of electroporating cells of claim 13, wherein the electrodes are between 25 μ

m to 2 mm apart.

15. The method of electroporating cells of claim 1, wherein the FTEP device further comprises at least one filter disposed within the flow channel.

16. The method of electroporating cells of claim 15, wherein the filter is integrally-formed as part of the FTEP device.

17. The method of electroporating cells of claim 15, wherein the filter is a gradient filter.

18. The method of electroporating cells of claim 17, wherein the gradient comprises large pores proximal to the inlet channel, and small pores proximal to the electrodes.

19. The method of electroporating cells of claim 1, wherein the FTEP device comprises a second inlet and a second inlet channel and further comprises a reservoir connected to the second inlet for introducing exogenous material into the FTEP device.