Cellular poration using laser radiation

Patent metadata

Field	Value
Publication number	US-10829729-B2
Application number	US-201916250714-A
Country	US
Kind code	B2
Filing date	Jan 17, 2019
Priority date	Nov 3, 2016
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

In one aspect, a method of cell processing is disclosed, which includes disposing a plurality of cells on a substrate across which a plurality of projections are distributed and an electrically conductive layer at least partially coating said projections, exposing the cells to a cargo to be internalized by the cells, irradiating the substrate surface (and in particular the projections) with continuous wave or pulsed laser radiation. For example, one or more laser pulses having a pulse width in a range of about 1 ns to about 1000 ns can be applied so as to facilitate uptake of the cargo by at least a portion of the cells (e.g., the cells positioned in the vicinity of the projections (e.g., within hundreds of nanometer (such as less than 100 nm) of the projections)). In some embodiments, the laser pulses have a pulse width in a range of about 10 ns to about 500 ns, e.g., in a range of about 5 ns to about 50 ns.

First claim

What is claimed is:

1. A method of cell processing, comprising:
disposing a plurality of cells on a substrate having a plurality of projections and an electrically conductive layer at least partially coating said projections,
exposing the cells to a cargo to be internalized by the cells,
irradiating the projections with one or more laser pulses having a pulse width in a range of about 5 ns to about 1000 ns so as to facilitate uptake of the cargo by at least a portion of said cells,
wherein the laser pulses are applied with a fluence in a range of about 40 mJ/cm 2 to about 90 mJ/cm 2 .

2. The method of claim 1 , wherein said laser pulses have a pulse width in a range of about 10 ns to about 100 ns.

3. The method of claim 1 , wherein said laser pulses have a pulse width in a range of about 20 ns to about 500 ns.

4. The method of claim 1 , wherein the laser pulses are applied at a repetition rate of at least about 1 Hz.

5. The method of claim 4 , wherein the laser pulses are applied to the cells at a repetition rate in a range of about 0.1 Hz to about 100 GHz.

6. The method of claim 1 , wherein said cargo comprises any of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a plasmid, a protein, a dye, a polymer, a quantum dot, a nanoparticle, and a protein complex.

7. The method of claim 6 , wherein said protein complex comprises a Cas9-gRNA complex.

8. The method of claim 1 , wherein said cargo has a size in a range of about 10 kDa to about 1000 kDa.

9. A method of cell processing, comprising
disposing a plurality of cells on a substrate having a plurality of projections distributed across the substrate and an electrically conductive layer at least partially coating said projections,
exposing the cells to a macromolecular cargo to be internalized by the cells, said cargo having a size ranging from about 10 kDa to about 1000 kDa,
irradiating the projections with one or more laser pulses having a pulse width in range of about 5 ns to about 1000 ns and a fluence sufficient to facilitate uptake of the macromolecular cargo by at least a portion of said cells, and
wherein the laser pulses are applied with a fluence in a range of about 40 mJ/cm 2 to about 90 mJ/cm 2 .

10. The method of claim 9 , wherein the laser pulses have a pulse width in a range of about 10 ns to about 1000 ns.

11. The method of claim 9 , wherein the projections have a pyramidal shape.

12. The method of claim 11 , wherein said pyramidal shape is in the form of a pyramid extending from a base to an apex.

13. A method of delivering a genetic cargo to a cell, comprising:
disposing a plurality of cells on a substrate having a plurality of projections of pyramidal shape, and an electrically conductive layer at least partially coating said projections,
exposing the cells to a genetic cargo to be internalized by the cells, and
irradiating the projections with laser radiation pulses having a pulse width in a range of about 5 ns to about 1000 ns and a fluence in a range of about 40 mJ/cm 2 to about 90 mJ/cm 2 to induce a transient change in permeability of the cells' membrane to facilitate uptake of the genetic cargo by at least a portion of said cells.

14. The method of claim 13 , wherein the genetic cargo comprises a nucleic acid sequence.

15. The method of claim 13 , wherein the genetic cargo comprises a Cas9-gRNA complex.

16. The method of claim 13 , wherein said pyramidal shape is in the form of a pyramid extending from a base to an apex.

17. The method of claim 1 , wherein said plurality of projections have a pyramidal shape.

18. The method of claim 1 , wherein said plurality of pyramidal projections have a height in a range of about 0.1 μm to about 20 μm.

19. The method of claim 1 , wherein said plurality of pyramidal projections have a height in a range of about 1 μm to about 10 μm.

20. The method of claim 9 , wherein said plurality of projections is distributed across the substrate as a regular array.

Assignees

• Harvard College

Inventors

• Mazur Eric
• Saklayen Nabiha
• Madrid Marinna
• Huber Marinus
• Nuzzo Valeria

Classifications

• C12M35/02 Primary

  Electrical or electromagnetic means, e.g. for electroporation or for cell fusion · CPC title

• C12N15/87

  Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation · CPC title