# US 11,306,284 B2 · Method for mechanical and hydrodynamic microfluidic transfection

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- Number: US 11,306,284 B2
- Document ID: US11306284B2
- Status: Granted
- Kind: B2
- Application number: 16/100,158
- Filed: 2018-08-09
- Published: 2018-12-06
- Granted: 2022-04-19
- Earliest claimed: 2015-01-07
- Assignees: Indee. Inc.
- Applicants: Indee. Inc.
- Inventors: Ryan Pawell
- Web page: https://patentgrep.com/patent/US11306284B2

## CPC

- C12M 35/04: {Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli}
- B81B 1/00: Devices without movable or flexible elements, e.g. microcapillary devices
- C12M 23/16: {Microfluidic devices; Capillary tubes (integrated microfluidic structures B01L3/5027; microreactors B01J19/0093)}
- C12M 27/18: {Flow directing inserts}
- C12M 35/00: {Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion (machines for extracting juice from animal or plant tissue by electroplasmolysis A23N1/006, processes employing electric or wave energy B01J19/08; treatment of microorganisms or enzymes with electrical or wave energy C12N13/00; methods for cell fusion C12N15/02; introduction of foreign genetic material C12N15/87)}
- C12N 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- B81B 2201/058: Microfluidics not provided for in B81B2201/051 - B81B2201/054
- B81B 2203/0338: Channels
- C12N 2510/00: Genetically modified cells
- C12N 2521/00: Culture process characterised by the use of hydrostatic pressure, flow or shear forces
- C12N 2740/15043: viral genome or elements thereof as genetic vector
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- B81B: MICROSTRUCTURAL DEVICES OR SYSTEMS, e.g. MICROMECHANICAL DEVICES (piezoelectric, electrostrictive or magnetostrictive elements per se H10N30/00, H10N35/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Methods for introducing exogenous material into a cell are provided, which include exposing the cell to a transient decrease in pressure in the presence of the exogenous material. Also provided are devices for performing the method of the invention.

## Claims

### Claim 1

A method introducing an exogenous material into a cell, comprising:
inducing a first condition around the cell where there is a first ambient pressure insufficient to lyse the cell;
inducing a second condition around the cell where a second ambient pressure is less than the first ambient pressure; and
inducing a third condition around the cell where a third ambient pressure is greater than the second ambient pressure, the third condition being induced while the cell is in an unsteady flow, which creates a permeabilized cell membrane, allowing the exogenous material to be introduced into the cell.

### Claim 2 (depends on 1)

The method of claim 1, wherein the second condition is induced in less time than the third condition.

### Claim 3 (depends on 1)

The method of claim 1, wherein at least one of the second and third conditions is induced under unsteady flow conditions, which temporarily cause an extracellular pressure that is greater than an intracellular pressure and permeabilises a cell membrane.

### Claim 4 (depends on 1)

The method of claim 1, wherein the third condition is induced by pulsing the cell with an unsteady flow.

### Claim 5 (depends on 1)

The method of claim 1, further comprising repeatedly pulsing the cell with an unsteady flow to facilitate delivery of the exogenous material into the cell.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cell is viable after the introduction of the exogenous material into the cell.

### Claim 7 (depends on 1)

The method of claim 1, wherein the exogenous material is selected from the group consisting of an organic molecule, a physiologically acceptable organic molecule derivative, a biomolecule, a physiologically acceptable biomolecule derivative, a physiologically acceptable biomolecule analogue, an inorganic molecule, physiologically acceptable inorganic molecule derivative, a quantum dot, a carbon nanotube, a nanoparticle, and a gold particle.

### Claim 8 (depends on 7)

The method of claim 7, wherein the biomolecule is selected from the group consisting of a carbohydrate, a lipid, an amino acid, a peptide, a protein, a nucleotide, and a nucleic acid.

### Claim 9 (depends on 8)

The method of claim 8, wherein the nucleic acid is selected from the group consisting of a deoxyribonucleic acid and a ribonucleic acid.

### Claim 10 (depends on 8)

The method of claim 8, wherein the nucleic acid is included in an expression vector.

### Claim 11 (depends on 10)

The method of claim 10, wherein the expression vector is a plasmid.

### Claim 12 (depends on 10)

The method of claim 10, wherein the expression vector comprises at least one regulatory sequence.

### Claim 13 (depends on 12)

The method of claim 12, wherein the at least one regulatory sequence is selected from the group consisting of an enhancer region and a promoter region.

### Claim 14

A method for introducing exogenous material into a cell, comprising:
exposing the cell to a first pressure change under unsteady flow conditions, where the extracellular pressure is different than the intracellular pressure, to temporarily permeabilise the cell membrane without the cell becoming lysed; and
exposing the cell to a second pressure change under unsteady flow to introduce the exogenous material into the cell, the first pressure change being a negative pressure change, the second pressure change being a positive pressure change along with unsteady flow.

### Claim 15 (depends on 14)

The method of claim 14, wherein the first pressure change decreases extracellular pressure relative to the first pressure of surrounding the cell.

### Claim 16 (depends on 14)

The method of claim 14, wherein the second pressure change increases the extracellular pressure relative to the intracellular pressure of the cell.

### Claim 17 (depends on 14)

The method of claim 14, wherein the second pressure change is induced by pulsing the cell with an unsteady flow.

### Claim 18 (depends on 14)

The method of claim 14, further comprising repeatedly pulsing the cell—with an unsteady flow to facilitate delivery of the exogenous material into the cell.

### Claim 19 (depends on 14)

The method of claim 14, wherein the cell is viable after the introduction of the exogenous material into the cell.

### Claim 20 (depends on 14)

The method of claim 14, wherein the exogenous material is selected from the group consisting of an organic molecule, a physiologically acceptable organic molecule derivative, a biomolecule, a physiologically acceptable biomolecule derivative, a physiologically acceptable biomolecule analogue, an inorganic molecule, physiologically acceptable inorganic molecule derivative, a quantum dot, a carbon nanotube, a nanoparticle, and a gold particle.

### Claim 21 (depends on 20)

The method of claim 20, wherein the biomolecule is selected from the group consisting of a carbohydrate, a lipid, an amino acid, a peptide, a protein, a nucleotide, and a nucleic acid.

### Claim 22 (depends on 21)

The method of claim 21, wherein the nucleic acid is selected from the group consisting of a deoxyribonucleic acid and a ribonucleic acid.

### Claim 23 (depends on 21)

The method of claim 21, wherein the nucleic acid is included in an expression vector.

### Claim 24 (depends on 23)

The method of claim 23, wherein the expression vector is a plasmid.

### Claim 25 (depends on 23)

The method of claim 23, wherein the expression vector comprises at least one regulatory sequence.

### Claim 26 (depends on 25)

The method of claim 25, wherein the at least one regulatory sequence is selected from the group consisting of an enhancer region and a promoter region.

### Claim 27 (depends on 1)

The method of claim 1, further comprising introducing the exogenous material into the cell during the third condition.

### Claim 28 (depends on 1)

The method of claim 1, wherein the cell is transported in a liquid medium while the first, second, and third conditions are induced.