# US 10,479,979 B2 · Method for making and using cold atmospheric plasma stimulated media for cancer treatment

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- Number: US 10,479,979 B2
- Document ID: US10479979B2
- Status: Granted
- Kind: B2
- Application number: 15/392,620
- Filed: 2016-12-28
- Published: 2017-06-29
- Granted: 2019-11-19
- Earliest claimed: 2015-12-28
- Assignees: US Patent Innovations, LLC
- Applicants: U.S. Patent Innovations, LLC
- Inventors: Michael Keidar; Jerome Canady; Dayun Yan
- Web page: https://patentgrep.com/patent/US10479979B2

## CPC

- C12N 5/0693: {Tumour cells; Cancer cells}
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- H05H 1/2443: {the plasma fluid flowing through a dielectric tube}
- H05H 1/245: {the plasma being activated using internal electrodes}
- H05H 1/246: {the plasma being activated using external electrodes (H05H1/245 takes precedence)}
- H05H 1/2465: {the plasma being activated by inductive coupling, e.g. using coiled electrodes}
- C12N 2500/02: Atmosphere, e.g. low oxygen conditions
- H05H 2240/10: at atmospheric pressure
- H05H 2240/20: Non-thermal plasma
- H05H 2245/32: Surgery, e.g. scalpels, blades or bistoury; Treatments inside the body
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- H05H: PLASMA TECHNIQUE (apparatus or processes specially adapted for producing X-rays H05G2/00); PRODUCTION OF ACCELERATED ELECTRICALLY-CHARGED PARTICLES OR OF NEUTRONS; PRODUCTION OR ACCELERATION OF NEUTRAL MOLECULAR OR ATOMIC BEAMS

## Abstract

A method for preparing cold atmospheric plasma stimulated cell culture media with a cold atmospheric plasma system having a delivery port out of which an inert gas flows. The inert gas may be helium. The method comprises the steps of placing a cell culture media in a first well, the first well having a bottom and having a diameter greater than 20 mm; wherein the cell culture media placed in the first well has a volume of 4 ml or less, treating the cell culture media in the first well with cold atmospheric plasma, wherein the treating is performed with a gap between the delivery port and the bottom of the first well is between 2.5 cm and 3.5 cm, and transferring a portion of the treated media to cultured cancer cells in a second well. The cold atmospheric plasma may be applied for 0.5 minutes to 2 minutes.

## Claims

### Claim 1

A method for preparing stable cold atmospheric plasma stimulated cell culture media with a cold atmospheric plasma system having a delivery port out of which inert gas flows, the method comprising the steps of:
placing a cell culture media in a first well, said first well having a bottom and having a diameter greater than 20 mm; wherein said cell culture media placed in said first well has a volume of 4 ml or less, said cell culture media comprising an amino acid solution not having cysteine or tryptophan;
treating said cell culture media in said first well with cold atmospheric plasma, wherein said treating is performed with a gap between said delivery port and said bottom of said first well is between 2.5 cm and 3.5 cm; and
applying a portion of said treated media to cancer cells.

### Claim 2 (depends on 1)

The method according to claim 1 wherein said gap is 3 cm.

### Claim 3 (depends on 1)

The method according to claim 1 wherein said step of treating said cell culture media comprises applying cold atmospheric plasma to said cell culture media for 0.5 minutes to 2 minutes.

### Claim 4 (depends on 1)

The method according to claim 1 wherein said step of treating said cell culture media comprises applying cold atmospheric plasma to said cell culture media for 1.5 minutes or longer.

### Claim 5 (depends on 1)

The method according to claim 1 wherein said cell culture media further comprises Dulbecco's modified Eagle's medium (DMEM).

### Claim 6 (depends on 1)

The method according to claim 1 wherein said media further comprises Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) antibiotic (penicillin and streptomycin) solution.

### Claim 7 (depends on 1)

The method according to claim 1 wherein said cell culture media placed in said first well has a volume of 2 ml or less.

### Claim 8 (depends on 1)

The method according to claim 1 wherein the inert gas comprises helium from which the cold atmospheric plasma is formed is helium.