# US 9,434,936 B2 · Method for removing cells

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- Number: US 9,434,936 B2
- Document ID: US9434936B2
- Status: Granted
- Kind: B2
- Application number: 13/638,795
- Filed: 2011-03-28
- Published: 2013-01-24
- Granted: 2016-09-06
- Earliest claimed: 2010-04-02
- Assignees: National Institute of Advanced Industrial Science and Technology
- Applicants: Kimio Sumaru; Toshiyuki Takagi; Kyoko Kikuchi; Taku Sato; Manae Yamaguchi; Toshiyuki Kanamori
- Inventors: Kimio Sumaru; Toshiyuki Takagi; Kyoko Kikuchi; Taku Sato; Manae Yamaguchi; Toshiyuki Kanamori
- Web page: https://patentgrep.com/patent/US9434936B2

## CPC

- C12N 13/00: Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
- C12M 23/20: {Material Coatings (immunocoatings C12M25/00)}
- C12M 25/06: {Plates; Walls; Drawers; Multilayer plates}
- C12M 33/00: {Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus (chemical or physical laboratory apparatus in general B01L, devices for taking cell samples A61B10/0045, withdrawing or distributing predetermined quantities of fluid B01L99/00)}
- C12M 47/04: {Cell isolation or sorting (purging biological preparations of unwanted cells C12N5/0081, determining the presence or kind of microorganism C12Q1/04)}
- C12N 1/02: Separating microorganisms from their culture media
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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}

## Abstract

A method for separating cells which includes: adhering cells to the surface of a cell culture substrate containing a photo-acid generator that generates an acidic substance upon irradiation with active energy rays, and irradiating only a partial region of the cell culture substrate with the active energy rays to selectively remove the cells within the partial region, thereby separating the cells within the partial region and cells in other regions.

## Claims

### Claim 1

A method for removing target cells from a cell culture substrate, the method comprising:
providing a cell culture substrate including a substrate and a layer containing a photo-acid generator formed on a surface of the substrate:
adhering cells to the surface of the layer containing the photo-acid generator which generates an acidic substance upon irradiation with light,
generating the acidic substance by irradiating only a region of the substrate containing the photo-acid generator layer beneath target cells to be removed, and
removing the target cells from the region of the irradiated substrate by the action of the acidic substance generated from the irradiated layer containing the photo-acid generator beneath the target cells.

### Claim 2 (depends on 1)

The method for removing target cells according to claim 1, wherein the removal is conducted by killing the target cells by the action of the acidic substance generated from the irradiated layer containing the photo-add generator beneath the removal target cells.

### Claim 3 (depends on 1)

The method for removing cells according to claim 1, wherein the substrate comprises a polyvinylpyridine-based resin.

### Claim 4 (depends on 1)

The method for removing target cells according to claim 1, wherein
a layer containing an adhesion inhibitor is formed on only a partial region of the surface of the layer containing the photo-acid generator, and
the layer containing the photo-acid generator, which has the target cells adhered thereto in a region in which the layer containing an adhesion inhibitor is not formed, is irradiated with light.

### Claim 5 (depends on 1)

The method for removing cells according to claim 1, wherein prior to irradiation with the light, a portion of the cells are labeled, and a region to be irradiated with light is determined based on positioning information for the labeled cells.

### Claim 6 (depends on 1)

The method for removing cells according to claim 1, wherein
the photo-acid generator is at least one member selected from the group consisting of a naphthaleneimide-based sulfonic acid derivative, a thioxanthone-based sulfonic acid derivative, 1,8-naphthalenedicarboxylic acid imidomethylsulfonate, 1,8-naphthalenedicarboxylic acid imidotosylsulfonate, and sulfonium salts or iodonium salts having an anion which is tetrafluoroborate (BF4−) or hexafluorophosphate (PF6−).

### Claim 7 (depends on 1)

The method for removing cells according to claim 1, wherein
the photo-acid generator is at least one member selected from the group consisting of a compound represented by formula (1) and a compound represented by formula (2) shown below:

### Claim 8 (depends on 1)

The method for removing cells according to claim 1, wherein
the photo-acid generator includes a compound which generates an acidic substance by irradiation with the light, and
the acidic substance is a polymeric compound.

### Claim 9 (depends on 8)

The method for removing cells according to claim 8, wherein the photo-acid generator is at least one member select from the group consisting of a compound represented by formula (3a), a compound represented by formula (3b), a compound represented by formula (3c) and a compound represent by formula (3d):
wherein
for the compound represented by formula 3(a): R1 is Me and R2 is OMe;
for the compound represented by 3(b): R1 is H and R2 is OMe;
for the compound represented by 3(c): R1 is H and R2 is NH-iPr; and
for the compound represented by 3(d): R1 is H and R2 is NH-iPr; wherein x and y are integers of 1 or greater.

### Claim 10 (depends on 1)

The method for removing cells according to claim 1, wherein the light has a wavelength of 200 to 600 nm.

### Claim 11 (depends on 1)

The method for removing cells according to claim 1, wherein a light source of the light is an ultraviolet lamp or a visible light lamp.

### Claim 12 (depends on 1)

The method for removing target cells according to claim 1, wherein the step of removing the target cells is by washing the irradiated substrate with a culture medium or a buffer solution thereby detaching the target cells.