# US 2019/0316092 A1 · Methods for isolating and culturing living cells using method of permeabilizing cell membrane

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- Number: US 2019/0316092 A1
- Document ID: US20190316092A1
- Status: Published application
- Kind: A1
- Application number: 16/382,267
- Filed: 2019-04-12
- Published: 2019-10-17
- Earliest claimed: 2018-04-13
- Applicants: Seoul National University Hospital
- Inventors: Hyo-Soo Kim; Han-Mo Yang; Joo-Eun Lee; Ju-Young Kim
- Web page: https://patentgrep.com/patent/US20190316092A1

## CPC

- C12N 5/0658: {Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts}
- C12N 5/0634: {Cells from the blood or the immune system}
- C12N 5/0644: {Platelets; Megakaryocytes}
- C12N 5/0661: {Smooth muscle cells}
- C12N 5/069: {Vascular Endothelial cells}
- C12N 5/0691: {Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels}
- C12N 5/0696: {Artificially induced pluripotent stem cells, e.g. iPS}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided are methods of isolating and culturing various cells in a living state including peripheral blood mononuclear cells (PBMCs) isolated from peripheral blood, which use a cell membrane permeabilization method. The methods use a streptococcal hemolytic exotoxin, which binds to cholesterol present in a cell membrane so as to make a pore therein, thereby allowing an exogenous protein to be permeated into the cell, and is a technique of isolating and culturing desired cells in a living state by probing a specific intracellular protein and performing flow cytometry (FACS). According to the methods, various intracellular proteins that could not previously be isolated and cultured from a patient's blood as well as various tissues may be targeted, and homogeneous cells may be obtained with high purity and high efficiency. Therefore, it is expected that the methods will highly contribute to many applications targeting a specific intracellular protein and research on mechanisms of various diseases and treatment thereof.

## Claims

### Claim 1

A method of isolating target cells in a living state, comprising:
(s1) treating a test material including target cells with a streptococcal hemolytic exotoxin, i.e., Streptolysin O (SLO);
(s2) treating the test material with an antibody against a target intracellular protein of the target cells; and
(s3) isolating the target cells in a living state, which have a positive response to the antibody.

### Claim 2 (depends on 1)

The method of claim 1, wherein the target cells include peripheral blood mononuclear cells (PBMCs), skeletal myoblasts (SMBs), smooth muscle cells (SMCs) and human induced pluripotent stem cells.

### Claim 3 (depends on 1)

The method of claim 1, wherein, in the step (s1), the streptococcal hemolytic exotoxin, i.e., SLO, is treated at a concentration of 5 U/ml or less.

### Claim 4 (depends on 1)

The method of claim 1, wherein, in the step (s2), the target intracellular protein is a marker protein of the target cells.

### Claim 5 (depends on 4)

The method of claim 4, wherein the marker protein of the target cells includes calponin, smooth muscle actin (SMA), nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and dextran.

### Claim 6 (depends on 4)

The method of claim 4, wherein the marker protein of the target cells is NANOG for human induced pluripotent stem cells or Tom20 of cardiomyocytes.

### Claim 7

A method of isolating target cells in a living state, comprising:
(s1) treating a test material including target cells with a streptococcal hemolytic exotoxin, i.e., Streptolysin O (SLO);
(s2) treating the test material with an antibody against a target intracellular protein of the target cells; and
(s3) isolating the target cells in a living state, which have a positive response to the antibody through fluorescence-activated cell sorting (FACS) analysis.

### Claim 8 (depends on 7)

The method according to claim 7, wherein, in the step (s2), a fluorescent dye-conjugated antibody is used.

### Claim 9 (depends on 7)

The method according to claim 7, wherein, in the step (s2), an antibody against target intracellular protein of the target cells is used as a primary antibody, and a fluorescent dye-conjugated secondary antibody is used.

### Claim 10 (depends on 9)

The method according to claim 9, wherein the fluorescent dye-conjugated secondary antibody has a size of 150 kDa or less.

### Claim 11 (depends on 7)

The method according to claim 7, wherein the fluorescent dye is one or more selected from the group consisting of FITC, PE, Alexa Fluor 488, DyLight 488, PerCP, PerCP-Cy5.5, Alexa Fluor 555, Alexa Fluor 633, Alexa Fluor 700, Alexa Fluor 405, Cy3 and Cy5.

### Claim 12 (depends on 1)

The method according to claim 1, further comprising suspending the target cells in a calcium chloride (CaCl2)-added cell culture to regenerate a cell membrane.

### Claim 13 (depends on 7)

The method according to claim 7, further comprising suspending the target cells in a calcium chloride (CaCl2)-added cell culture to regenerate a cell.