METHODS FOR ISOLATING AND CULTURING LIVING CELLS USING METHOD OF PERMEABILIZING CELL MEMBRANE

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

1-12. (canceled)

13. A method of isolating target cells in a living state, comprising: (s1) treating a test material including target cells with a streptococcal hemolytic exotoxin; (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, and wherein the target cells are skeletal myoblasts (SMBs) or human induced pluripotent stem cells.

14. The method of claim 13, wherein the streptococcal hemolytic exotoxin is Streptolysin O (SLO).

15. The method of claim 13, wherein, in the step (s2), the target intracellular protein is a marker protein of the target cells.

16. The method of claim 15, wherein the marker protein of the target cells includes calponin, smooth muscle actin (SMA), and CD31.

17. The method of claim 15, wherein the marker protein of the target cells is NANOG for human induced pluripotent stem cells or Tom20 of cardiomyocytes.

18. The method according to claim 13, wherein, in the step (s2), a fluorescent dye-conjugated antibody is used.

19. The method according to claim 13, 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.

20. The method according to claim 19, wherein the fluorescent dye-conjugated secondary antibody has a size of 150 kDa or less.

21. The method according to claim 13, 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.

22. The method according to claim 13, further comprising suspending the target cells in a calcium chloride (CaCl.sub.2)-added cell culture to regenerate a cell.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0066] The above and other objects, features and advantages of the present invention will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the accompanying drawings, in which:

[0067] FIG. 1A shows the confocal imaging results and a graph for comparing FITC-dextran positive cells, after PBMCs are isolated from the peripheral blood of a donor and treated with 5U of a streptococcal hemolytic exotoxin, and then permeabilized cells are treated with various sizes of FITC-dextran;

[0068] FIG. 1B shows the results of detecting the fluorescence of a variety of FITC-dextran, after PBMCs are isolated from the peripheral blood of a donor and treated with a streptococcal hemolytic exotoxin at various concentrations, using a flow cytometer;

[0069] FIG. 2A shows the apoptosis and necrosis analysis results (top), and graphs for comparing the death of cells in which the cell membrane is regenerated (bottom), after PBMCs are isolated from the peripheral blood of a donor and then the cell membranes of the cells permeabilized by the treatment with a streptococcal hemolytic exotoxin at various concentrations are regenerated, using a flow cytometer;

[0070] FIG. 2B shows cell death analysis results (top), and graphs for comparing the death of cells in which the cell membrane is regenerated (bottom), after PBMCs are isolated from the peripheral blood of a donor, and then the cell membranes of the cells permeabilized by the treatment with a streptococcal hemolytic exotoxin at various concentrations are regenerated and treated with PI, using a flow cytometer;

[0071] FIG. 3A shows graphs (top) of the comparative analysis of NFATc1 targeting an endomembrane between a group in which a target expressed in the endomembrane is treated with a flow cytometry fixation and permeabilization buffer kit (R&D, FC009, USA) and a BD fixation and permeabilization solution (554722,BD,USA) (saponin concentration: 0.02-0.1%) and a group treated with 5 U of a streptococcal hemolytic exotoxin, after PBMCs are isolated from the peripheral blood of a donor, and then the cells are killed, and a graph (down) showing that there is no difference in efficiency between the conventional method and a method targeting an endomembrane in a living state;

[0072] FIG. 3B shows graphs (top) of the comparative analysis of CiMS expression rates (top) between a group in which a target expressed in the endomembrane is treated with a flow cytometry fixation and permeabilization buffer kit (R&D, FC009, USA) and a BD fixation and permeabilization solution (554722,BD,USA) (saponin concentration: 0.02-0.1%) and a group treated with 5 U of a streptococcal hemolytic exotoxin, after PBMCs are isolated from the peripheral blood of a donor, and then the cells are killed, and a graph (down) showing that there is no difference in efficiency between the conventional method and the method of the present invention;

[0073] FIG. 4A shows graphs (left) for analyzing fluorescence expression rates and graphs (right) for comparatively analyzing the most suitable secondary antibodies, when skeletal myoblast cells are treated with a streptococcal hemolytic exotoxin, treated with representative markers, calponin and SMA, and then treated with various sizes of secondary antibodies;

[0074] FIG. 4B shows results obtained by isolating PBMCs from the peripheral blood of a donor, treating the cells with a streptococcal hemolytic exotoxin, treating the cells with an antibody targeting NFATc1 expressed in the cells, and then sorting NFATc1 positive and negative cells using a flow cytometer;

[0075] FIG. 4C shows that only calponin positive cells are isolated using a flow cytometer after culturing a rat artery using an enzyme reaction, treating the tissue with a streptococcal hemolytic exotoxin, and treating the tissue with calponin, which is a representative intracellular expression marker, to isolate vascular smooth muscle, and then cross-stained with smooth muscle actin to prove that the cells are vascular smooth muscle cells;

[0076] FIG. 4D shows that calponin positive cells are isolated and cultured using a flow cytometer, after skeletal myoblasts are treated with a streptococcal hemolytic exotoxin and treated with calponin, which is an intracellular expression marker;

[0077] FIG. 5 shows the results of confirming cell death when PBMCs are isolated from the peripheral blood of a donor, treated with a streptococcal hemolytic exotoxin, stained with dextran, subjected to flow cytometry to sort dextran positive and negative cells after isolating and culturing for 48 hours, and graphs for comparatively analyzing the cell death;

[0078] FIG. 6A is a set of live confocal images showing that human induced pluripotent stem cells are alive, after NANOG, which is a representative marker of a stem cell, is treated with a streptococcal hemolytic exotoxin;

[0079] FIG. 6B is a set of images proving that human induced pluripotent stem cells differentiate into human cardiomyocytes, are treated with a streptococcal hemolytic exotoxin, are stained with Tom20, which is a representative cytoplasmic marker of mitochondria, followed by beating; and

[0080] FIG. 7 schematically illustrates an overall method of the present invention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

[0081] Hereinafter, the present invention will be described in further detail with reference to examples. However, the following examples are merely exemplary, and thus the present invention is not limited to following examples.

Example 1. Isolation of PBMCs from Peripheral Blood

[0082] 10 cc of blood of a normal group was well mixed in 20 mL of phosphate-buffered saline (PBS, Invitrogen, NY, USA), and 12 mL of Ficoll-Paque (GE Health Care, Piscataway, NJ) was slowly added thereto from the bottom so as to separate layers. Following centrifugation at 2,500 rpm for 30 minutes, the serum was removed from the uppermost layer of the four layers, and only PBMCs present in the middle of the layers were isolated using a pipette and mixed in PBS. Afterward, the PBMCs were washed twice by centrifugation at 1,800 rpm for 10 minutes, suspended in PBS at 1×10.sup.6/100 .Math.L, and then dispensed into EP tubes.

Example 2. Permeability of Sreptococcal Hemolytic Exotoxin (Streptolysin O; SLO)

[0083] An experiment for determining an exact concentration for making a pore in a cell membrane using a streptococcal hemolytic exotoxin (Streptolysin O; SLO; Sigma-Aldrich, MO, USA) was performed. In a permeabilization step, the isolated PBMCs or cell lines were centrifuged in cold PBS (Invitrogen, NY, USA) at 1,800 rpm for 5 minutes to collect the cells. 1×10.sup.6/100 .Math.L of the cells were suspended in PBS, and dispensed into EP tubes. Each cell line was treated with a suitable concentration of a streptococcal hemolytic exotoxin (Streptolysin O; SLO) in a 37 □ water bath for 50 minutes and then incubated on ice for 1 minute, 1 mL of PBS was added to the sample, and then the cells collected by centrifugation at 4 □ for 3 minutes at 1,800 rpm were resuspended in an ATP regeneration solution. The ATP regeneration solution was prepared by adding 1 mM ATP, 10 mM creatine phosphate, 25 .Math.g/mL creatine kinase, 100 .Math.M GTP (Sigma-Aldrich, MO, USA) and 1 mM dNTP (Applied Biosystems, CA, USA) to PBS.

Example 3. Analysis of Permeabilization Efficiency Using Dextran-FITC

Example 3-1. Analysis of Permeabilization Efficiency Using Confocal Imaging

[0084] To analyze permeabilization efficiency, uptake using various molecular sizes of fluorescein isothiocyanate (FITC)-dextran (Sigma-Aldrich, MO, USA) was confirmed by FACS and confocal imaging.

[0085] Specifically, the permeabilized cell line was dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L, and the cells were treated with FITC-dextran 10 KMW, 40 KMW, 70 KMW, 250 KMW, or 500 KMW (Sigma-Aldrich, MO, USA) by size, and incubated at 37 □ for 1 hour. After staining, to regenerate the cell membrane of a cell, 2 mM CaCl.sub.2 was added to the same volume of the cell culture solution, and the cells were incubated at 37 □ for 2 hours and centrifuged at 4 □ for 3 minutes at 1,800 rpm, followed by washing. The cells were resuspended in the cell culture solution to which 2 mM CaCl.sub.2 was added and mounted on a confocal dish (Ibidi, Germany), fluorescence was visualized using a confocal laser scanning microscope 710 (Zeiss, Germany) and analyzed using Zeiss Zen software, and FITC positive cells were statistically analyzed using Image J. It was confirmed that, according to the above-described method, up to FITC-dextran 250 K among those of various molecular sizes was permeated into the cells using SLO. FIG. 1A shows confocal images of FITC positive cells, and the result is shown by a graph.

Example 3-2. Permeabilization Efficiency Assay Using FACS

[0086] To see permeabilization efficiency, using various molecular sizes of the FITC-dextran (Sigma-Aldrich, MO, USA), uptake was confirmed by FACS. Specifically, the permeabilized cell line was dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L, FITC-dextran 10 KMW, 40 KMW, 70 KMW, 250 KMW or 500 KMW (Sigma-Aldrich, MO, USA) were added to the cells by size, and the cells were incubated at 37 □ for 1 hour and stained. To regenerate the cell membrane of a cell, 2 mM CaCl.sub.2 was added to the same volume of the cell culture solution, and the cells were incubated at 37 □ for 2 hours, and centrifuged at 4 □ for 3 minutes at 1,800 rpm, followed by washing. The cells were resuspended in a 2 mM CaCl.sub.2-added cell culture solution, and a fluorescent material was detected using FACS Canto II (BD Biosciences, CA, USA) (all data measured by FACS was analyzed using BD FACSDiva software). While adding various concentrations of SLO, various molecular sizes of FITC-dextran were uptaken, and the ratio of permeated positive cells is shown in FIG. 1B.

Example 4. Method of Confirming Cell Death of Cell Line Treated with Streptococcal Hemolytic Exotoxin (Streptolysin O; SLO)

Example 4-1. Method of Confirming Necrosis and Apoptosis

[0087] Permeabilized cells were resuspended in cold PBS (Invitrogen, NY, USA), and to regenerate the cell membrane of a cell, 2 mM CaCl.sub.2 was added to the same volume of the cell culture solution, and then the cells were incubated at 37 □ for 2 hours, and centrifuged at 4 □ for 3 minutes at 1,800 rpm, followed by washing. The cells were resuspended in the 2 mM CaCl.sub.2-added cell culture solution, and dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L. The cells were mixed with 7AAD (BD Biosciences, CA. USA) and ANNEXIN V (BD Biosciences, CA. USA), cultured at room temperature for 15 minutes, and stained. Afterward, the cells were centrifuged in 2.5% fetal bovine serum, (FBS 16000, Gibco, NY, USA)-added PBS (Invitrogen, NY, USA) at 1,800 rpm for 5 minutes for washing, resuspended in 100 .Math.L of washing buffer, and analyzed using FACS Canto II (BD Biosciences, CA, USA). All data measured by FACS was analyzed using BD FACSDiva software. As shown in FIG. 2A, it can be seen that necrotic and apoptotic cells increased according to an increase in the concentration of SLO.

Example 4-2. PI Staining Method

[0088] Permeabilized cells were resuspended in cold PBS (Invitrogen, NY, USA), and to regenerate the cell membrane of a cell, 2 mM CaCl.sub.2 was added to the same volume of the cell culture solution, and then the cells were incubated at 37 □ for 2 hours, and centrifuged at 4 □ for 3 minutes at 1,800 rpm, followed by washing. The cells were resuspended in the 2 mM CaCl.sub.2-added cell culture solution, and dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L. The cells were mixed with propidium iodine (PI, BD Biosciences, CA. USA), incubated at room temperature for 15 minutes and stained. Then, the cells were centrifuged in 2.5% fetal bovine serum (FBS 16000, Gibco, NY, USA)-added PBS (Invitrogen, NY, USA) at 1,800 rpm for 5 minutes for washing, resuspended in 100 .Math.L of washing buffer, and analyzed using FACS Canto II (BD Biosciences, CA, USA). All data measured by FACS was analyzed using BD FACSDiva software. As shown in FIG. 2B, it can be confirmed that as the SLO concentration increased, PI positive cells increased, and it can be seen that the most suitable concentration was 5U.

Example 5. Methods of FACS and Sorting for SLO-Treated Cell Line

Example 5-1. Comparative Analysis of Difference in Expression of NFATc1 and CiMS Cells in PBMCs According to Permeabilization Buffer

[0089] Using PBMCs permeabilized using SLO and commercially available R&D, a BD Permeabilization buffer, a flow cytometry fixation and permeabilization buffer kit (R&D, FC009, USA) (saponin concentration: 0.02-0.1%) and a BD fixation and permeabilization solution (554722, BD, USA), the permeabilized PBMCs were resuspended in cold PBS (Invitrogen, NY, USA), and the cells were dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L. 5 .Math.L each of allophycocyanin (APC) conjugated-mouse anti-human CD45 (BD Biosciences, CA, USA), APC conjugated-mouse anti-human CD3 (BD Biosciences, CA, USA), Alexa 488 conjugated-anti-human NFATc1 (Santa Cruz Biotechnology, CA, USA) and phycoerythrin (PE) conjugated-mouse anti-human CD31 (BD Biosciences, CA, USA) was added to each cell-dispensed tube, and the cells were incubated at room temperature for 30 minutes and stained. Afterward, to regenerate the cell membrane of the cell, 2 mM CaCl.sub.2 was added to the same volume of the cell culture solution, and the cells were incubated at 37 □ for 2 hours, and centrifuged in 2.5% fetal bovine serum (FBS 16000, Gibco, NY, USA)-added PBS (Invitrogen, NY, USA) at 1,800 rpm for 5 minutes for washing. The collected cells were resuspended in the 2 mM CaCl.sub.2-added cell culture solution, and analyzed using FACS Canto II.

[0090] As a result, as shown in FIG. 3A, under conditions in which the commercially available permeabilization buffer and 5U SLO are used, it was proven that there was no difference in expression of NFATc1 positive cells, and as shown in FIG. 3B, it was demonstrated that, under conditions in which the commercially available permeabilization buffer and 5U SLO are used, there was no difference in expression level of PBMC-derived circulatory multipotent stem cells (CiMS cells) (NFATcl+/CD31+/CD3+/CD45+), either.

Example 5-2. Calponin and SMA FACS for SMB Cell Line

[0091] Permeabilized SMBs were resuspended in cold PBS (Invitrogen, NY, USA), and the cells were dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L. 5 .Math.L each of mouse-anti-calponin (Sigma) and mouse anti-SMA (Abcam) was added to each tube, and incubated at room temperature for 30 minutes. After the first staining, as a secondary antibody, 2 .Math.L each of a fluorescein isothiocyanate (FITC)-conjugated antibody (BD Biosciences, CA, USA), a PerCP-conjugated antibody (BD Biosciences, CA, USA), and a Cy3-conjugated antibody (BD Biosciences, CA, USA) were added to the resulting cells, and then incubated at room temperature for 30 minutes. After the second staining, to regenerate the cell membrane of each cell, 2 mM CaCl.sub.2 was added to the same volume of SK-GMV (Lonza), the cells were incubated at 37 □ for 2 hours, and centrifuged in 2.5% fetal bovine serum (FBS 16000, Gibco, NY, USA)-added PBS (Invitrogen, NY, USA) at 1,800 rpm for 5 minutes for washing. The collected cells were resuspended in 2 mM CaCl.sub.2-added SK-GMV (Lonza), analyzed using FACS Canto II and then represented by a graph shown in FIG. 4A. When the size of the secondary fluorescent antibody was 150 kDa or more (APC, PE), it did not permeate, showing that it is not suitable for the analysis method using SLO.

Example 5-3. Detection of NFATc1 Positive Cells in PBMCs

[0092] Permeabilized PBMCs were resuspended in cold PBS (Invitrogen, NY, USA), and the cells were dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L. 5 .Math.L of Alexa 488-conjugated anti-human-NFATcl (Santa Cruz Biotechnology, CA, USA) was added to each tube, incubated at room temperature for 30 minutes, and stained. Afterward, to regenerate the cell membrane of the cell, 2 mM CaCl.sub.2 was added to the same volume of cell culture solution, cells were incubated at 37 □ for 2 hours, and centrifuged in 2.5% fetal bovine serum (FBS 16000, Gibco, NY, USA)-added PBS (Invitrogen, NY, USA) at 1,800 rpm for 5 minutes for washing. The collected cells were resuspended in 2 mM CaCl.sub.2-added cell culture solution, NFATc1-stained cells were filtrated through a mesh, and NFATc1 positive and negative cells were concentrated in PBS using a FACSAria cell sorter (BD Biosciences, CA, USA) and then centrifuged at 1,800 rpm for 10 minutes. The collected cells were resuspended in 2 mM CaCl.sub.2-added EGM2-MV (Lonza, USA), and seeded in a 10 .Math.g/mL fibronectin (Sigma-Aldrich, MO, USA)-coated Corning 6-well plate. The medium was changed daily, and after 5 days, the cells were able to be identified as shown in FIG. 4B.

Example 5-4. Method of Culturing Primary Rat Artery

[0093] After six-week-old rats were killed by inhalation anesthesia with CO.sub.2, arteries of the rats were harvested and put into cold PBS, and then cells were obtained by a method using an enzyme reaction.

[0094] More specifically, after blood removal, the harvested rat arteries were cut into small pieces, added to a conical flask containing 5 mL of 0.05% collagenase type II (Invitrogen, NY, USA) in high glucose DMEM, and left at 37 □ on a shaking incubator for 30 minutes. To terminate enzyme treatment, 10% FBS and the sample were suspended and centrifuged at 1000 rpm for 10 minutes. The collected cells were resuspended in DMEM, and centrifuged again for washing. The collected cells were seeded in a 1.5% gelatin (Sigma-Aldrich, MO, USA)-coated Coming® 60-mm dish containing high glucose DMEM which contains 10% FBS and antibiotics, and the medium was changed every other day. After seven days, when mixed cells were observed, they were subjected to cell permeabilization using SLO by the same method as described in Example 5-2, and as shown in FIG. 4C, only calponin positive cells were sorted and grown.

Example 5-4-1. Immunocytochemistry

[0095] To confirm that the cells sorted in FIG. 4C were calponin positive cells, the cells were seeded in a 35-mm confocal dish (Ibidi, Germany), 1.5 mL of cold 100% methanol was added thereto, and the cells were maintained at -20 □ for 10 minutes. The cells fixed with methanol were washed with a washing buffer containing 0.05% Tween 20 in PBS twice, and to reduce a non-specific reaction, 0.1% BSA was mixed in PBS, and a blocking buffer filtrated through a 0.22-mm mesh was added thereto, followed by maintaining at room temperature for 30 minutes. Mouse anti-SMA (rat smooth muscle actin; Abcam) antibodies were suspended in a 1:200 antibody diluent (Invitrogen, NY, USA) and then the resulting suspension was added to the blocked cells, followed by maintaining at 4 □ overnight. After the first staining, the cells were washed with a washing buffer containing 0.05% Tween 20 in PBS three times, a secondary antibody, i.e., Alexa Fluor 488 anti-mouse IgG, was suspended in a 1:200 antibody diluent (Invitrogen, NY, USA), and then the resulting suspension was added to the cells, followed by maintaining at room temperature for 1 hour. After the second staining, the cells were washed with a washing buffer containing 0.05% Tween 20 in PBS three times, and to stain the nucleus, and DAPI (Sigma-Aldrich) was suspended in a 1:1000 antibody diluent (Invitrogen, NY, USA), and then the resulting suspension was added to the cells, followed by maintaining at room temperature for 10 minutes. The nucleus-stained sample was treated with mounting media, covered with a cover glass, observed using a confocal laser scanning microscope 710 (Zeiss, Germany) to visualize fluorescence, and then analyzed using Zeiss Zen software (SMA-stained image of FIG. 4C).

Example 5-4-2. Detection of Calponin and CD31 in SMB Cell Line

[0096] SMB cells were resuspended in cold PBS (Invitrogen, NY, USA), and dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L .Math.L. 5 .Math.L each of mouse-anti-calponin (Sigma) and PE-conjugated rat CD31 (BD Biosciences, CA, USA) was added to each tube, and incubated at room temperature for 30 minutes. After the staining, to regenerate the cell membrane of the cell, 2 mM CaCl.sub.2 was added to the same volume of high glucose DMEM (Invitrogen, NY, USA) containing 10% fetal bovine serum (FBS 16000, Gibco, NY, USA), the cells were incubated at 37 □ for 2 hours, and centrifuged in 2.5% fetal bovine serum (FBS 16000, Gibco, NY, USA)-added PBS (Invitrogen, NY, USA) at 1,800 rpm for 5 minutes for washing. The collected cells were resuspended in 2 mM CaCl.sub.2-added high glucose DMEM (Invitrogen, NY, USA) containing 10% fetal bovine serum (FBS 16000, Gibco, NY, USA), the stained cells were filtrated through a mesh, and then calponin positive cells were concentrated in PBS using a FACSAria cell sorter (BD Biosciences, CA, USA) and then centrifuged at 1,800 rpm for 10 minutes. The collected cells were resuspended in 2 mM CaCl.sub.2-added high glucose DMEM (Invitrogen, NY, USA) containing 10% fetal bovine serum (FBS 16000, Gibco, NY, USA), and seeded in a 1.5% gelatin (Sigma-Aldrich, MO, USA)-coated Coming® 6-well plate. The medium was changed daily, and after 10 days, as shown in FIG. 4D, it was observed that, compared with calponin negative cells, in calponin positive cells, actin was more highly formed, showing that the calponin positive cells were successfully isolated and cultured.

Example 5-3. Detection of Dextran and Confirmation of Cell Death in PBMCs

[0097] Permeabilized PBMCs were resuspended in cold PBS (Invitrogen, NY, USA), and the cells were dispensed into round-bottom tubes (BD Biosciences, CA, USA) at 5×10.sup.5/100 .Math.L. 5 .Math.L each of FITC conjugated-Dextran (Invitrogen, NY, USA) was added to each tube, and incubated at room temperature for 30 minutes. After the staining, to regenerate the cell membrane of the cell, 2 mM CaCl.sub.2 was added to the same volume of a cell culture solution, the cells were incubated at 37 □ for 2 hours, and centrifuged in 2.5% fetal bovine serum (FBS 16000, Gibco, NY, USA)-added PBS (Invitrogen, NY, USA) at 1,800 rpm for 5 minutes for washing. The collected cells were resuspended in 2 mM CaCl.sub.2-added EGM2-MV (Lonza, USA) and filtrated through a mesh, and dextran positive and negative cells were concentrated in PBS using a FACSAria cell sorter (BD Biosciences, CA, USA) and centrifuged at 1,800 rpm for 10 minutes. The collected cells were resuspended in 2 mM CaCl.sub.2-added EGM2-MV (Lonza, USA) and seeded in a 10 .Math.g/mL fibronectin (Sigma-Aldrich, MO, USA)-coated Coming® 6-well plate, and after 48 hours, the cell line was analyzed by the method of confirming the cell death of a streptococcal hemolytic exotoxin (Streptolysin O; SLO)-treated cell line, described in Example 4. As a result, as shown in FIG. 5, like dextran negative cells, in dextran positive cells, ratios of necrotic and apoptotic cells and PI positive cells were merely less than 5%, showing that dextran has substantially no effect on cell death.

Example 6. Preparation of Induced Pluripotent Stem Cells Using Fibroblasts

Example 6-1. Preparation of Retrovirus and Human Induced Pluripotent Stem Cells and NANOG Fluorescent Staining

[0098] Retroviruses expressing four genes, called Yamanaka factors, such as SOX2, c-MYC, OCT4 and KLF4, respectively, were prepared to be used for the preparation of induced pluripotent stem cells. To this end, 293T cells were cultured in high glucose DMEM containing 10% FBS and an antibiotic until reaching 90% of the area of a culture container. Meanwhile, 800 .Math.L of basal DMEM was dispensed into each 1.5-mL Eppendorf tube, a plasmid in which one of the four genes prepared herein was cloned and packaging vectors, i.e., pVSV-G and pGag-Pol, were added at 10 .Math.g/plasmid, 60 .Math.L of 1 mg/mL polyethyleneimine (PEI) stock was added and well mixed, and the cell mixture was maintained at room temperature for 30 minutes. During this process, to increase transfection efficiency, the 293T cells were washed with 5 mL of basal DMEM twice, and then 10 mL of high glucose DMEM (containing 10% FBS) without an antibiotic was added thereto, and then the cells were stored in a 37 □ incubator in a state in which an antibiotic was removed. Thirty minutes after the preparation of a plasmid DNA-PEI mixed solution, the antibiotic-removed 293T cells were extracted, and each plasmid DNA-PEI mixed solution per gene type was gently stirred once using a pipette and then dropped on the 293T cells for transfection. After 18 hours, the transfected 293T cells were taken out from the incubator, heated to 37 □, and rinsed twice with 5 mL of basal DMEM to remove the excess plasmid DNA-PEI mixed solution. 10 mL of fresh high glucose DMEM containing 10% FBS and an antibiotic was added to the cells, and incubated in a 37 □ incubator. Here, this process was very carefully performed so as not to detach the cells. After 48 hours, to recover the produced retroviruses, only the culture of 293T cells was collected in a 15 mL tube and centrifuged at 2500 rpm for 15 minutes, and then only the supernatant was harvested so that detached cells and debris, which were precipitated on the bottom, were not included, filtrated through a 0.22-.Math.m filter, and subjected to ultracentrifugation at 4 □ for 1.5 hours at 25,000 rpm to concentrate the retroviruses included in the supernatant. The retroviruses expressing four different genes, which were precipitated in pellets were resuspended in 100 .Math.L of EBM-2, and stored at -70 □ before use. The concentrated Yamanaka 4F were inoculated into fibroblasts, thereby preparing human induced pluripotent stem cells.

[0099] Afterward, the prepared human induced pluripotent stem cells were treated with 5U SLO, thereby obtaining permeabilized cells. A 1:100 dilution of mouse-anti-NANOG (Cell Signaling) was incubated at 37 □, a 1:100 dilution of secondary antibodies, i.e., anti-mouse-Alexa Fluor 488, was incubated at 37 □. To regenerate the cell membrane of a cell, the stem cells which had been subjected to the antibody reaction were seeded in a Matrigel coated dish after 2 mM CaCl.sub.2 was added to the same volume of hES media. As shown in FIG. 6A, confocal imaging was conducted for the cells in a living state, showing that the nucleus was stained by the transcription factor NANOG, and through Z-stack, it was demonstrated that staining is carried out using NANOG which has permeated a cell membrane.

Example 6-2. Differentiation of Fibroblast-iPSC Into Cardiomyocytes and Confirmation of Staining with Tom 20

[0100] For differentiation into cardiomyocytes, an ES medium of an embryonic body of fibroblast-iPSC during culture was removed, 0.5 mg/mL of dispase was dissolved in a bFGF-free ES medium, the cells were treated with 1 mL of the resulting ES medium and then incubated at 37 □ for 1 hour, such that fibroblast-iPSC colonies were separated from feeder cells. Suspended fibroblast-iPSC colonies were harvested and transferred to a 15-mL tube, and then the fibroblast-iPSCs were washed twice with a bFGF-free ES medium. On the following day, 3 .Math.M of CHIR99021, 25 ng/mL of BMP4, 50 .Math.g/mL of vitamin C, and 100 ng/mL of Activin A were added to an RPMI1640 medium containing B27-minus insulin (RPMI1640+B27 minus insulin), and the fibroblast-iPSC colonies were incubated for exactly 24 hours. Afterward, 10 ng/mL of BMP4 and 10 ng/mL of VEGF, which were diluted concentrations, were added to the RPMI1640+B27 minus insulin medium, followed by further incubation for 3 days, and then the cells were rinsed with the RPMI1640+B27 minus insulin, followed by further incubation for 1 day. On the following day, 5 .Math.M IWP2, which is a low molecular substance for inhibiting a Wnt signaling pathway, was added to the cells, followed by culturing for 2 days and continuous culturing in a RPMI1640+B27 medium, thereby obtaining beating cardiomyocytes (CMCs). The obtained CMCs were treated with 5 U SLO, thereby obtaining permeabilized cells, and the permeabilized cardiomyocytes were treated with mouse-anti-Tom20 (Santa Cruz), which is a representative marker of mitochondria, in a 1:100 dilution at 37 □ for 30 minutes, a secondary antibody, anti-mouse-Alexa Fluor 488, was incubated in a 1:100 dilution at 37 □ for 30 minutes. To regenerate the cell membrane of the cardiomyocytes which had been subjected to the antibody reaction, 2 mM CaCl.sub.2 was added to the same volume of the cell culture solution, followed by incubation at 37 □. As shown in FIG. 6A, the cardiomyocytes were visualized by live imaging in a living state, confirming that mitochondria were stained with Tom20, and through Z-stack, it was demonstrated that the cells were stained with Tom20 which had permeated through the cell membrane. The results are illustrated in FIG. 6B.

[0101] According to the method of the present invention, although a target protein is present in the cell, it can be detected and analyzed by a simple method using an antibody. In addition, isolation and culturing can be performed easily using a flow cytometer, and further, specific cells which are isolated and cultured can be attached to enable subculture and cryopreservation. In addition, conventionally, cells were able to be isolated and cultured only by targeting an extracellular membrane protein, but according to the method of the present invention, target cells of interest can be obtained more rapidly and accurately by forming a pore in various cell membranes using a streptococcal hemolytic exotoxin only with a frequently used flow cytometer and antibodies without limiting a specific protein target. Moreover, while, in the case of primary culture, cells can be isolated and cultured by targeting only a specific extracellular membrane protein with several complicated steps for a long time, the most important thing is, when the method of the present invention is used, homogeneous cells of interest, as well as various types of cells, can be obtained with a higher purity and a higher efficiency than the conventional method by targeting both the extracellular membrane and a specific intracellular protein without using complicated steps and taking a long time in primary culture. In addition, the present invention is a technique of isolating, analyzing and culturing desired cells by permeating an intracellular protein into the living cells using a commercially available monoclonal antibody without the manufacture of a probe such as a molecular beacon. By using these advantages, cells having various intracellular proteins, which were limited in research since they could not be isolated and cultured from a patient’s blood or various tissues in the past, can be easily detected, isolated and cultured, and the present invention is expected to highly contribute to research on the mechanisms of various diseases and treatment thereof, as well as various applications targeting a specific intracellular protein.

[0102] It will be apparent to those skilled in the art that various modifications can be made to the above-described exemplary embodiments of the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention covers all such modifications provided they come within the scope of the appended claims and their equivalents.