WO2008004752A1

WO2008004752A1 - Method for preparing mesenchymal stem cells by ultrasonic treatment

Info

Publication number: WO2008004752A1
Application number: PCT/KR2007/002031
Authority: WO
Inventors: Byoung-Hyun Min; So Ra Park; Woo-Hee Choi
Original assignee: Regenprime Co., Ltd.
Priority date: 2006-07-07
Filing date: 2007-04-25
Publication date: 2008-01-10
Also published as: KR100808546B1; KR20080004881A

Abstract

The present invention relates to a method for improving the efficiency of obtaining mesenchymal stem cells (MSCs), more particularly, a method for obtaining bone marrow-derived mesenchymal stem cells, the method comprising enhancing cell adhesion capacity and proliferation of mesenchymal stem cells (MSCs) by treating MSC-containing cells isolated from MSC-containing tissues with ultrasound. According to the present invention, a great amount of pure MSCs having a normal differentiation capacity can be easily obtained from a group of cells containing MSCs, compared to the existing methods.

Description

METHOD FOR PREPARING MESENCHYMAL STEM CELLS BY ULTRASONIC TREATMENT

TECHNICAL FIELD

The present invention relates to a method for improving the efficiency of obtaining mesenchymal stem cells (MSCs), more particularly, a method for obtaining bone marrow-derived MSCs, the method comprising treating MSC- containing cells isolated from MSC-containing tissues with ultrasound to enhance the ability of MSCs to adhere to culture plates and to proliferate well.

BACKGROUND ART

Bone marrow-derived MSCs are stem cells which co-exist with hematopoietic stem cells (HSCs) in the bone marrow. They can proliferate and expand well in an in vitro culture, and have the ability to differentiate into various cells such as osteocytes, chondrocytes, adipocytes, myocytes, hepatocytes, cardiac muscle cells, and neuronal cells if appropriate culture conditions are given (Pittenger, M.F. et al., Science, 284:143, 1999; Deans, RJ. et al., Exp. Hematol., 28: 875, 2000). With these reasons, studies on bone marrow-derived MSCs for tissue regeneration have been much progressed in the field of cell biology and tissue engineering, and clinical studies on myocardial damage and brain damage have been carried out through stem cell transplantation (Horwitz, E.M. et al., Nat. Med., 5:309, 1999; Quarto, R. et al, N. Engl. J. Med., 344:385, 2001).

Recently, as a method for obtaining pure bone marrow-derived MSCs, FACS analysis using cell surface markers has been mainly used (Baddoo, M. et al., J. Cell. Biochem., 89: 1235, 2003; Tondreau, T. et al., Stem Cells, 23: 1105, 2005). This method is a method performed by separating cells expressing a specific cell surface marker from mononuclear cells isolated by Ficoll density gradient and culturing them, and cells obtained by the method are more homogeneous than cells isolated using only Ficoll. However, the method has disadvantages in that extra cost is incurred as well as the number of bone marrow-derived MSCs that can be obtained in the initial state is low. Above all, it has a huge limitation since cell surface markers capable of confirming the initial bone marrow-derived MSCs have not been revealed (Ishii, M. et al, Biochem. Biophys. Res. Commun., 332:297, 2005; Wang, X. et al, Stem Cells, 24:482, 2006).

Mesenchymal stem cells derived from bone marrow are initially recognized by colony-forming unit-fibroblasts (CFU-Fs) because the bone marrow-derived MSCs form colonies and have features similar to fibroblasts while being cultured in vitro. It is known that one CFU-F (colony) is derived from a single bone marrow-derived MSC (Castro-Malaspina, H. et al, Blood, 56:289, 1980; Friedenstein, A. et al, Cell Tissue Kine., 3:393, 1970; Owen, M. et al, Ciba. Found. Symp., 136:42, 1988). Since the number of CFU-Fs refers to the number of bone marrow- derived MSCs, CFU-Fs are much used for studies on bone marrow-derived MSCs. A large number of bone marrow-derived MSCs can be obtained as the number of CFU-Fs increases.

However, cells in the bone marrow are very heterogeneous (Bianco, P. et al, Stem Cells, 19: 180, 2001 ; Baksh, D. et al, J. Cell MoI. Med., 8:301, 2004) and the rate of MSCs in the bone marrow is one per l * 10⁵ bone marrow cells, which is considerably low (Galotto, M. et al, Exp Hematol., 27: 1460, 1999). It is believed that a separate group of cells exists having capacity to form CFU-Fs among the low rate of bone marrow-derived MSCs (Gronthos, S. et al, J. Cell Science, 116: 1827, 2003; Kortesidis, A. et al, Blood, 105:3793, 2005). Also, it has been ascertained that the rate of bone marrow-derived MSCs in the bone marrow decreases as patient's age increases (D'ippolito, G. et al, J. Bone Mine. Re., 14: 1115, 1999; Fehrer, C. et al, Exp. Gerontol., 40:926, 2005).

Although bone marrow-derived MSCs having the self-renewal capacity easily proliferate in an in vitro culture, their proliferation capacity becomes lower rapidly during the repeated subcultures, which limits obtaining a large amount of pure MSCs (Bonab, M.M. et al, BMC Cell Biol., 7: 14, 2006). In order to overcome this limitation, studies on increasing the MSCs proliferation capacity have been being conducted by controlling cell culture conditions, such as adding specific growth factors like bFGF (basic Fibroblast growth factor) (Bianchi, G. et al., Exp. Cell Res., 287:98, 2003) or plotting plating cell densities in culture (Sekiya, I. et al., Stem Cells, 20:530, 2002), and the like (Matsubara, T. et al., Biochem. Biophys. Res. Commun., 313:503, 2004; Ogura, N. et al., J. Oral Sci., 46:207, 2004: Sotiropoulou, P.A. et al., Stem Cells, 24:462, 2006).

Recently, studies on controlling cell culture conditions through physical or mechanical stimuli have increased, other than treating growth factors or cytokines (chemical stimuli). The physical or mechanical stimuli include stretch, shear stress, electric stimuli, ultrasound stimuli and so forth. Particularly, in vitro experiments revealed that these stimuli cause changes in the activity of integrins on cell membrane and the expression of cell surface markers, thus resulting in a change in signal transmission so that cell proliferation or differentiation is affected. Ultrasound stimuli can accelerate the differentiation of stem cells into chondrocytes or osteocytes and are widely used for fracture healing in real clinical practices (Pilla, A.A. et al., J. Orthop. Trauma, 4:246, 1990; Hadjiargyrou, M. et al, Clin. Orthop. Relat. Res., 355:216, 1998). In order to know how ultrasound stimuli have an effect on cells, recent studies on finding factors involved in signaling pathway are being actively conducted (Shyy, J.Y. et al., Cir. Res., 91 :769, 2002; Zhou, S. et al, J. Biol Chem., 279:54463, 2004; Yang, R.S. et al, Bone, 36:276, 2005). As a result, it was found that factors involved in signaling processes of cell adhesion, migration and proliferation increased, but up to now, studies on how ultrasound stimuli affect stem cells at the initial culture have not been reported yet.

Thus, the present inventors have made extensive efforts to obtain a large amount of MSCs forming large number of CFU-Fs by enhancing adhesion capacity of MSCs, as a result, have found that a great amount of pure bone marrow- derived MSCs can be obtained in a short period of time at the initial culture by improving adhesion capacity of bone marrow-derived MSCs through ultrasound stimuli, thereby completing the present invention.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for enhancing the efficiency of obtaining MSCs, the method comprising improving adhesion capacity of MSCs by treating cells containing MSCs with ultrasound in order to obtain a large amount of pure MSCs.

In order to achieve the above object, the present invention provides a method for obtaining MSCs from a group of cells containing MSCs, the method comprising the steps of: (a) culturing a group of cells containing MSCs in a culture vessel while treating the cells or the culture vessel with ultrasound; and (b) removing cells which are not attached to the culture vessel and obtaining MSCs attached to the culture vessel, which form CFU-Fs.

Another features and embodiments of the present invention will be more clarified from the following detailed description and the appended claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram illustrating how bone marrow-derived MSCs are treated with ultrasound.

FIG. 2 shows the number of CFU-Fs of bone marrow-derived MSCs according to intensity of ultrasonic treatment; (A) is the result of crystal violet staining, and (B) is the number of the formed CFU-Fs, quantitatively represented in a histogram.

FIG. 3 shows the results obtained by crystal violet staining of CFU-FS of bone marrow-derived MSCs formed by ultrasonic treatment at the intensity of 100 mW/cπf.

FIG. 4 is a graph showing the number of CFU-Fs of bone marrow-derived MSCs formed by ultrasonic treatment at the intensity of 100 mW/cnf.

FIG. 5 and 6 represent the results of FACS analyses of cell surface marker expression.

FIG. 7 shows the expression of osteogenic differentiation markers when bone- marrow-derived MSCs differentiate into osteocytes; (A) is the result of Alizarin Red S staining, and (B) is the result of RT-PCR.

FIG. 8 shows the expression of adipogenic differentiation markers when bone- marrow-derived MSCs differentiate into adipocytes; (A) is the result of Oil Red O staining, and (B) is the result of RT-PCR.

DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED EMBODIMENTS

The present invention relates to a method for enhancing the efficiency of obtaining MSCs, the method comprising improving adhesion capacity of MSCs by treating cells containing MSCs with ultrasound. More particularly, the present invention relates to a method for obtaining MSCs from a group of cells containing MSCs, the method comprising the steps of: (a) culturing a group of cells containing MSCs in a culture vessel while treating the cells or the culture vessel with ultrasound; and (b) removing cells which are not attached to the culture vessel and obtaining MSCs attached to the culture vessel, which form CFU-Fs.

In the present invention, the ultrasonic wave is preferably treated for 5-50 minutes per day for 1-12 days from initial culture, and preferably treated at the intensity of 10-1000 mW/cnf.

In the present invention, the ultrasonic wave is preferably delivered as either pulsed or continuous application, and the bottom of the culture vessel is preferably coated with collagen or fibronectin. In the present invention, the cells are preferably cultured while being attached to a cell carrier, and the culture vessel is preferably a bioreactor.

The method for obtaining MSCs according to the present invention can be applied not only to cells isolated from tissues such as umbilical cord blood, adipocytes, bone marrow and the like where it is known to have MSCs but also to MSCs which will be developed at a later time.

In the present invention, it was found that the ultrasonic wave accelerated cell adhesion and proliferation during the initial culture of MSCs. Bone marrow mononuclear cells were obtained from bone marrow by Ficoll density gradient and it was confirmed that the attached cells are bone marrow-derived stem cells due to an increase in the expression of CD29 (Integrin β_\ chain), CD90 (Thy-1), and CD 106 (VCAM-I) through FACS analysis right after isolation and 12 days after culture, respectively. In the examples of the present invention, ultrasonic wave was delivered as continuous application, but adhesion capacity of MSCs could be improved even though ultrasonic wave was delivered at a given interval as pulsed application.

In the present invention, the culture vessel used for cell adhesion can be coated with collagen and fϊbronectin to facilitate cell adhesion. Collagen coating is carried out by dispensing type I collagen solution obtained by adding 50 μg/ml type I collagen to 0.02M acetic acid into a culture vessel and leaving it to stand at ambient temperature for 1 hour to use. In case of fϊbronectin coating, 50-100 /zg/m-C fibronectin is added to PBS in order to prepare fϊbronectin solution, which is dispensed into a culture vessel and then this solution is left to stand for 1 hour to use.

In order to examine whether or not ultrasonic treatment affects cell adhesion during the initial culture of MSCs, CFU-Fs were analyzed in ultrasound treatment group and untreatment group 12 days after incubation. Since one MSC proliferates and forms colonies, thus resulting in CFU-Fs in vitro, the number of CFU-Fs refers to the number of MSCs. In the present invention, the number and size of CFU-Fs were analyzed by crystal violet staining to find the difference between the ultrasound treatment group and the untreatment group. In the ultrasonic treatment group at the initial culture, CFU-Fs showed about 50% increase in number as well as approximately 50% increase in number of over 5- mm long CFU-Fs.

The aforementioned results suggest that ultrasonic treatment accelerates adhesion and proliferation of MSCs so that it is effective to obtain a large number of MSCs at the initial culture.

Mesenchymal stem cells have an excellent proliferation capacity in culture and they are multipotent cells capable of differentiating into different lineages such as osteocytes, adipocytes, and chondrocytes if proper culture conditions are given. In the present invention, in order to obtain a large number of MSCs at the initial culture, FACS analysis and tests for differentiation capacity into many cell types were performed to confirm whether ultrasound-treated cells maintain sternness and multipotent capacity. Although definite cell surface markers of MSCs were not known yet, FACS analysis was carried out using cell surface markers of MSCs, which have been reported so far.

In the present invention, there is no limitation for types of cell carriers as long as they are generally used carriers such as high molecular polymer beads, scaffolds, ECM membranes and the like.

In the present invention, MSCs isolated from tissues can be cultured by transferring them into a bioreactor containing the cell carriers, and after contacting a group of cells with cell carriers, they can be cultured by transferring to a bioreactor. If the bioreactor is a bioreactor for cell culture, it can be used without limitations. It is preferable to use a bioreactor for stem cell culture.

In the present invention, cells obtained from ultrasonic treatment and untreatment groups at 12 days after culture were collected to carry out FACS analysis of cell surface markers CD29, CD90, CD 106, and CD45. As a result, it was seen that there was no difference of the expression of cell surface markers in ultrasound treatment group and untreatment group. Also, in order to examine multipotent capacity of MSCs obtained from the two groups, the cells were subcultured 2 times to induce osteogenic and adipogenic differentiation. It was confirmed through cell morphological test, cytological examination, and RT-PCR that differentiation into each cell type under proper culture conditions was successfully induced in both groups. These results revealed that ultrasonic treatment at the initial culture has positive effects on adhesion and proliferation of bone marrow-derived MSCs but has no effect on maintaining properties of sternness and multipotent capacity.

Examples

Hereinafter, the present invention will be described in more detail by examples. It will be obvious to a person skilled in the art, however, that these examples are for illustrative purpose only and are not construed to limit the scope of the present invention.

Example 1: Isolation and culture of mesenchymal stem cells

After white rats (weight 250~300g, male, Sprague-Dawleyrate rat, orient, Korea) were sacrificed by carbon dioxide inhalation, the fur was completely removed from both hind limbs and sterilized with povidone iodine and 70% ethanol to dissect skin, thereby aseptically isolating the femur. In a sterile room, a syringe sterilized by 6 ml of PBS (Gibco, 21600-010, Canada) containing antibiotics [lOOU/mfc of penicillin G (Sigma, P-7794) and 100 μg/ml of streptomycin (Sigma, S-9137)] was inserted into the femur to extract bone marrow aspirates, and then coagulated bone marrow was dissolved using a pipette. Cells were filtered using a 100-μm-pore-size cell strainer (Falcon, USA), and then centrifuged at 2000rpm for 30 minutes at a ratio of 1 :3(2 ml of Ficoll (Amersham Biosciences, 17-1440-02, Sweden) to 6 ml of cell wash buffer), followed by separating a yellow-brown buffy coat layer to obtain mouse mononuclear cells. The isolated cells were suspended in 10 ml of PBS and centrifuged at 1500rpm for 5 minutes. The cell pellets were resuspended in 10 ml of an α-MEM (ct- minimum essential medium, Sigma, M-0644, USA) with antibiotics. Some of the cells were stained with Trypan Blue (Gibco, 15250-061, Canada) to count cell number using a hemocytometer under a microscope. The cells suspended in culture broth were plated at a concentration of 8x 10⁴/cπf on a 60mm culture dish(TPP, Switzerland) coated with collagen or fibronectin and cultured in a α-MEM medium with 10% FBS (fetal bovine serum, Hyclone, Canada) and antibiotics at 37 ^"C under 5% CO₂. At day 6, the culture was replenished with a fresh medium to remove non-adherent cells and the adherent MSCs were maintained by continuously replacing the medium once every three days. When the secondary subcultures of MSCs (passage 2) were 80%~90% confluent, subculture was carried out and second passage cells were used for differentiation experiments.

Example 2; Ultrasonic treatment

Noblelife (Duplogen, Korea) was used for ultrasonic treatment, and a culture dish was tightly fixed using a gel for ultrasonic apparatus to prevent air gap formation between the bottom of the 60mm culture dish and the transducer of Noblelife upon ultrasonic treatment (FIG. 1).

In order to look for the optimum ultrasonic intensity for colony formation of bone marrow-derived MSCs, bone marrow- derived MSCs obtained from example 1 were treated with ultrasonic wave at an intensity of 0 mW/cπf, 100 mW/cπf and 200 mW/cπf, respectively, for 10 minutes once a day for 6 days starting on the first day of culture as a continuous application. The non-adherent, suspended cells were removed by new medium replacement at the sixth day and then the remaining cells were cultured for an additional 6 days with medium replacement once every three days. After changing a medium, ultrasonic treatment was not performed.

12 days after the culture, the cells were fixed with 95% ethanol and CFU-Fs formation was compared between each group through crystal violet staining (FIG. 2). Consequently, the ultrasonic treatment group formed more colonies than the ultrasonic untreatment group and 3~5mm colonies were found more at an intensity of 100 mW/cπf, over 5mm colonies were found more at an intensity of 200 mW/cπf in comparing ultrasonic intensity difference (100 mW/cnf and 200 mW/cπf). However, since total colony numbers between two groups are almost same, all ultrasonic treatments in the following examples were carried out at an intensity of 100 mW/cπf.

Example 3: Effect of ultrasonic treatment on CFU-Fs formation of MSCs

Crystal violet staining was performed using the same method described in Example 2 to examine the number of CFU-Fs in the ultrasound-treated experimental group and the untreated control group of bone marrow-derived MSCs. In order to check CFU-Fs, the 12-day cultured cell layer was washed with PBS twice and fixed with 95% ethanol for 2 minutes at ambient temperature. The fixed cells are washed with PBS three times, and then 5g of 5% crystal violet (Sigma, C-3886, USA) and 100 ml of methanol were added to dye the cells for 5 minutes, followed by washing them with water to dry at room temperature. The number of CFU-Fs with a diameter of over 3mm was counted and compared in the ultrasound-treated experimental group and the non-treated control group.

As a result, the total number of CFU-Fs in the ultrasound-treated group showed 1.5-fold increase compared to the non-treated group. This result revealed that ultrasonic treatment at the initial culture is effective for adhesion of bone marrow-derived MSCs and formation of CFU-Fs (FIG. 3 and 4).

Furthermore, the expression marker of the initial osteogenic differentiation, ALP, provides information on osteogenic differentiation of bone marrow-derived MSCs but was rarely expressed in both the ultrasound-treated experimental group and the ultrasound-untreated control group. Example 4; Fluorescence-Activated Cell Sorting (FACS) analysis

FACS analysis was performed in order to investigate a change of cell surface markers in the ultrasound- treated group and the ultrasound-untreated group. The 12-day cultured cells were detached using 0.25% Trypsin- EDTA and washed with PBS containing 2% FBS twice, and then l * 10⁶ cells were incubated for 40 minutes at 4 °C in a dark room by treating 0.5 μg of antibody [(FITC-conjugated Hamster Anti-Rat CD29 (Integrin /3j chain), PE-Cy5-conjugated Mouse Anti-Rat CD45 (Leukocyte Common Antigen), R-PE-conjugated Mouse Anti-Rat CD 106 (VCAM-I) and PerCP-conjugated Mouse Anti-Rat CD90 (Thy- 1)], respectively. After reacting with antibodies, cells of the control group and experimental group were washed again with PBS containing 2% FBS twice and the expression of cell surface markers was examined using FACS Vantage (Beckon Dickinson, USA).

As a result of FACS analysis using antibodies of CD29 (Integrin β_\ chain), CD90 (Thy-1) and CD 106 (VCAM-I), which are well known as cell surface markers of bone marrow-derived MSCs, all the markers were expressed in both the ultrasound-treated group and the ultrasound-untreated group and there was no difference between the two groups. On the other hand, CD45 (Leukocyte Common Antigen), a cell surface marker of hematopoietic stem cell, which was used for a negative control was hardly expressed in both groups (FIG. 5 and 6). From the result, it could be confirmed that ultrasonic treatment at the initiate culture was effective for enhancing cell adhesion capacity but did not largely affect sternness and a change of cell surface markers in bone marrow- derived MSCs.

Example 5: Differentiation of Bone Marrow-derived Mesenchymal Stem Cells (BMMSCs)

Differentiation experiments were performed to examine whether or not ultrasound treatment affects differentiation capacity of MSCs. Ultrasound-treated bone marrow-derived MSCs (experimental group) and ultrasound-untreated bone marrow-derived MSCs (control group) were induced to differentiate into osteoblasts and adipocytes after second subculture (the cells at passage 2).

5* 10⁴ BMMSCs (2x l0³cells/cm²) were cultured using α-MEM containing 10% FBS added with 50 μg/πύ ascorbate-2 phosphate (Sigma, A-8960) and O.lμM dexamethasone (Sigma, D-8893) in a 60mm culture dish, for 3 weeks to induce osteogenic differentiation.

Also, 5x lO⁵ BMMSCs (2^χ l0⁴cells/cm²) were cultured using α-MEM containing 10% FBS added with 0.5mM IBMX (Sigma, 1-5879, USA), lO μg/ml insulin (Sigma, 1-9278, USA), 0.ImM indomethacin (Sigma, 1-8280) and lμM dexamethasone, in the same culture dish as the above for 3 weeks to induce adipogenic differentiation (Pittenger, M.F. et al., Science, 284: 143, 1999). The medium for differentiation induction was replaced once every three days, and the cells at week 3 after culture were selected and fixed for RT-PCR and cytological examination.

RT-PCR was carried out as follows: TRIzol (Invitrogen, 15596-018, USA) was used to extract RNA from the cultured cells. After quantifying total RNAs, 1 μg of total RNA was taken for reverse transcription (RT), which comprises the steps of; (1) synthesizing cDNA using 1^st stand cDNA Synthesis Kit (AMV) (Roche, 1- 483-188, Germany), (2) adding 0.5 μg of synthesized cDNA, 1 μi of each specific primer [GAPDH, osteopontin, Lipoprotein lipase, peroxisome proliferator activated receptor gamma 2; Table 1] and DEPC(diethyl pyrocarbonate)-treated water to PCR PreMix (Bioneer, K-2016, Korea) to a total volume of 20 μi, and (3) performing PCR according to the condition shown in Table 2. Table 1 : Primer lists of molecular markers used in Example

Table 2: PCR condition according to each molecular marker

Cytological characteristics were examined by the following method using staining. In order to confirm osteogenic differentiation of MSCs, the cells at week 3 after differentiation induction were washed with PBS two times and fixed with 4% paraformaldehyde, followed by washing them with PBS again twice. Next, the cells were stained with Alizarin Red S (Sigma, A-5533) solution (4OmM, pH 4.2) for 5 minutes and washed with ultra-pure water three times.

In order to observe adipogenic differentiation, Oil Red O (Sigma, O-0625) was used. The cultured cells were washed with PBS two times and fixed with Formal Calcium solution (40% Formalin : 10% CaCl₂ : ultra-pure water = 1 : 1 :8) for 1 hour, and then washed with 60% isopropanol once. The cells were stained with 0.18% Oil Red O solution for 15 minutes and washed quickly with 60% isopropanol two times, thereby observing the stained cells with a microscope. After bone marrow-derived MSCs of the ultrasound-treated group and the ultrasound-untreated group (control) were induced to differentiate into osteoblasts and adipocytes, cytological examinations and RT-PCR were performed. As a result, the differentiation was found in both groups and there was no distinct difference between the differentiation level of experimental group and that of control group(FIG. 7 and 8). From the result, it was found that ultrasonic treatment at the initial culture for enhancing cell adhesion capacity does not affect differentiation capacity and sternness of bone marrow-derived MSCs.

INDUSTRIAL APPLICABILITY

As described in detail above, the present invention has an effect to provide a method for improving the efficiency of obtaining mesenchymal stem cells (MSC), the method comprising enhancing cell adhesion capacity by treating cells containing MSCs with ultrasound. According to the present invention, a large amount of pure MSCs having normal differentiation capacity can be easily obtained from cells containing MSCs.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMSWhat is Claimed is:

1. A method for obtaining MSCs from a group of cells containing MSCs, the method comprising the steps of:

(a) culturing the group of cells containing MSCs in a culture vessel while treating the cells or the culture vessel with ultrasound; and

(b) removing cells which are not attached to the culture vessel and obtaining MSCs attached to the culture vessel, which form colonies(CFU-Fs).

2. The method for obtaining MSCs from a group of cells containing MSCs according to claim 1 , wherein the ultrasonic wave is treated for 5-50 minutes per day for 1-12 days starting from the initial culture.

3. The method for obtaining MSCs from a group of cells containing MSCs according to claim 1, wherein the ultrasonic wave is treated at an intensity of 10-1000 mW/cπf

4. The method for obtaining MSCs from a group of cells containing MSCs according to claim 1, wherein the ultrasonic wave is delivered as a pulsed or continuous application.

5. The method for obtaining MSCs from a group of cells containing MSCs according to claim 1, wherein the bottom of the culture vessel is coated with collagen or fibronectin.

6. The method for obtaining MSCs from a group of cells containing MSCs according to claim 1 , wherein the cells are cultured while being attached to a cell carrier, and the culture vessel is a bioreactor.