KR20080004881A - Method for preparing of mesenchymal stem cell by ultrasound treatment - Google Patents

Abstract

A method for preparing mesenchymal stem cells is provided to improve the adhesion capability of the mesenchymal stem cells by treating cells including the mesenchymal stem cells with ultrasonic waves, thereby easily mass-obtaining the pure mesenchymal stem cells having the normal differentiation capability from the cell including the mesenchymal stem cells. A method for preparing mesenchymal stem cells from a cell group containing the mesenchymal stem cells comprises the steps of: (a) culturing the cell group containing the mesenchymal stem cells in a culture medium with treating the cell or the culture medium with 5-50 minutes/day of supersonic waves having the intensity of 10-1,000 mW/cm^2 for 1-12 days; and (b) removing the cell not attached to the culture medium and obtaining colony(CFU-Fs) forming mesenchymal stem cells attached to the culture medium.

Description

Method for obtaining mesenchymal stem cells by ultrasonication {Method for Preparing of Mesenchymal Stem Cell by Ultrasound Treatment}

1 is a schematic diagram showing the ultrasonic treatment method of bone marrow-derived mesenchymal stem cells.

Figure 2 shows the number of CFU-Fs of bone marrow-derived mesenchymal stem cells according to the intensity of ultrasonic treatment, (A) shows the crystal violet staining results, (B) shows the number of CFU-Fs formed in a graph .

Figure 3 shows the results of staining the CFU-Fs of the bone marrow-derived mesenchymal stem cells formed with crystal violet when ultrasonic treatment at an intensity of 100 ㎽ / ㎠.

Figure 4 is a graph showing the number of CFU-Fs of bone marrow-derived mesenchymal stem cells formed when the ultrasound treatment at an intensity of 100 ㎽ / ㎠.

Figure 5 shows the results of flow cytometry (FACS) of the cell surface expression factor.

Figure 6 shows the results of flow cytometry (FACS) of the cell surface expression factor.

Figure 7 shows the results of confirming the expression of osteoblast differentiation markers in cells differentiated into bone marrow-derived mesenchymal stem cells, (A) shows the results of Alizarin Red S staining, (B ) Shows the results of the RT-PCR.

FIG. 8 shows the results of confirming the expression of adipocyte differentiation markers in cells differentiated from bone marrow-derived mesenchymal stem cells into adipocytes, (A) shows the results of Oil Red O staining, and (B ) Shows the results of the RT-PCR.

The present invention relates to a method for improving the yield efficiency of mesenchymal stem cells, and more particularly, the mesenchymal stem cell-containing cells adhered to the mesenchymal stem cell-containing cells isolated from the tissue containing mesenchymal stem cells. The present invention relates to a method for obtaining bone marrow-derived mesenchymal stem cells, characterized by improving ability and proliferation ability.

Bone marrow mesenchymal stem cells are stem cells that are present in bone marrow together with hematopoietic stem cells.Bone marrow mesenchymal stem cells are capable of proliferation and expansion during in vitro culture and, given appropriate culture environment, It has the ability to differentiate into cells of various lineages such as chondrocytes, adipocytes, myoblasts, hepatocytes, cardiomyocytes and neurons (MF Pittenger et al., 1999; RJ Deans et al., 2000). For this reason, many researches on bone marrow-derived mesenchymal stem cells for tissue regeneration have been conducted in the fields of cell biology and tissue engineering. Also, clinical trials have been conducted through transplantation of bone marrow-derived mesenchymal stem cells to myocardial and brain injury. (EM Horwitz et al., 1999; Quarto R et al., 2001).

The method of separating bone marrow-derived mesenchymal stem cells from the extracted bone marrow is as follows. In the process of culturing mononuclear cells separated by Ficoll density gradient from bone marrow in the culture flask, non-stick cells were continuously removed through medium exchange and homogenous bone marrow-derived mesenchymal stem cells were obtained through passage of cells adhered to the culture flask. You can get it. This method can easily separate the cells, but it is difficult to obtain pure bone marrow-derived mesenchymal stem cells, which has the disadvantage of obtaining homogenous cells through multiple passages. In order to remedy these shortcomings, as a method of obtaining more pure bone marrow-derived mesenchymal stem cells, a recent flow cytometry (FACS) analysis using cell surface markers (Melody Baddoo et al., 2003; Tatiana Tondreau) Et al., 2005). This method isolates and cultures only cells expressing specific cell surface markers from mononuclear cells separated by Ficoll density gradient. Cells isolated by this method are more homogeneous than cells isolated using Ficoll alone, but are disadvantageous because they are more expensive and have fewer bone marrow-derived mesenchymal stem cells initially available. Above all, however, the cell surface markers for identifying early bone marrow-derived mesenchymal stem cells are not known (Masakazu Ishii et al. 1005; Xiaoli Wang et al., 2005).

Since bone marrow-derived mesenchymal stem cells isolated from bone marrow are cultured in vitro and form a colony-like feature similar to fibroblasts, they are colonized forming unit-fibroblasts (hereinafter referred to as CFU). CFU-Fs was found to form colonies by proliferation of a bone marrow-derived mesenchymal stem cell (Castro-Malaspina H et al., 1980; Friedenstein A et al., 1970; Owen M et al. 1988). In other words, the number of CFU-Fs means the number of bone marrow-derived mesenchymal stem cells. The larger the number of CFU-Fs, the more bone marrow-derived mesenchymal stem cells can be obtained. It is used. However, the cells in the bone marrow are very heterogeneous (Paolo Bianco et al., 2001; D. Baksh et al., 2004), where the proportion of bone marrow-derived mesenchymal stem cells is quite low, one of the 1 × 10 ⁵ bone marrow cells ( Galotto M et al., 1999), among a small proportion of myeloid-derived mesenchymal stem cells, is known to have a group of CFU-Fs (Gronthos S et al. 2003; Kortesidis A et al. 2005). It is also known that the proportion of bone marrow-derived mesenchymal stem cells in the bone marrow decreases as the patient ages (Gianluca D'ippolito et al., 1999; Christine fehrer et al., 2005). As described above, bone marrow-derived mesenchymal stem cells have a self renewal capacity during in vitro culture and are easy to proliferate, but as the subculture progresses, the proliferation capacity decreases (Mandana Mohyeddin Bonab et al., 2006). There are limitations in obtaining large amounts of stem cells. In order to overcome these limitations, specific growth factors such as fibroblast growth factor (FGF) are added in culture (Giordano Bianch et al., 2003), or low vs high plating densities in culture ( Sekiya I et al., 2002) Many studies have been conducted to increase cell proliferation by regulating the cell culture environment (Takehiro Matsubara et al. 2003; Naomi Ogura et al. 2004: Panagiota A et al. 2005).

In addition to the treatment of growth factors or other cytokines in controlling the cell culture environment (chemical stimuli), a lot of research has recently been conducted to observe the changes in cells through physical and mechanical stimulation. Physical and mechanical stimuli used in the research include stretch, shear stress, electrical stimulation, and ultrasonic stimulation. In particular, in vitro experiments, such stimuli may be applied to integrin and cell surface markers of cells. It is known to affect the proliferation or differentiation of cells due to the change and the change in signaling. Ultrasonic stimulation can promote the differentiation of stem cells into chondrocytes or osteoblasts and is effective for fractures in clinical practice (Pilla AA et al., 1990; Hadjiargyrou M et al., 1998). ). Recently, many studies have been conducted to identify factors involved in signal transduction in order to understand the effect of ultrasound stimulation on cells (John Y.-J. Shyy et al. 2002; Zhou S et al. 2004; Yang RS et al. 2005). Although the factors involved in the signaling process involved in cell adhesion, migration, and proliferation were increased, so far, no studies have been conducted on the effects of ultrasound stimulation on the stem cells in the early stage of culture.

The present inventors have made diligent efforts to improve the adhesion of mesenchymal stem cells to obtain mesenchymal stem cells that form more CFU-Fs. Ultrasonic stimulation increases the adhesion of bone marrow-derived mesenchymal stem cells. The present invention was completed by confirming that a pure and large amount of bone marrow-derived mesenchymal stem cells can be obtained in a short period.

An object of the present invention is to obtain a pure and large amount of mesenchymal stem cells, the mesenchymal stem cells of the mesenchymal stem cells, characterized in that to improve the adhesion of the mesenchymal stem cells by treating the cells containing mesenchymal stem cells It is to provide a method for improving the yield efficiency.

In order to achieve the above object, the present invention comprises the steps of (a) culturing a group of cells containing mesenchymal stem cells in the incubator, while culturing while treating the cells or the incubator with ultrasound; And (b) removing the cells not attached to the incubator, and obtaining the mesenchymal stem cells from the mesenchymal stem cell-containing cell population comprising the step of obtaining colony (CFU-Fs) forming mesenchymal stem cells attached to the incubator. Provide a method.

In the present invention, the ultrasound may be characterized in that the treatment for 5 to 50 minutes / day from the beginning of the culture for 1 to 12 days, the ultrasonic wave may be characterized in that the treatment with 10 ~ 1000 ㎽ / ㎠ intensity.

In the present invention, the ultrasonic wave may be characterized in that it is treated in a pulsed type (continuous type) or continuous (continuous type), the incubator may be characterized in that the bottom is coated with collagen or fibronectin.

In the present invention, the cells are cultured in a state attached to the cell carrier, the incubator may be characterized in that the bioreactor (bioreactor).

The method for obtaining mesenchymal stem cells of the present invention can be applied not only to cells isolated from tissues in which mesenchymal stem cells such as cord blood, adipocytes, bone marrow, etc. exist, but also to be developed later.

In the present invention, the ultrasound was confirmed to promote the adhesion and proliferation of cells in the initial culture of mesenchymal stem cells. Bone marrow mononuclear cells were obtained from bone marrow by density gradient using Ficoll. CD29 (Integrin β ₁ chain), CD90 (Thy-1), CD29 (Thy-1), through flow cytometry (FACS) immediately after isolation and 12 days after culture. Expression of CD106 (VCAM-1) was increased, confirming that the attached cells were bone stem stem cells.

In the embodiment of the present invention, but the ultrasonic treatment was treated in a continuous (continuous type), the treatment ability of the mesenchymal stem cells could be improved even if the treatment at regular intervals in the pulsed type (pulsed type).

In the present invention, the incubator used for cell attachment can be used by coating with collagen or fibronectin to facilitate cell attachment. The collagen coating can be used by dispensing the Type I collagen solution added with 50 μg / ml concentration of 5 type I collagen in 0.02M acetic acid in the incubator and leaving it at room temperature for 1 hour. The fibronectin coating is applied to fibronectin in PBS. Fibronectin solution added at 50-100 μg / ml may be dispensed into the incubator and left for 1 hour before use.

CFU-Fs were analyzed after 12 days of culturing in the ultrasonic and non-treated groups to determine whether ultrasound affected the cell adhesion in the initial culture of mesenchymal stem cells. In vitro , one mesenchymal stem cell proliferates to form colonies and forms CFU-Fs. Thus, the number of CFU-Fs means the number of mesenchymal stem cells. In the present invention, the number and size of CFU-Fs were analyzed by crystal violet staining to confirm the difference between the two groups. In the initial culture group, the number of CFU-Fs was increased by about 50%, and the number of CFU-Fs over 5 mm was also increased by 50% in the ultrasonic group.

The above results indicate that the ultrasound treated in the present invention promotes the attachment and proliferation of mesenchymal stem cells, and as a result, is effective in obtaining a large number of mesenchymal stem cells in the early stage of culture.

Mesenchymal stem cells are proliferative in culture, and given an appropriate culture environment, cells having multipotents capable of differentiation into other strains such as bone cells, adipocytes, and chondrocytes. In the present invention, flow cytometry and differentiation into various cells were examined in order to confirm whether the ultrasonically treated cells maintain the characteristics of stemness and multipotency in order to obtain a large number of mesenchymal stem cells during initial culture. Although no clear cell surface factor for mesenchymal stem cells is known, flow cytometry was performed using cell surface factors for mesenchymal stem cells.

In the present invention, the cell carrier may be used without limitation as long as it is a cell carrier that can be used commonly such as polymer polymer beads, scaffolds, ECM membranes.

In the present invention, the mesenchymal stem cells isolated from the tissue can be transferred to the bioreactor containing the cell carrier and cultured, and the cell group can be transferred to the bioreactor and cultured after contacting the cell carrier.

The bioreactor may be used without limitation as long as the bioreactor for cell culture, and preferably, a bioreactor for stem cell culture is used.

In the present invention, cells obtained from the ultrasonic treatment group and the non- ultrasound treatment group at 12 days of culture were collected and subjected to flow cytometry for the cell surface factors of CD29, CD90, CD106, and CD45. It was confirmed that there is no difference in the expression of cell surface factors. In addition, to confirm the multipotency of the mesenchymal stem cells obtained from the two groups after two passages, the differentiation into osteoblasts and adipocytes was induced. Differentiation into individual cells under appropriate culture conditions was well induced in both groups by cell morphology, cytology, and RT-PCR. It was confirmed that the ultrasound treated early in culture had a good effect on the adhesion and proliferation of bone marrow-derived mesenchymal stem cells and did not affect the maintenance of stemness and multipotent characteristics.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Isolation and Culture of Mesenchymal Stem Cells

Inhalation of carbon dioxide to euthanize rats ( 250-300 g, male, Sprague-Dawley rat , Orient, Korea), remove the hind leg hair, disinfect the skin with povidin and 70% ethanol and dissecting the femur aseptically Separated. 6 ml of phosphate-buffered saline (PBS; Gibco, 21600-010, containing antibiotics [100 U / ml penicillin G (sigma, P-7794) and 100 µg / ml strepto-mycin (sigma, S-9137)) in a clean room The bone marrow was extracted using a syringe sterilized with Canada) to extract bone marrow, and then the pipette was used to completely release the bone marrow. Cells were filtered using a 100 μm pore size cell strainer (Falcon, USA), and 2 ml of Ficoll (Amersham Biosciences, 17-1440-02, SWEDEN) and 6 ml of cell wash solution were 1: 3. The cells were centrifuged at 2000 rpm for 30 minutes, and then only the yellowish brown layer of the buffy coat was separated to obtain monmonuclear cells. The separated cells were suspended in 10 ml of phosphate buffered saline, centrifuged at 1500 rpm for 5 minutes, and then resuspended in 10 ml of α-MEM (sigma, M-0644, USA) containing antibiotics. After staining with Blue (Gibco, 15250-061, Canada), the cell number was measured using a haemo-cytometer under an optical microscope.

Cells suspended in the culture medium were dispensed in a 60 mm culture vessel (TPP, Switzerland) coated with collagen or fibronectin at a concentration of 8 × 10 ⁴ / cm 2, followed by 10% FBS (fetal) in a 37 ° C., 5% CO ₂ incubator. bovine serum, Hyclone, Canada) and antibiotics added α-MEM. After 6 days of culture, the medium was continuously changed for 3 days after removing the suspended cells without adhered to the culture vessel through medium exchange.

Subculture was performed when the cells were filled with 80-90% of the culture vessel, and differentiation experiments were carried out after two passages.

Example 2: Treatment of Ultrasound

Sonication was performed using Noblelife ^™ (Noblelife ^™ , Duplogen, Korea), and the incubator was tightly fixed using an ultrasonic gel to prevent air contact between the bottom of the 60 mm incubator and the water wave during the ultrasonic treatment (FIG. 1). ).

In order to find the optimal ultrasound intensity for colony formation of bone marrow-derived mesenchymal stem cells, ultrasound was applied to the bone marrow-derived mesenchymal stem cells obtained in Example 1 from the first day of culture at 0 ㎽ / ㎠, 100 ㎽ / ㎠ and 200 ㎽ / Once a day at an intensity of cm 2, it was treated for 6 days in a continuous (continuous type) for 10 minutes. After 6 days of culture, cells suspended without attachment were removed by exchanging with fresh medium and further cultured for 6 days while exchanging with fresh medium once every 3 days. Ultrasound was not treated after medium exchange. At 12 days of culture, the cells were fixed with 95% ethanol and compared with CFU-Fs formation between each group through crystal violet staining (FIG. 2). As a result, more colonies were formed in the ultrasonic treatment group than in the ultrasonic treatment group, and the colonies of 3 to 5 mm in size were different from the ultrasonic intensity of 100 ㎽ / ㎠ and 200 ㎽ / ㎠ at 200 ㎽ / ㎠ and over 5 mm in size. Colonies were more at 100 μs / cm 2. However, since there was almost no difference in the total number of colonies between the two groups, in the following examples, all the sonication proceeded at an intensity of 100 mW / cm 2.

 Example 3: Confirmation of the effect of ultrasound on the CFU-Fs formation of mesenchymal stem cells

The number of CFU-Fs in the bone marrow-derived mesenchymal stem cell experimental group treated with the method of Example 2 and the control group not treated with ultrasound was confirmed by crystal violet staining. To check the CFU-Fs 12 days after the culture, the cultured cell layer was washed twice with phosphate buffered saline and fixed in 95% ethanol at room temperature for about 2 minutes. The fixed cells were washed three times with phosphate buffered saline, and then 5% crystal violet solution [crystal violet (sigma, C-3886, USA) 5g, methanol 100ml] was added, stained for 5 minutes, washed with water, and then at room temperature. Dried. The number of CFU-Fs with a diameter of 3 mm or more in the sonicated group and the control group was counted and compared.

The total number of CFU-Fs in the ultrasound-treated group was increased by 1.5 times more than the non- ultrasound-treated group. Through these results, the initial ultrasound treatment was effective for the attachment of bone marrow-derived mesenchymal stem cells and the formation of CFU-Fs. It can be seen that (Fig. 3 and 4).

In addition, bone marrow-derived mesenchymal stem cells, ALP, an early expression factor for osteoblast differentiation, which is known to differentiate into bone cells, were hardly expressed in both the ultrasonic treatment group and the control group.

 Example 4: Fluoresence-Activated Cell Sorting Analysis

Flow cytometry was performed to confirm the change of cell surface factors of the sonicated and control groups.

After washing the cells twice in 12 days of culture in 0.25% Trypsin-EDTA were removed by 2% FBS-containing phosphate-buffered saline, 1 × 10 ⁶ 0.5㎍ of Antibody per cell [(FITC-conjugated Hamster Anti- Rat CD29 (Integrin β ₁ chain), PE-Cy5-conjugated Mouse Anti-Rat CD45 (Leukocyte Common Antigen), R-PE-conjugated Mouse Anti-Rat CD106 (VCAM-1) and PerCP-conjugated Mouse Anti-Rat CD90 (Thy -1)] and reacted for 40 minutes in the dark at 4 ° C. After the attachment of the antibody, each cell was washed twice with phosphate buffered saline containing 2% FBS, followed by flow cytometry (FACS Vantage, Beckon). Dickinson, USA) was used to confirm the difference in cell surface factor expression.

Flow cytometry (FACS) was performed with antibodies of CD 29 (Integrin β ₁ chain), CD 90 (Thy-1), and CD 106 (VCAM-1), which are widely known as cell surface factors of bone marrow-derived mesenchymal stem cells. As a result, it was expressed in both the ultrasonic treatment group and the control group, and there was almost no difference between the two groups. It was a cell surface factor of hematopoietic stem cells and used as negative control (Leukocyte Common Antigen). ) Was found to be rarely expressed in both groups (FIGS. 5 and 6). According to the above results, it can be seen that the ultrasonic wave treated at the early stage of culture is effective in increasing the adhesion rate of cells, but does not significantly affect the change and stemness of the cell surface factors of bone marrow-derived mesenchymal stem cells.

 Example 5: Differentiation of BMSSCs

Differentiation experiments were performed to determine whether sonication affects the differentiation capacity of mesenchymal stem cells.

Ultrasonically treated bone marrow-derived mesenchymal stem cells and control group bone marrow-derived mesenchymal stem cells were induced to differentiate into osteoblasts and adipocytes after two µg passages.

Induction of differentiation into osteoblasts was carried out using 5 × 10 ⁴ bone marrow-derived mesenchymal stem cells (2 × 10 ³ cells / cm ² ) in a 60 mm diameter incubator with 50 μg / ml ascorbate-2 in α-MEM containing 10% FBS. After addition of phosphate (sigma, A-8960) and 0.1 μM dexamethasone (sigma, D-8893) was performed by incubation for 3 weeks. Induction of differentiation into adipocytes was carried out in the same culture vessel as described above, 5 × 10 ⁵ cells (2 × 10 ⁴ cells / cm ² ) in 0.5 mM IBMX (sigma, I-5879) in α-MEM containing 10% FBS, 10 μg / ml insulin (sigma, I-9278), 0.1 mM indomethacin (sigma, I-8280) and 1 μM dexamethasone were also added and cultured for 3 weeks (Mark F. Pittenger et al., 1999). The media for differentiation induction were exchanged once every 3 days, and cells were harvested or fixed at 3 weeks of culture for RT-PCR and cytology.

RT-PCR was performed by the following method.

RNA was extracted from the cells cultured using TRIzol ^ㄾ (Invitrogen, 15596-018, USA) and quantified, and 1 μg of total RNA was used for reverse transcription (RT). RT was synthesized cDNA using 1 ^st stand cDNA Synthesis Kit (AMV) (Roche, 1-483-188, Germany), 0.5 μg of cDNA synthesized in PCR PreMix (Bioneer, K-2016, Korea), each 1 μl of specific primer [GAPDH, osteopontin, Lipoprotein lipase, peroxisome proliferator activated receptor gamma 2; Table 1], after adding DEPC (diethyl pyrocarbonate) -treated water (total 20ul), PCR was carried out under the conditions shown in Table 2.

Molecular Marker Primers Used in Examples primer order PCR product GAPDH S: 5'-GGCATTGCTCTCAATGACAA-3 '(SEQ ID NO: 1) A: 5'-TGTGAGGGAGATGCTCAGTG-3' (SEQ ID NO: 2) 223bp Osteopontin S: 5'-AGAGGAGAAGGCGCATTACA-3 '(SEQ ID NO: 3) A: 5'-GCAACTGGGATGACCTTGAT-3' (SEQ ID NO: 4) 500 bp LPL S: 5'-CAGCTGGGCCTAACTTTGAG-3 '(SEQ ID NO: 5) A: 5'-TGCTGGGGTTTTCTTCATTC-3' (SEQ ID NO: 6) 305 bp PPARγ2 S: 5'-CCCTGGCAAAGCATTTGTAT-3 '(SEQ ID NO: 7) A: 5'-ACTGGCACCCTTGAAAAATG-3' (SEQ ID NO: 8) 222 bp

PCR conditions for each differentiation factor GAPDH Osteopontin LPL PPARγ2 Denaturation  94 ℃, 5 ' Denaturation  94 ° C, 1 ' Annealing 55 ℃, 1 ' 57 ℃, 1 ' 53 ℃, 1 ' 55 ℃, 1 ' Extension 72 ° C., 1 ' Cycle 30 27 33 30 Extension 72 ℃, 5 '

Cytological examination was confirmed by staining in the following manner.

To confirm the differentiation of mesenchymal stem cells differentiated into osteoblasts into osteoblasts, cells at the 3rd week of differentiation were washed twice with phosphate buffered saline, fixed with 4% paraformaldehyde, and then washed twice with phosphate buffered saline. After staining with Alizarin Red S (sigma, A-5533) solution (40mM, pH 4.2) for 5 minutes and washed three times with 3 times.

Oil Red O (sigma, O-0625) staining was performed to confirm the differentiation into adipocytes. The cultured cells were washed twice with phosphate buffered saline, fixed in Formal Calcium solution (40% Formalin: 10% CaCl ₂ : 3rd order = 1: 1: 8) for 1 hour, and washed once with 60% isopropanol. It was. After staining with 0.18% Oil Red O solution for 15 minutes, the cells were washed twice with 60% isopropanol and stained with an optical microscope.

The bone marrow-derived mesenchymal stem cells of the ultrasonic treatment group and the control group were analyzed by cytological examination and RT-PCR after induction of differentiation into osteoblasts and adipocytes. Was found to be absent (FIGS. 7 and 8). From the above results, it can be seen that the ultrasound treated in the early stage of culture to increase the adhesion rate of the cells does not affect the differentiation capacity and stemness of bone marrow-derived mesenchymal stem cells.

The present invention has the effect of providing a method for improving the efficiency of obtaining mesenchymal stem cells, characterized in that to improve the adhesion of mesenchymal stem cells by treating the cells containing mesenchymal stem cells. According to the present invention, pure mesenchymal stem cells having normal differentiation ability can be easily obtained in large quantities from cells including mesenchymal stem cells.

Having described the specific parts of the present invention in detail, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. will be.

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Claims (6)

Method for obtaining mesenchymal stem cells from the mesenchymal stem cell-containing cell population comprising the following steps: (A) culturing a group of cells containing mesenchymal stem cells in the incubator, while culturing while treating the cells or incubator with ultrasound; And (b) removing cells not attached to the incubator and obtaining colony (CFU-Fs) forming mesenchymal stem cells attached to the incubator. The method of claim 1, wherein the ultrasonic wave is treated for 5 to 50 minutes / day from the beginning of the culture for 1 to 12 days. The method of claim 1, wherein the ultrasonic wave is treated at an intensity of 10 to 1000 mW / cm 2. The method of claim 1, wherein the ultrasonic waves are processed in a pulsed type or a continuous type. The method of claim 1, wherein the incubator is coated with collagen or fibronectin at the bottom. The method of claim 1, wherein the cells are cultured with a cell carrier attached and the incubator is a bioreactor.

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