KR100808546B1 - Method for Preparing of Mesenchymal Stem Cell by Ultrasound Treatment - Google Patents

Method for Preparing of Mesenchymal Stem Cell by Ultrasound Treatment Download PDF

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KR100808546B1
KR100808546B1 KR1020060063752A KR20060063752A KR100808546B1 KR 100808546 B1 KR100808546 B1 KR 100808546B1 KR 1020060063752 A KR1020060063752 A KR 1020060063752A KR 20060063752 A KR20060063752 A KR 20060063752A KR 100808546 B1 KR100808546 B1 KR 100808546B1
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박소라
최우희
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Abstract

본 발명은 중간엽 줄기세포의 수득 효율을 증진시키는 방법에 관한 것으로, 더욱 자세하게는 중간엽 줄기세포를 함유하는 조직에서 분리된 중간엽 줄기세포 함유 세포군에 초음파를 처리하여 중간엽 줄기세포의 세포부착능력 및 증식능력을 향상시키는 것을 특징으로 하는 골수유래 중간엽 줄기세포의 수득 방법에 관한 것이다.The present invention relates to a method for enhancing the yield efficiency of mesenchymal stem cells, and more specifically, to the mesenchymal stem cell-containing cell group isolated from the tissue containing mesenchymal stem cells by ultrasonic treatment of cell adhesion of 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.

본 발명에 따르면, 중간엽 줄기세포를 함유하는 세포군으로부터 정상적인 분화능력을 가지는 중간엽 줄기세포를 기존의 방법에 비하여 대량으로 수득할 수 있다.According to the present invention, mesenchymal stem cells having normal differentiation ability can be obtained in large quantities from the cell group containing mesenchymal stem cells as compared with the conventional methods.

중간엽 줄기세포, CFU-Fs, 세포 증식, 세포부착능력, 세포분화 Mesenchymal stem cells, CFU-Fs, cell proliferation, cell adhesion, cell differentiation

Description

초음파 처리에 의한 중간엽 줄기세포의 수득방법{Method for Preparing of Mesenchymal Stem Cell by Ultrasound Treatment}Method for obtaining mesenchymal stem cells by ultrasonication {Method for Preparing of Mesenchymal Stem Cell by Ultrasound Treatment}

도 1은 골수유래 중간엽 줄기세포의 초음파 처리 방법을 나타낸 모식도이다.1 is a schematic diagram showing the ultrasonic treatment method of bone marrow-derived mesenchymal stem cells.

도 2 초음파 처리 강도에 따른 골수유래 중간엽 줄기세포의 CFU-Fs의 수를 나타낸 것으로, (A)는 크리스탈 바이올렛 염색 결과를 나타낸 것이고, (B)는 형성된 CFU-Fs의 수를 그래프로 나타낸 것이다.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 .

도 3은 100㎽/㎠의 세기로 초음파를 처리하였을 때, 형성된 골수유래 중간엽 줄기세포의 CFU-Fs를 크리스탈바이올렛으로 염색한 결과를 나타낸 것이다.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 ㎽ / ㎠.

도 4는 100㎽/㎠의 세기로 초음파를 처리하였을 때, 형성된 골수유래 중간엽 줄기세포의 CFU-Fs의 수를 나타낸 그래프이다.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 ㎽ / ㎠.

도 5는 세포표면발현인자의 유세포분석(FACS) 결과를 나타낸 것이다.Figure 5 shows the results of flow cytometry (FACS) of the cell surface expression factor.

도 6은 세포표면발현인자의 유세포분석(FACS) 결과를 나타낸 것이다.Figure 6 shows the results of flow cytometry (FACS) of the cell surface expression factor.

도 7은 골수유래 중간엽 줄기세포를 골세포로 분화시킨 세포에서의 골세포분화표식인자의 발현을 확인한 결과를 나타낸 것으로, (A)는 Alizarin Red S 염색을 수행한 결과를 나타낸 것이고, (B)는 RT-PCR을 수행한 결과를 나타낸 것이다.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.

도 8은 골수유래 중간엽 줄기세포를 지방세포로 분화시킨 세포에서의 지방세포분화표식인자의 발현을 확인한 결과를 나타낸 것으로, (A)는 Oil Red O 염색을 수행한 결과를 나타낸 것이고, (B)는 RT-PCR을 수행한 결과를 나타낸 것이다.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.

본 발명은 중간엽 줄기세포의 수득 효율을 증진시키는 방법에 관한 것으로, 더욱 자세하게는 중간엽 줄기세포를 함유하는 조직에서 분리된 중간엽 줄기세포 함유 세포에 초음파를 처리하여 중간엽 줄기세포의 세포부착능력 및 증식능력을 향상시키는 것을 특징으로 하는 골수유래 중간엽 줄기세포의 수득 방법에 관한 것이다.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.

골수유래 중간엽 줄기세포(Bone marrow mesenchymal stem cell)는 조혈모세포(Hematopoietic stem cell)와 함께 골수 내에 존재하는 줄기세포로, in vitro 배양시 증식 및 팽창이 가능하고 적절한 배양환경이 주어지면 골세포, 연골세포, 지방세포, 근아세포, 간세포, 심근세포, 신경세포 등 다양한 계통의 세포로 분화할 수 있는 능력을 지니고 있다(M.F. Pittenger 등, 1999 ; R.J. Deans 등, 2000). 이러한 이유로 조직재생을 위한 골수유래 중간엽 줄기세포의 연구가 세포생물학, 조직공학 분야 등에서 많이 진행되었으며, 또한 심근손상과 뇌손상에 골수유래 중간엽 줄기세포의 이식을 통한 임상시험 연구도 이루어지고 있다(E.M. Horwitz 등, 1999 ; Quarto R 등, 2001).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).

현재 사용되고 있는 추출된 골수로부터 골수유래 중간엽 줄기세포를 분리하는 방법은 다음과 같다. 골수로부터 Ficoll 밀도구배에 의해 분리된 단핵구세포를 배양 플라스크에서 배양하는 과정에서 붙지 않은 세포를 배지교환을 통해 계속 제거해주고, 배양 플라스크내 붙은 세포의 계대배양을 통해 동질한 골수유래 중간엽 줄기세포를 얻을 수 있다. 이 방법은 손쉽게 세포를 분리할 수는 있으나 순수한 골수유래 중간엽 줄기세포를 얻기는 힘들기에 여러 번의 계대배양을 통하여 동질한 세포를 얻어야 한다는 단점이 있다. 이러한 단점을 개선하기 위해, 더 순수한 골수유래 중간엽 줄기세포를 얻는 방법으로서 최근 세포표면표지(cell surface marker)를 이용한 유세포 분석(FACS analysis)이 많이 이루어지고 있다(Melody Baddoo 등, 2003 ; Tatiana Tondreau 등, 2005). 이 방법은 Ficoll 밀도구배에 의해 분리된 단핵세포에서 특정 세포표면표지가 발현된 세포만을 분리하여 배양하는 방법이다. 이러한 방법으로 분리된 세포는 Ficoll만을 이용하여 분리된 세포보다 더 동질이지만, 비용이 많이 들고, 초기에 얻을 수 있는 골수유래 중간엽 줄기세포의 수가 적은 것이 단점이다. 그러나 무엇보다도 초기의 골수유래 중간엽 줄기세포를 확인할 수 있는 세포표면표지가 밝혀지지 않고 있으므로 큰 한계점을 가지고 있다(Masakazu Ishii 등 1005; Xiaoli Wang 등, 2005).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).

골수에서 분리된 골수유래 중간엽 줄기세포는 in vitro 상태에서 콜로니(colony)를 형성하면서 섬유아세포(fibroblast)와 유사한 특징을 이루며 배양되므로 이를 콜로니 형성 단위-섬유아세포(colony forming unit-fibroblasts, 이하 CFU-Fs)라고 명하였는데 CFU-Fs는 하나의 골수유래 중간엽 줄기세포가 증식되어서 콜로니를 이루는 것으로 밝혀졌다(Castro-Malaspina H 등, 1980 ; Friedenstein A 등, 1970 ; Owen M 등 1988). 즉 CFU-Fs의 수는 골수유래 중간엽 줄기세포의 수를 의미하는 것으로 이 CFU-Fs의 수가 많을수록 많은 양의 골수유래 중간엽 줄기세포를 얻을 수 있으므로 골수유래 중간엽 줄기세포에 대한 연구에 많이 사용되고 있다. 하지만 골수내의 세포들은 매우 이질적(heterogeneous) 상태(Paolo Bianco 등, 2001 ; D. Baksh 등, 2004)로 그 중 골수유래 중간엽 줄기세포의 비율은 1×105의 골수세포 중 하나로 상당히 낮으며(Galotto M 등, 1999), 낮은 비율의 골수유래 중간엽 줄기세포 중에서도 CFU-Fs를 형성하는 능력을 지닌 집단이 따로 있는 것으로 알려져 있다(Gronthos S 등 2003 ; Kortesidis A 등 2005). 또한 환자의 나이가 증가함에 따라 골수 내의 골수유래 중간엽 줄기세포의 비율은 감소하는 것으로 알려져 있다(Gianluca D'ippolito 등, 1999 ; Christine fehrer 등, 2005). 위에서 설명한바와 같이 골수유래 중간엽 줄기세포는 in vitro 배양시 자가 복제능(self renewal capacity)이 있어 증식에 용이하지만 계대배양이 진행될수록 증식능력이 감소(Mandana Mohyeddin Bonab 등, 2006)하여 결과적으로 순수하고 많은 양의 줄기세포를 얻는 데는 한계점이 있다. 이러한 한계점을 극복하기 위해 배양시 FGF(Fibroblast growth factor)와 같은 성장인자(specific growth factors)를 첨가하거나(Giordano Bianch 등, 2003) 배양시의 세포밀도를 달리(low vs high plating densities)하는 등(Sekiya I 등, 2002) 세포배양환경을 조절하여 세포의 증식력을 증가시키려는 많은 연구가 진행되고 있다(Takehiro Matsubara 등 2003 ; Naomi Ogura 등 2004 : Panagiota A 등 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).

세포배양환경을 조절하는데 있어 성장인자나 다른 사이토카인 등을 처리하는 것(chemical stimuli) 외에 최근 물리적, 기계적 자극을 통한 세포의 변화를 관찰하는 연구가 많이 진행되고 있다. 연구에 있어 이용되는 물리적, 기계적 자극 중에는 stretch, shear stress와 전기자극, 초음파자극 등이 있으며, 특히 in vitro 실험을 통하여 이와 같은 자극이 세포의 인테그린(integrin), 세포표면표지(cell surface marker)에 변화를 주고 그에 의한 신호전달의 변화로 인해 세포의 증식이나 분화에 영향을 주는 것으로 알려져 있다. 초음파자극을 줄 경우 줄기세포를 연골세포 또는 골세포로의 분화를 촉진시킬 수 있으며 실제 임상에 있어서도 골절치료에 효과적이어서 치료의 목적으로 많이 이용되고 있다(Pilla AA 등, 1990 ; Hadjiargyrou M 등, 1998). 최근 초음파자극이 세포에 미치는 영향을 알기위해 신호전달에 관여하는 인자들을 확인하는 연구가 많이 진행되고 있으며(John Y.-J. Shyy 등 2002 ; Zhou S 등 2004 ; Yang RS 등 2005), 그 결과 세포의 부착, 이동, 증식에 관여하는 신호전달 과정 중의 인자들이 증가한 것을 확인하였으나, 아직까지 초음파 자극이 배양초기의 줄기세포에 미치는 영향에 관한 연구는 이루어지지 않았다.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.

본 발명자들은 중간엽 줄기세포의 부착능력을 향상시켜, 보다 많은 CFU-Fs를 형성하는 중간엽 줄기세포를 얻기 위하여 예의 노력한 결과, 초음파 자극이 골수유래 중간엽 줄기세포의 부착능력을 증가시켜 배양초기 짧은 기간동안 순수하고 많은 양의 골수유래 중간엽 줄기세포를 얻을 수 있다는 것을 확인하고 본 발명을 완성하게 되었다.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.

본 발명의 목적은 순수하고 많은 양의 중간엽 줄기세포를 얻기 위하여, 중간엽 줄기세포를 포함하는 세포에 초음파를 처리하여 중간엽 줄기세포의 부착능력을 향상시키는 것을 특징으로 하는 중간엽 줄기세포의 수득 효율을 증진시키는 방법을 제공하는데 있다.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.

상기 목적을 달성하기 위하여, 본 발명은 (a) 배양기에서 중간엽 줄기세포를 함유한 세포군을 배양하되, 배양 중에 상기 세포 또는 배양기에 초음파를 처리하면서 배양하는 단계; 및 (b) 배양기에 부착되지 않은 세포를 제거하고, 배양기에 부착된 콜로니(CFU-Fs) 형성 중간엽 줄기세포를 수득하는 단계를 포함하는 중간엽 줄기세포 함유 세포군로부터 중간엽 줄기세포를 수득하는 방법을 제공한다.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.

본 발명에 있어서, 상기 초음파는 5~50분/일로 배양초기부터 1~12일간 처리하는 것을 특징으로 할 수 있고, 상기 초음파는 10~1000㎽/㎠ 강도로 처리하는 것을 특징으로 할 수 있다.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.

본 발명에 있어서, 초음파는 단속적(pulsed type) 또는 연속적(continuous type)으로 처리하는 것을 특징으로 할 수 있고, 상기 배양기는 바닥이 콜라겐 또는 피브로넥틴으로 코팅되어 있는 것을 특징으로 할 수 있다.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.

본 발명에 있어서, 상기 세포는 세포담체에 부착된 상태로 배양되고, 상기 배양기는 바이오리액터(bioreactor)인 것을 특징으로 할 수 있다.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).

본 발명의 중간엽 줄기세포의 수득방법은 기존에 알려진 제대혈, 지방세포, 골수 등 중간엽 줄기세포가 존재하는 조직에서부터 분리된 세포뿐만 아니라, 추후 개발될 중간엽 줄기세포에도 적용될 수 있다. 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.

본 발명에서는 초음파가 중간엽 줄기세포의 초기배양에 있어 세포의 부착과 증식을 촉진시키는 것을 확인하였다. Ficoll을 이용한 밀도구배에 의해 골수로부터 단핵구세포(Bone marrow Mononuclear Cell)를 얻었으며, 분리 직후 및 배양 12일 후의 유세포 분석(FACS)를 통해 CD29(Integrin β1 chain), CD90(Thy-1), CD106(VCAM-1)의 발현이 증가되어, 부착된 세포가 골유래 줄기세포임을 확인하였다. 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.

본 발명의 실시예에서는 초음파처리를 연속적(continuous type)으로 처리하였으나, 단속적(pulsed type)으로 일정 간격을 두면서 처리하여도 중간엽 줄기세포의 부착능을 향상시킬 수 있었다.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).

본 발명에 있어서, 세포부착에 사용되는 배양기는 세포부착을 용이하게 하기 위하여 콜라겐 또는 피브로넥틴(fibronectin)으로 코팅하여 사용할 수 있다. 콜라겐 코팅은 0.02M의 아세트산에 5 타입I 콜라겐을 50㎍/㎖농도로 첨가한 타입I 콜라겐 용액을 배양기에 분주하고 1시간 동안 실온에 방치하여 사용할 수 있으며, 피브 로넥틴 코팅은 PBS에 피브로넥틴을 50~100㎍/㎖로 첨가한 피브로넥틴 용액을 배양기에 분주하고 1시간 동안 방치한 뒤에 사용할 수 있다.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.

초음파가 중간엽 줄기세포의 초기배양에 있어 세포의 부착에 영향을 미치는지를 확인하기 위하여 초음파 처리군과 비처리군을 12일간의 배양한 후, CFU-Fs를 분석하였다. In vitro 상태에서 하나의 중간엽 줄기세포는 증식하여 콜로니를 이루며 CFU-Fs를 형성하게 되므로, CFU-Fs의 수는 곧, 중간엽 줄기세포의 수를 의미한다. 본 발명에서는 CFU-Fs를 크리스탈 바이올렛 염색을 통해 그 수와 크기를 분석하여 두 그룹간의 차이를 확인하였다. 배양초기 초음파 처리군에서 CFU-Fs의 수가 약 50%정도 증가하였으며, 5mm 이상의 CFU-Fs의 수 역시 초음파 처리군에서 50%정도 증가한 것을 확인하였다CFU-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.

상기 결과는 본 발명에서 처리한 초음파가 중간엽 줄기세포의 부착 및 증식을 촉진시켜 결과적으로 배양 초기 많은 수의 중간엽 줄기세포를 얻는데 효과적이라는 것을 나타내는 것이다.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.

중간엽 줄기세포는 배양 시 증식력이 뛰어나고, 적절한 배양환경이 주어지면 골세포, 지방세포, 연골세포 등 다른 계통으로의 분화가 가능한 다분화능(multipotent)을 지닌 세포이다. 본 발명에서는 초기배양 시 많은 수의 중간엽 줄기세포를 얻기 위해 초음파가 처리된 세포가 stemness와 다분화능의 특징을 유지하는지 확인하기 위하여 유세포분석과 다양한 세포로의 분화능을 검사하였다. 중간엽 줄기세포에 대한 확실한 세포표면인자는 알려져 있지 않으나, 현재까지 알려진 중간엽 줄기세포에 대한 세포표면인자를 이용하여 유세포 분석검사를 시행하였다. 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.

본 발명에서, 세포담체는 고분자 폴리머 비드, 스캐폴드, ECM 막 등 통상적으로 사용할 수 있는 세포 담체라면 제한없이 사용될 수 있다. 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.

본 발명에서 조직에서 분리된 중간엽 줄기세포는 상기 세포담체를 포함하는 바이오리액터에 옮겨 배양시킬 수 있으며, 세포군을 세포담체에 접촉시킨 후 바이오리액터로 옮겨 배양시킬 수 있다.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.

상기 바이오리액터는 세포배양용 바이오리액터라면 제한없이 사용할 수 있으며, 바람직하게는 줄기세포 배양용 바이오리액터를 사용하는 것이 바람직하다.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.

본 발명에서는 배양 12일째 초음파 처리군 및 초음파 비처리군으로부터 얻어진 세포를 수거하여 CD29, CD90, CD106, CD45의 세포표면인자에 대한 유세포 분석를 하였으며, 그 결과, 초음파 처리군 및 초음파 비처리군 모두에서 세포표면인자의 발현에 차이는 없다는 것을 확인하였다. 또한 상기 두 그룹에서 얻어진 중간엽 줄기세포의 다분화능을 확인하기 위하여 2번의 계대배양을 거친 후, 골세포, 지방세포로의 분화를 유도하였다. 적절한 배양환경 아래 각 세포로의 분화는 세포의 형태학, 세포학적인 검사, RT-PCR을 통해 두 그룹 모두에서 잘 유도되었음을 확인하였다. 이것으로 배양 초기 처리한 초음파가 골수유래 중간엽 줄기세포의 부착과 증식에는 좋은 영향을 미치며, stemness와 다분화능한 특성을 유지하는데 영향을 미치지 않는다는 것을 확인하였다. 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.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다. 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.

실시예 1: 중간엽 줄기세포의 분리 및 배양Example 1: Isolation and Culture of Mesenchymal Stem Cells

이산화탄소를 흡입시켜 흰쥐(체중 250~300g, 수컷, Sprague-Dawley rat, 오리엔트, 대한민국)를 안락사시킨 후 뒷다리의 털을 제거하고 포비딘과 70% 에탄올로 소독하여 피부를 절개하고 대퇴골을 무균적으로 분리하였다. 무균실에서 항생제[100U/㎖ penicillin G(sigma, P-7794) 및 100㎍/㎖ strepto-mycin(sigma, S-9137)]가 포함된 6㎖의 인산완충식염수(PBS ; Gibco, 21600-010, Canada)로 소독된 주사기를 이용하여 대퇴골 내에 삽입하여 골수를 추출한 후, 피펫을 이용하여 뭉쳐있는 골수를 완전히 풀어주었다. 100㎛ 기공크기의 세포 여과기(cell strainer, Falcon, USA)를 이용하여 세포들을 걸러주고 2㎖의 Ficoll(Amersham Biosciences, 17-1440-02, SWEDEN)과 세포세척액 6㎖을 1:3의 비율로 하여 2000rpm에서 30분간 원심분리 한 후 황갈색의 연막(buffy coat) 층만을 분리함으로서 쥐의 단핵구세포(mononuclear cell)를 얻었다. 분리된 세포들은 10㎖의 인산완충식염수에 부유시킨 후, 1500rpm에서 5분간 원심분리한 후 항생제가 첨가된 10㎖의 α-MEM(sigma, M-0644, USA) 배양액에 재부유하여 일부는 Trypan Blue(Gibco, 15250-061, Canada)로 염색 후 광학현미경하에서 혈구계수판(haemo-cytometer)을 이용하여 세포수를 측정하였다.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.

배양액에 부유된 세포를 콜라겐 또는 피브로넥틴으로 코팅된 60㎜ 배양용 기(TPP, Switzerland)에 8×104/㎠의 농도로 분주한 후 37℃, 5% CO2 인큐베이터에서 10%의 FBS(fetal bovine serum, Hyclone, Canada)와 항생제가 첨가된 α-MEM으로 배양하였다. 배양 6일째에 배지교환을 통해 배양용기에 부착되지 않고 부유된 세포를 제거한 후 3일에 한번씩 배양기간 동안 지속적으로 배지를 갈아주었다.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.

계대배양은 세포가 배양용기의 80~90%정도가 채워졌을 때 이루어졌으며, 분화 실험은 2번의 계대배양 후 진행하였다.Subculture was performed when the cells were filled with 80-90% of the culture vessel, and differentiation experiments were carried out after two passages.

실시예 2: 초음파의 처리Example 2: Treatment of Ultrasound

초음파처리는 노블라이프(Noblelife, 듀플로젠, 대한민국)을 사용하였으며, 초음파 처리 시 60㎜ 배양기의 바닥과 송수파기 사이에 공기가 접하지 않도록 초음파기기용 젤을 사용하여 배양기를 밀착고정 하였다 (도 1). 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). ).

골수유래 중간엽 줄기세포의 콜로니 형성에 최적인 초음파 강도를 찾기 위하여, 실시예 1에서 수득한 골수유래 중간엽 줄기세포에 배양 첫째날부터 초음파를 각각 0㎽/㎠, 100㎽/㎠ 및 200㎽/㎠의 세기로 하루에 한번, 연속적(continuous type)으로 10분씩 6일 동안 처리하였다. 배양 6일째 부착하지 않고 부유된 세포들은 새 배지로 교환하면서 제거하였으며 3일에 한번 새 배지로 교환하면서 6일을 추가적으로 배양하였다. 배지교환 이후로는 초음파를 처리하지 않았다. 배양 12일째 세포는 95% 에탄올로 고정되었으며 크리스탈 바이올렛 염색을 통하여 각 그룹간의 CFU-Fs 형성을 비교하였다 (도 2). 그 결과 초음파 비처리군보다 초음파 처리군에 서 더 많은 콜로니가 형성되었으며, 초음파 강도 100㎽/㎠와 200㎽/㎠의 차이에 있어 3~5mm 크기의 콜로니는 200㎽/㎠에서, 5mm 이상 크기의 콜로니는 100㎽/㎠에서 더 많았다. 그러나 두 그룹사이의 전체 콜로니 수의 차이는 거의 없었으므로, 이 후의 실시예에서 모든 초음파처리는 100㎽/㎠의 강도에서 진행하였다.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.

실시예 3: 초음파가 중간엽 줄기세포의 CFU-Fs 형성에 미치는 영향확인 Example 3: Confirmation of the effect of ultrasound on the CFU-Fs formation of mesenchymal stem cells

실시예 2의 방법으로 초음파 처리한 골수유래 중간엽 줄기세포 실험군 및 초음파를 처리하지 않은 대조군의 CFU-Fs의 수를 크리스탈 바이올렛 염색을 통하여 확인하였다. CFU-Fs를 확인하기 위해 배양 후 12일째, 배양된 세포층을 인산완충식염수로 2회 세척하고 상온에서 약 2분간 95% 에탄올로 고정하였다. 고정된 세포를 인산완충식염수로 3회 세척한 후, 5% 크리스탈 바이올렛 용액[crystal violet(sigma, C-3886, USA) 5g, 메탄올 100ml]을 첨가한 후 5분 동안 염색하고 물로 세척 후 실온에서 건조하였다. 초음파 처리군 및 대조군에서 지름 3mm 이상의 CFU-Fs 수를 계수하여 비교하였다.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.

초음파를 처리군에서 전체 CFU-Fs의 수는 초음파 비처리군보다 1.5배 정도 더 증가하였으며, 상기 결과를 통하여 배양 초기 초음파의 처리가 골수유래 중간엽 줄기세포의 부착과 CFU-Fs의 형성에 효과적임을 알 수 있다 (도 3 및 도 4).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).

또한 골수유래 중간엽 줄기세포의 골세포로의 분화여부를 알 수 있는 골세포 분화초기 발현인자인 ALP는 초음파 처리군 및 대조군 모두에서 거의 발현되지 않았다. 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.

실시예 4: 유세포 분석( Fluoresence-Activated Cell Sorting Analysis) Example 4: Fluoresence-Activated Cell Sorting Analysis

초음파처리군 및 대조군의 세포표면인자의 변화를 확인하기 위해 유세포 분석을 시행하였다.Flow cytometry was performed to confirm the change of cell surface factors of the sonicated and control groups.

배양 12일째의 세포를 0.25% Trypsin-EDTA로 떼어내어 2% FBS가 함유된 인산완충식염수로 2회 세척한 후, 1×106의 세포 당 0.5㎍의 Antibody[(FITC-conjugated Hamster Anti-Rat CD29(Integrin β1 chain), PE-Cy5-conjugated Mouse Anti-Rat CD45(Leukocyte Common Antigen), R-PE-conjugated Mouse Anti-Rat CD106(VCAM-1) 및 PerCP-conjugated Mouse Anti-Rat CD90(Thy-1)]를 각각 처리하여 4℃의 암실에서 40분간 반응시켰다. Antibody의 부착 후 각각의 세포는 다시 2% FBS가 함유된 인산완충식염수로 2회 세척한 후, 유세포분석기(FACS Vantage, Beckon Dickinson, U.S.A)를 이용하여 세포표면인자 발현의 차이를 확인하였다.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.

골수유래 중간엽 줄기세포의 세포표면인자로 널리 알려진 CD 29(Integrin β1 chain), CD 90(Thy-1), CD 106(VCAM-1)의 항체(Antibody)로 유세포 분석(FACS)를 시행한 결과 초음파처리군과 대조군 모두에서 발현이 되었으며, 두 그룹간의 차이는 거의 없었으며, 조혈모세포(hematopoietic stem cell)의 세포표면인자이며, 음성컨트롤(negative control)로 사용된 CD 45(Leukocyte Common Antigen)는 두 그룹 모두에서 거의 발현되지 않는 것으로 확인되었다 (도 5 및 도 6). 상기 결과에 의하여, 배양 초기 처리한 초음파가 세포의 부착율을 증가시키는데 효과적이나, 골수 유래 중간엽 줄기세포의 세포표면인자의 변화와 stemness에 크게 영향을 주지 않는다는 것을 알 수 있다.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.

실시예 5: 중간엽 줄기세포의 분화유도 (Differentiation of BMSSCs) Example 5: Differentiation of BMSSCs

초음파처리가 중간엽 줄기세포의 분화능에 영향을 미치는지를 확인하기 위해 분화 실험을 시행하였다. Differentiation experiments were performed to determine whether sonication affects the differentiation capacity of mesenchymal stem cells.

초음파처리한 골수유래 중간엽 줄기세포 및 대조군의 골수유래 중간엽 줄기세포를 두㎍ 번의 계대배양 후 골세포(osteoblast) 및 지방세포(adipocyte)로 분화를 유도하였다. 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.

골세포로의 분화유도는 지름 60mm 배양기에 5×104의 골수유래 중간엽 줄기세포(2×103cells/cm2)를 10% FBS가 포함된 α-MEM에 50㎍/ml ascorbate-2 phosphate(sigma, A-8960) 및 0.1μM dexamethasone(sigma, D-8893)을 첨가한 후 3주 동안 배양하여 수행하였다. 지방세포로의 분화유도는 상기와 동일한 배양용기에 5×105의 세포(2×104cells/cm2)를 10% FBS가 포함된 α-MEM에 0.5mM IBMX(sigma, I-5879), 10㎍/ml insulin(sigma, I-9278), 0.1mM indomethacin (sigma, I-8280) 및 1μM dexamethasone을 첨가한 후 역시 3주 동안 배양하였다 (Mark F. Pittenger 등, 1999). 분화 유도를 위한 배지는 각각 3일에 한 번 교환되었으며, 배양 3주 째에 RT-PCR과 세포학검사를 위해 세포를 수거 또는 고정하였다.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-PCR은 하기의 방법으로 수행하였다.RT-PCR was performed by the following method.

TRIzol(Invitrogen, 15596-018, USA)을 이용하여 배양된 세포로부터 RNA를 추출 후 정량하여 1㎍씩의 total RNA를 Reverse transcription(RT)에 사용하였다. RT는 1st stand cDNA Synthesis Kit(AMV)(Roche, 1-483-188, Germany)를 이용하여 cDNA를 합성하고, PCR PreMix(Bioneer, K-2016, Korea)에 합성된 cDNA 0.5㎍, 각각의 특이 프라이머 1㎕[GAPDH, osteopontin, Lipoprotein lipase, peroxisome proliferator activated receptor gamma 2; 표 1], DEPC(diethyl pyrocarbonate)-처리수를 넣은 후(전체 20㎕), 표 2에 나타낸 조건에서 PCR을 실시하였다.RNA was extracted from the cells cultured using TRIzol (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.

실시예에서 사용한 분자표식인자 프라이머Molecular Marker Primers Used in Examples 프라이머primer 서열order PCR산물PCR product GAPDHGAPDH S: 5'-GGCATTGCTCTCAATGACAA-3'(서열번호 1) A: 5'-TGTGAGGGAGATGCTCAGTG-3'(서열번호 2)S: 5'-GGCATTGCTCTCAATGACAA-3 '(SEQ ID NO: 1) A: 5'-TGTGAGGGAGATGCTCAGTG-3' (SEQ ID NO: 2) 223bp223bp OsteopontinOsteopontin S: 5'-AGAGGAGAAGGCGCATTACA-3'(서열번호 3) A: 5'-GCAACTGGGATGACCTTGAT-3'(서열번호 4)S: 5'-AGAGGAGAAGGCGCATTACA-3 '(SEQ ID NO: 3) A: 5'-GCAACTGGGATGACCTTGAT-3' (SEQ ID NO: 4) 500bp500 bp LPLLPL S: 5'-CAGCTGGGCCTAACTTTGAG-3'(서열번호 5) A: 5'-TGCTGGGGTTTTCTTCATTC-3'(서열번호 6)S: 5'-CAGCTGGGCCTAACTTTGAG-3 '(SEQ ID NO: 5) A: 5'-TGCTGGGGTTTTCTTCATTC-3' (SEQ ID NO: 6) 305bp305 bp PPARγ2PPARγ2 S: 5'-CCCTGGCAAAGCATTTGTAT-3'(서열번호 7) A: 5'-ACTGGCACCCTTGAAAAATG-3'(서열번호 8)S: 5'-CCCTGGCAAAGCATTTGTAT-3 '(SEQ ID NO: 7) A: 5'-ACTGGCACCCTTGAAAAATG-3' (SEQ ID NO: 8) 222bp222 bp

각 분화표식인자에 따른 PCR 조건PCR conditions for each differentiation factor GAPDHGAPDH OsteopontinOsteopontin LPLLPL PPARγ2PPARγ2 DenaturationDenaturation 94℃, 5' 94 ℃, 5 ' DenaturationDenaturation 94℃, 1' 94 ° C, 1 ' AnnealingAnnealing 55℃,1'55 ℃, 1 ' 57℃,1'57 ℃, 1 ' 53℃,1'53 ℃, 1 ' 55℃,1'55 ℃, 1 ' ExtensionExtension 72℃, 1'72 ° C., 1 ' CycleCycle 3030 2727 3333 3030 ExtensionExtension 72℃, 5'72 ℃, 5 '

세포학적 검사는 다음의 방법으로 염색을 통하여 확인하였다.Cytological examination was confirmed by staining in the following manner.

골세포로 분화시킨 중간엽 줄기세포의 골세포로의 분화를 확인하기 위하여 분화 3주째의 세포를 인산완충식염수로 2회 세척하고 4% paraformaldehyde로 고정 후 다시 인산완충식염수로 2회 세척하였다. 후에 Alizarin Red S(sigma, A-5533) 용액(40mM, pH 4.2)으로 5분간 염색하고 3차수로 3회 세척하였다. 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.

지방세포로의 분화를 확인하기 위해 Oil Red O(sigma, O-0625)염색을 하였다. 배양된 세포를 인산완충식염수로 2회 세척한 후 Formal Calcium 용액(40% Formalin :10% CaCl2 :3차수= 1:1:8)으로 1시간동안 고정하고 60% 이소프로파놀로 1회 세척하였다. 그리고 0.18%의 Oil Red O 용액으로 15분간 염색 후, 60% 이소프로파놀로 빠르게 2회 세척하고 광학현미경에서 염색된 세포를 확인하였다.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.

초음파처리군 및 대조군의 골수유래 중간엽 줄기세포를 골세포와 지방세포로의 분화 유도 후 세포학적 검사와 RT-PCR을 통해 분석한 결과, 상기 두 군 모두에서 분화가 이루어 졌으며, 분화정도에도 거의 차이가 없는 것으로 나타났다(도 7 및 도 8). 상기 결과로부터 세포의 부착율을 높이기 위해 배양 초기에 처리한 초음파가 골수유래 중간엽 줄기세포의 분화능과 stemness에 영향을 미치지 않는다는 것을 알 수 있다.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.

본 발명은 중간엽 줄기세포를 포함하는 세포에 초음파를 처리하여 중간엽 줄 기세포의 부착능력을 향상시키는 것을 특징으로 하는 중간엽 줄기세포의 수득 효율을 증진시키는 방법을 제공하는 효과가 있다. 본 발명에 따르면, 중간엽 줄기세포를 포함하는 세포로부터 정상적인 분화능력을 가지는 순수한 중간엽 줄기세포를 손쉽게 대량으로 수득할 수 있다.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.

이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다.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.

서열목록 전자파일 첨부 Attach sequence list electronic file

Claims (6)

다음의 단계를 포함하는 중간엽 줄기세포 함유 세포군로부터 중간엽 줄기세포를 수득하는 방법:Method for obtaining mesenchymal stem cells from the mesenchymal stem cell-containing cell population comprising the following steps: (a) 배양기에서 중간엽 줄기세포를 함유한 세포군을 배양하되, 배양 중에 상기 세포 또는 배양기에 초음파를 5~50분/일로 배양초기부터 1~12일간 처리하면서 배양하는 단계; 및(A) culturing a group of cells containing mesenchymal stem cells in the incubator, while culturing the cells or incubator while treating the cells or incubator for 1 to 12 days from the beginning of incubation at 5 to 50 minutes / day; And (b) 배양기에 부착되지 않은 세포를 제거하고, 배양기에 부착된 콜로니(CFU-Fs) 형성 중간엽 줄기세포를 수득하는 단계.(b) removing cells not attached to the incubator and obtaining colony (CFU-Fs) forming mesenchymal stem cells attached to the incubator. 삭제delete 제1항에 있어서, 초음파는 10~1000㎽/㎠ 강도로 처리하는 것을 특징으로 하는 방법.The method of claim 1, wherein the ultrasonic wave is treated at an intensity of 10 to 1000 mW / cm 2. 제1항에 있어서, 초음파는 단속적(pulsed type) 또는 연속적(continuous type)으로 처리하는 것을 특징으로 하는 방법.The method of claim 1, wherein the ultrasonic waves are processed in a pulsed type or a continuous type. 제1항에 있어서, 배양기는 바닥이 콜라겐 또는 피브로넥틴으로 코팅되어 있는 것을 특징으로 하는 방법.The method of claim 1, wherein the incubator is coated with collagen or fibronectin at the bottom. 제1항에 있어서, 세포는 세포담체에 부착된 상태로 배양되고, 배양기는 바이오리액터(bioreactor)인 것을 특징으로 하는 방법.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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