KR100827660B1 - Methods for selection and culture of mesenchymal stem cell expressing CD9 - Google Patents
Methods for selection and culture of mesenchymal stem cell expressing CD9 Download PDFInfo
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Abstract
본 발명은 동물혈청 없이 인간혈청을 포함하는 세포 배양 배지 조성물에 관한 것으로, 더욱 구체적으로 50~60 %(v/v) DMEM , 40~50 %(v/v) IMDM, 4~6 μg/ml 인슐린 (insulin), 4~6 μg/ml 트랜스페린 (transferrin), 4~6 μg/ml 셀레니움 (selenium), 10-7~10-9 M 덱사메타손 (dexamethasone), 80~120 U 페니실린 (penicillin)/800~1200 U 스트렙토마이신 (streptomycin), 5~15 ng/ml FGF, 및 1~10 % 인간혈청을 포함하는 것을 특징으로 하는 인간 중간엽 줄기세포 배양 배지 조성물에 관한 것이다.The present invention relates to a cell culture medium composition comprising human serum without animal serum, more specifically 50-60% (v / v) DMEM, 40-50% (v / v) IMDM, 4-6 μg / ml insulin (insulin), 4 ~ 6 μg / ml transferrin (transferrin), 4 ~ 6 μg / ml selenium (selenium), 10 -7 ~ 10 -9 M dexamethasone (dexamethasone), 80 ~ 120 U penicillin (penicillin) / 800 ˜1200 U streptomycin, 5-15 ng / ml FGF, and 1-10% human serum.
본 발명에 따르면, 인간 중간엽 줄기세포의 배양에 사용하는 자가혈청의 농도를 감소시키기 위하여 F-12를 대체하는 배지를 검색한 결과 DMEM/F-12 대신 DMEM/IMDM 배지를 사용하였을 때 인체혈청농도를 5%로 감소시켜도 세포증식능이 대조군 (DMEM/F12, 10% HS)과 유사한 수준으로 유지되어 인체혈청사용을 최소화하며 생체 안전성을 확보한 배양방법을 확립하였다. 또한, CD9 표지인자의 발현이 중간엽줄기세포의 증식능, 부착능 및 생체생존능을 증가시킴과 DMEM/IMDM배지사용시 CD9발현이 증가됨을 발견하고 CD9 표지인자를 띄는 세포개체를 분리하여 DMEM/IMDM배지에 배양함으로써 중간엽 줄기세포의 세포치료효과를 증진시킬 수 있는 방법을 확립하였다.According to the present invention, as a result of searching for a medium replacing F-12 to reduce the concentration of autologous serum used for culturing human mesenchymal stem cells, human serum was used when DMEM / IMDM medium was used instead of DMEM / F-12. Even if the concentration was reduced to 5%, cell proliferation was maintained at a level similar to that of the control group (DMEM / F12, 10% HS), thereby minimizing human serum use and establishing a culture method that secured biosafety. In addition, the expression of CD9 markers increased mesenchymal stem cell proliferation, adhesion, and viability, and CDMEM expression was increased when using DMEM / IMDM media. By culturing in the established method that can enhance the cell therapeutic effect of mesenchymal stem cells.
중간엽 줄기세포, CD9, 인간혈청, IMDM, 세포치료 Mesenchymal stem cells, CD9, human serum, IMDM, cell therapy
Description
도 1은 DMEM/F-12 대신 DMEM/IMDM 배지를 사용하였을 때 인체혈청농도를 5%로 감소시켰을 때 세포 증식능이 대조군 (DMEM/F12, 10% 인체혈청)과 유사한 수준으로 유지됨을 나타낸 것이다. ADSC : 지방세포 유래 줄기세포(adipocyte derived stem cell), BMSC : 골수 유래 줄기세포 (Bone marrow stem cell).Figure 1 shows that when using DMEM / IMDM medium instead of DMEM / F-12, the cell proliferation capacity is maintained at a level similar to the control (DMEM /
도 2은 중간엽 줄기세포에서 발현하는 CD9의 비율을 확인한 것이다.Figure 2 confirms the ratio of CD9 expressed in mesenchymal stem cells.
도 3은 중간엽 줄기세포 중 세포군집 형성에 따라 CD9 표지인자의 발현비율을 확인한 것이다.Figure 3 confirms the expression rate of the CD9 marker according to the formation of the cell population of the mesenchymal stem cells.
도 4는 유세포분석을 통해 CD9 표지인자 비율을 확인하고 그 중 CD9를 띄는 세포를 분리해 내고 각각 세포의 모양과 형태를 비교한 것이다.Figure 4 shows the ratio of the CD9 marker factor through flow cytometry, the cells showing the CD9 of them are isolated and compared the shape and shape of each cell.
도 5은 CD9의 유무를 통해 분리한 세포의 증식능을 확인한 것으로 104개의세포를 시작으로 비교해 본 것과 cm2 당 1개의 세포로부터 형성되는 세포군집수와 세포군집당 세포수 비교로 확인해 보았다.FIG. 5 confirms the proliferative capacity of the cells isolated through the presence or absence of CD9. The comparison was made with 10 4 cells starting from the cell population formed from one cell per cm 2 and the number of cells per cell population.
도 6는 CD9의 유무를 통해 분리한 세포의 부착능을 확인한 것으로 105개의 세포를 심고 90분 후에 배양접시에 붙은 세포 수를 확인해 본 것이다.6 is to check the presence or absence of the separation through the CD9 cell adhesion ability to plant 10 5 cells to confirm the number of cells attached to the Petri dish after 90 minutes.
도 7는 DMEM/IMDM 배지에서 CD9의 발현이 DMEM/F12 배지에 비하여 높음을 확인한 것이다.7 shows that the expression of CD9 in DMEM / IMDM medium is higher than that of DMEM / F12 medium.
도 8은 nude mouse의 뇌에 2주후 CD9 발현세포가 CD9 미발현세포에 비하여 생체생존능이 현저히 높음을 나타내는 것이다.Figure 8 shows that after two weeks in the brain of nude mouse CD9 expressing cells are significantly higher in viability than CD9 non-expressing cells.
본 발명은 CD9를 발현하는 인간 중간엽 줄기세포 배양 배지 조성물에 관한 것으로, 더욱 구체적으로 특정 농도의 DMEM, IMDM, 인슐린-트랜스페린-셀레니움 (insulin-transferrin-selenium), FGF, 인간혈청 등을 포함하는 것을 특징으로 하는 인간 중간엽 줄기세포 배양 배지 조성물, 이를 이용한 CD9를 발현하는 인간 중간엽 줄기세포 선별 및 배양방법에 관한 것이다.The present invention relates to a human mesenchymal stem cell culture medium composition expressing CD9, and more specifically, comprises a specific concentration of DMEM, IMDM, insulin-transferrin-selenium, FGF, human serum, and the like. It relates to a human mesenchymal stem cell culture medium composition, characterized in that human mesenchymal stem cell selection and culture method for expressing CD9 using the same.
CD9는 4개의 putative transmembrane domain을 가지는 type III 막단백질로서, 24 kDa의 단일 사슬 (single chain) 단백질이다 (Boucheix C, et al., Ann Genet. 1985;28(1):19-24). CD9는 혈소판, B 세포, 활성화된 T 세포, eosinophil, 혈관내피세포 (endothelial cell), 뇌 (brain), 그리고 말초신경에서 발현된다. 이러한 표지인자는 세포발달, 활성, 성장, 운동 등을 조절하는 신호전달을 매개한다고 알려져 있으며 (Zheng R, et al., 2005;15(7-8):365-72; Murayama Y, et al., 2002 Apr 1;98(4):505-13; Hattori T, et al., J Cell Physiol. 2005 Jan;202(1):191-204), 이 표지인자에 의해 코드화된 단백질은 integrin 등과 복합체 (Yang XH, et al., J Biol Chem. 2006 May 5;281(18):12976-85)를 이루며 세포의 부착능과 이동, 혈소판의 응집과 활성 등을 유도한다 ( Shi W, et al., Blood. 2005 Apr 1;105(7):2852-61). CD9 is a type III membrane protein with four putative transmembrane domains and is a 24 kDa single chain protein (Boucheix C, et al., Ann Genet. 1985; 28 (1): 19-24). CD9 is expressed in platelets, B cells, activated T cells, eosinophils, endothelial cells, brains, and peripheral nerves. These markers are known to mediate signaling that regulates cell development, activity, growth, movement, and the like (Zheng R, et al., 2005; 15 (7-8): 365-72; Murayama Y, et al. , 2002 Apr 1; 98 (4): 505-13; Hattori T, et al., J Cell Physiol. 2005 Jan; 202 (1): 191-204), the protein encoded by this marker is complexed with integrin and the like. (Yang XH, et al., J Biol Chem. 2006 May 5; 281 (18): 12976-85) and induce cell adhesion and migration, platelet aggregation and activity (Shi W, et al. , Blood. 2005 Apr 1; 105 (7): 2852-61).
또한 이 항원은 pre-B cell 및 단핵구 혈소판에서 발현되며 단백질 활성화효소(protein kinase) 활성을 갖는다 (Zilber MT, et al., Semin Thromb Hemost. 2000;26(1):47-5; Nomura S, et al., Thromb Res. 1992 Jan 1;65(1):95-104). 게다가 CD9는 근육세포의 융합을 촉진시키며 근관의(myotube) 유지에도 관여한다(Tachibana I, et al., J Cell Biol. 1999 Aug 23;146(4):893-904).This antigen is also expressed in pre-B cells and monocyte platelets and has protein kinase activity (Zilber MT, et al., Semin Thromb Hemost. 2000; 26 (1): 47-5; Nomura S, et al., Thromb Res. 1992 Jan 1; 65 (1): 95-104). In addition, CD9 promotes fusion of muscle cells and is involved in myotube maintenance (Tachibana I, et al., J Cell Biol. 1999 Aug 23; 146 (4): 893-904).
생체에서와는 달리, 체외에서 인위적으로 동물 세포를 배양하기 위하여 배양 배지를 공급하려면, 혈장이나 림프액과 같은 체액을 근거한 생체의 조건에 가까운 영양분과 pH, 온도, 삼투압 등의 환경 조건을 충분히 만족시켜 주어야 한다. 따라서, 체액을 근거로 배양 배지 조성을 결정하고 이 결정된 배양 배지를 선택하여 혈청을 적정 비율로 세포에 맞게 첨가한 뒤 세포 배양에 이용하여 왔다. 이런 방법으로서, 배지 개발 초기에는 1950년경 모건 (Morgan) 등이 체액 조성을 근거로 M199 배지를 만들어 태아 세포 (primary chick)를 배양하였다 (MORGAN JF, et al., Proc Soc Exp Biol Med. 1950 Jan;73(1):1-8). 이어서 개발된 주요 배지로는, 이글 (Eagle)이 사람과 쥐의 태아 세포 배양에 이용한 기초 배양액 (basal medium)(Eagle H. Science. 1955; 122, 501), 쥐의 태아 세포 배양에 이용한 배양 액 (Dulbecco's modified eagles medium, Dulbecco, et al., 1959), Ham's F-12 (HAM RG, Proc Natl Acad Sci USA. 1965 Feb;53:288-93)와 모르 (Moore) 등이 햄스터 종양 세포 배양에 이용한 RPMI-1640 (MOORE GE, 1967 JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION 199(8):519) 등이 있다. 이러한 세포 배양시, 세포의 영양 요구성에 따라 상기의 기본 배지에 5 내지 20%의 동물혈청을 첨가하여 사용한다. 혈청에는 세포의 성장과 기능을 촉진하는 인슐린 또는 폴리펩티드계통의 다양한 호르몬과 세포의 분열과 기능을 조절하는 성장 인자들, 그리고 피브로넥틴과 같은 부착 및 확산 인자들, 결합 단백질로 트랜스페린이 있으며, 지질, 무기질 등 미량 요소들이 다수 포함되어 있다. 혈청은 또한 완충액으로서 배양액의 삼투압과 pH를 조절하며, 단백질 분해 저해 및 물리적 충격에서 세포를 보호할 수 있는 점성 작용도 가지고 있다 (Griffiths JB, Dev Biol Stand. 1987;66:155-60) 따라서, 기본 배지에 일정 농도의 혈청을 넣어 배양용 배지를 만들고 있다. 이처럼 다양한 기능을 가진 물질들이 포함된 혈청을 사용하여 동물 세포를 배양할 수 있다.Unlike in vivo, in order to supply culture medium to artificially cultivate animal cells in vitro, it is necessary to satisfactorily satisfy the nutrients close to the biological conditions based on body fluids such as plasma or lymph and environmental conditions such as pH, temperature and osmotic pressure. . Therefore, the culture medium composition was determined based on the body fluid, and the determined culture medium was selected and serum was added to the cells at an appropriate ratio, and then used for cell culture. In this way, early in the development of the medium, Morgan et al., About 1950, made M199 medium based on the fluid composition and cultured the primary chick (MORGAN JF, et al., Proc Soc Exp Biol Med. 1950 Jan; 73 (1): 1-8). Subsequently, the main medium developed was Eagle basal medium (Eagle H. Science. 1955; 122, 501) used for culturing fetal cells of humans and rats, and culture medium used for culturing fetal cells of rats. (Dulbecco's modified eagles medium, Dulbecco, et al., 1959), Ham's F-12 (HAM RG, Proc Natl Acad Sci USA. 1965 Feb; 53: 288-93), and More, were used to culture hamster tumor cells. RPMI-1640 (MOORE GE, 1967 JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION 199 (8): 519). In such cell culture, 5-20% of animal serum is added to the basal medium according to the nutritional requirements of the cells. The serum contains insulin, which promotes cell growth and function, various hormones of the polypeptide system, growth factors that regulate cell division and function, and adhesion and diffusion factors such as fibronectin, transferrin as a binding protein, lipids and minerals. Many trace elements are included. Serum also regulates the osmotic pressure and pH of the culture as a buffer, and has a viscous action that protects cells from proteolytic inhibition and physical shock (Griffiths JB, Dev Biol Stand. 1987; 66: 155-60). A certain concentration of serum is added to the basal medium to make a culture medium. Animal cells can be cultured using serums containing these various functions.
중간엽 줄기세포 (Mesenchymal Stem Cell, MSC)는 체외에서 쉽게 증식할 수 있으며, 여러 가지 세포형태 지방세포, 연골세포, 근육세포, 뼈세포)로 분화가 가능한 세포이다 (Caplan, A.I. 1991, J. Orthop. Res. 9, 641-650; Beresford, J. N. et al. (1992), J. Cell Sci. 102, 341-351; Pittenger, M.F., et al., (1999), Science 284, 143-147; Patricia, A. Z., et al. 2001, Tissue engineering. 7, 211-227). 따라서 중간엽 줄기세포는 유전자 치료와 세포치료에 있어서 유용한 타깃(target)으로 이용될 수 있으며 중간엽 줄기세포의 성장기전에 대한 이해는 다양 한 이를 이용한 세포치료를 위해 중요하다. 하지만 지금까지 중간엽 줄기세포에서 표지인자 CD9이 어떠한 영향을 미치는지 그 기능이 밝혀진 바가 없다. Mesenchymal Stem Cells (MSCs) are easily proliferated in vitro and are capable of differentiating into various cell-like adipocytes, chondrocytes, muscle cells, and bone cells (Caplan, AI 1991, J.). Orthop. Res. 9, 641-650; Beresford, JN et al. (1992), J. Cell Sci. 102, 341-351; Pittenger, MF, et al., (1999), Science 284, 143-147; Patricia, AZ, et al. 2001, Tissue engineering. 7, 211-227). Therefore, mesenchymal stem cells can be used as a useful target in gene therapy and cell therapy. Understanding the mechanism of growth of mesenchymal stem cells is important for various cell therapy. However, the function of marker marker CD9 in mesenchymal stem cells has not been known so far.
본 발명에서는 중간엽 줄기세포에서 CD9가 줄기세포의 성장?부착능에 중요한 역할을 하며, 이러한 표지인자만을 띄는 세포를 분리?이용하여 최소한의 자가혈청만을 이용, 최적배지 조성을 통한 세포재생 치료제 개발기술에 기반을 제공한다.In the present invention, CD9 plays an important role in the growth and adhesion of stem cells in mesenchymal stem cells, by using only a small number of autologous serum by separating and using cells with only these markers, and in developing cell regeneration therapeutics through optimal medium composition. Provide the foundation.
이에, 본 발명자들은 상기 종래기술들의 문제점들을 극복하기 위하여 예의 연구 노력한 결과, 본 발명의 배지조성물의 경우, 증식능과 부착능을 증가시키는 표지인자 CD9를 발현하는 인간 중간엽 줄기세포를 선별하고 배양할 수 있음을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the present inventors have made intensive studies to overcome the problems of the prior art, and as a result of the media composition of the present invention, it is possible to select and culture human mesenchymal stem cells expressing the marker factor CD9 that increases proliferative capacity and adhesion. After confirming that it can, the present invention was completed.
따라서, 본 발명의 주된 목적은 최소한의 인간혈청을 사용하여 생체 안전성을 확보한 인간 중간엽 줄기세포 배양 배지 조성물을 제공하는 데 있다.Accordingly, a main object of the present invention is to provide a human mesenchymal stem cell culture medium composition which ensures biosafety using minimal human serum.
본 발명의 다른 목적은 상기 배양배지 조성물을 이용한 CD9를 발현하는 인간중간엽 줄기세포의 선별 및 배양방법을 제공하는데 있다.Another object of the present invention is to provide a method for selecting and culturing human mesenchymal stem cells expressing CD9 using the culture medium composition.
본 발명의 한 양태에 따르면, 본 발명은 동물혈청 없이 인간혈청을 포함하는 세포 배양 배지 조성물에 있어서, 50~60 %(v/v) DMEM (Dulbeco's Modified Eagle's Medium), 40~50 %(v/v) IMDM (Iscove's Modified Dulbecco's Medium), 4~6 μg/ml 인슐린 (insulin), 4~6 μg/ml 트랜스페린 (transferrin), 4~6 μg/ml 셀레니움 (selenium), 10-7~10-9 M 덱사메타손 (dexamethasone), 80~120 U 페니실린 (penicillin)/800~1200 U 스트렙토마이신 (streptomycin), 5~15 ng/ml FGF, 및 1~10 % 인간혈청을 포함하는 것을 특징으로 하는 인간 중간엽 줄기세포 배양 배지조성물을 제공한다.According to one aspect of the invention, the present invention provides a cell culture medium composition comprising human serum without animal serum, 50 ~ 60% (v / v) DMEM (Dulbeco's Modified Eagle's Medium), 40 ~ 50% (v / v) IMDM (Iscove's Modified Dulbecco 's Medium), 4 ~ 6 μg / ml insulin (insulin), 4 ~ 6 μg / ml transferrin (transferrin), 4 ~ 6 μg / ml selenium (selenium), 10 -7 ~ 10 -9 Human mesenchymal comprising M dexamethasone, 80-120 U penicillin / 800-1200 U streptomycin, 5-15 ng / ml FGF, and 1-10% human serum A stem cell culture medium composition is provided.
본 발명의 배지조성물에서, DMEM (둘베코 변형 이글 배지)는 세포배양에 사용되는 기본배지로서, 본 발명의 실시예에서는 구체적으로 low-glucose DMEM (L-glutamine)(Life Technologies사)를 사용하였다.In the medium composition of the present invention, DMEM (Dulbecco's modified Eagle's medium) is a basic medium used for cell culture, and in the examples of the present invention, low-glucose DMEM (L-glutamine) (Life Technologies) was used. .
본 발명의 배지조성물에서, IMDM은 세포배양에 일반적으로 사용되는 각종 아미노산, 비타민, 무기염 등을 포함하는 혼합물로써, 이스코프 변형 둘베코 배지 (Iscove's Modified Dulbecco's Medium)(Life Technologies사)를 의미한다.In the medium composition of the present invention, IMDM is a mixture containing various amino acids, vitamins, inorganic salts, and the like commonly used in cell culture, and means Iscove's Modified Dulbecco's Medium (Life Technologies). .
본 발명의 배지조성물에서 인슐린 (insulin), 트랜스페린 (transferrin) 및 소디움셀레나이트 (sodium selenite)를 제공하기 위해, 실시예에서는 구체적으로 insulin-transferrin-selenium (Sigma사)를 사용하였다.In order to provide insulin, transferrin, and sodium selenite in the medium composition of the present invention, specifically, insulin-transferrin-selenium (Sigma) was used in the Examples.
본 발명의 조성물에서, 덱사메타손 (dexamethasone)이란 9-Fluoro-16-Methylprednisolone (C22H29FO5)를 의미하고, FGF는 섬유아세포성장호르몬 (Fibroblast Growth Factor)을 의미한다.In the composition of the present invention, dexamethasone (dexamethasone) means 9-Fluoro-16-Methylprednisolone (C 22 H 29 FO 5 ), FGF means Fibroblast Growth Factor (Fibroblast Growth Factor).
본 발명의 배지조성물에서, 상기 인간혈청은 중간엽 줄기세포의 기원이 되는 환자에서 채취한 자가혈액으로부터 얻은 것을 특징으로 하는 배지조성물인 것이 바 람직하다. 본 발명의 인간혈청은 혈액원 등에서 입수할 수도 있으나, 배양하고자 하는 세포의 기원이 되는 환자에서 채취한 자가혈액으로부터 원심분리에 의해 얻은 것이 바람직하다. 또한, 상기 인간 중간엽 줄기세포는 종래 알려진 방법에 따라 인간의 골수 및 지방으로부터 분리할 수 있다.In the medium composition of the present invention, the human serum is preferably a medium composition, characterized in that obtained from autologous blood collected from a patient of origin of mesenchymal stem cells. The human serum of the present invention may be obtained from a blood source or the like, but is preferably obtained by centrifugation from autologous blood collected from a patient of origin of the cell to be cultured. In addition, the human mesenchymal stem cells can be isolated from human bone marrow and fat according to a conventionally known method.
본 발명의 배지조성물에서, 종래 DMEM/F-12 대신 DMEM/IMDM을 사용할 경우 인간혈청을 1 ~ 10 % 범위에서 포함하는 경우에도 세포증식능에 있어 유사한 수준으로 유지되어 인체혈청사용을 최소화함으로써 생체 안전성을 확보할 수 있다.In the medium composition of the present invention, when using DMEM / IMDM instead of the conventional DMEM / F-12, even in the case of containing human serum in the range of 1 ~ 10% is maintained at a similar level in cell proliferation ability to minimize human serum use bio safety Can be secured.
본 발명의 조성물에서, 상기 중간엽 줄기세포는 부착능과 증식능을 증가시키는 표지인자 CD9를 발현하는 것을 특징으로 하는 배지조성물인 것이 바람직하다.In the composition of the present invention, the mesenchymal stem cells are preferably a medium composition characterized by expressing the marker factor CD9 to increase the adhesion and proliferative capacity.
본 발명의 조성물에서, CD9는 4개의 putative transmembrane domain을 가지는 type Ш 막단백질로서 24 kDa 의 단일 사슬 (single chain) 단백질이다. CD9는 혈소판, B 세포, 활성화된 T 세포, eosinophil, 혈관내피세포 (endothelial cell), 뇌(brain), 그리고 말초신경에서 발현된다. 이러한 표지인자는 세포발달, 활성, 성장, 운동 등을 조절하는 신호전달을 매개한다고 알려져 있으며, 이 표지인자에 의해 코드화된 단백질은 integrin 등과 복합체를 이루며 세포의 부착능과 이동, 혈소판의 응집과 활성등을 유도한다. 또한 이 항원은 pre-B cell 및 단핵구 혈소판에서 발현되며 단백질활성화효소 (protein kinase) 활성을 갖는다. 게다가 CD9는 근육세포의 융합을 촉진시키며 근관의 (myotube) 유지에도 관여한다.In the composition of the present invention, CD9 is a type Ш membrane protein with four putative transmembrane domains and is a 24 kDa single chain protein. CD9 is expressed in platelets, B cells, activated T cells, eosinophils, endothelial cells, brain, and peripheral nerves. These markers are known to mediate signal transduction that regulates cell development, activity, growth, and movement. Proteins encoded by these markers form complexes with integrins, and are capable of cell adhesion, migration, and platelet aggregation and activity. Induce. This antigen is also expressed in pre-B cells and monocyte platelets and has protein kinase activity. In addition, CD9 promotes muscle cell fusion and is involved in myotube maintenance.
본 발명의 다른 양태에 따르면, 본 발명은 상기 본 발명의 배지조성물을 이용하여 지방조직, 골수 및 제대혈 등에서 유래한 인간 중간엽 줄기세포를 배양하는 단계 및 CD9 표지인자를 발현하는 줄기세포를 분리하는 단계를 포함하는 증식능과 부착능이 증가된 인간 중간엽 줄기세포의 선별 및 배양방법을 제공한다. 본 발명에서는 지방세포 유래 줄기세포는 ADSC (adipocyte derived stem cell), 골수 유래 줄기세포는 BMSC (Bone marrow stem cell)를 의미한다.According to another aspect of the present invention, the present invention comprises the steps of culturing human mesenchymal stem cells derived from adipose tissue, bone marrow, umbilical cord blood, etc. using the media composition of the present invention and separating the stem cells expressing CD9 markers It provides a method for screening and culturing human mesenchymal stem cells with increased proliferative capacity and adhesion including the step. In the present invention, the adipocyte-derived stem cell means ADSC (adipocyte derived stem cell), and the bone marrow-derived stem cell means BMSC (Bone marrow stem cell).
본 발명에 있어서, 상기 분리단계는 CD9 항원을 갖는 세포를 분리할 수 있는 어떠한 방법, 예컨대 Fluorescent activated cell sorting system (FACS)와 Magnetic activated cell sorting system (MACS) 등이 이용될 수 있다.In the present invention, the separation step may be any method capable of separating cells having a CD9 antigen, such as Fluorescent activated cell sorting system (FACS) and Magnetic activated cell sorting system (MACS).
본 발명의 인간 중간엽 줄기세포 배양방법은, 바람직하게는 상기 세포 배양 배지조성물을 포함하는 배양용기에 인간 중간엽 줄기 세포를 접종하는 단계 및 상기 배양용기를 CO2 인큐베이터에 넣고 5 내지 15 %의 이산화탄소의 통기량으로 35 ~ 37 ℃에서 배양하는 단계를 포함한다.In the method of culturing human mesenchymal stem cells of the present invention, inoculating human mesenchymal stem cells in a culture vessel comprising the cell culture medium composition and putting the culture vessel in a CO 2 incubator, Culturing at 35 to 37 ° C. with aeration of carbon dioxide.
본 발명의 배양방법에서, 배양용기는 예컨대 100 mm tissue culture dish (IWAKI,Japan), 60 mm tissue culture dish (IWAKI,Japan), 6 well tissue culture dish (NuncolnTM , Denmark), 12 well tissue culture dish (TPP, Europe) 등 일반세포 배양용기를 사용할 수 있으며, CO2 인큐베이터는 예컨대 Forma 3130 CO2 incubator (Formascientific Inc, USA), SANYO CO2 incubator (Japan), NUAIR US Autoflow CO2 incubator (Hansol S.M. Korea)를 사용할 수 있다.In the culture method of the present invention, the culture vessel is, for example, 100 mm tissue culture dish (IWAKI, Japan), 60 mm tissue culture dish (IWAKI, Japan), 6 well tissue culture dish (Nuncoln ™ , Denmark), 12 well tissue culture dish General cell culture vessels such as (TPP, Europe) can be used, and CO 2 incubators can be used, for example, Forma 3130 CO 2 incubator (Formascientific Inc, USA), SANYO CO 2 incubator (Japan), NUAIR US Autoflow CO 2 incubator (Hansol SM Korea) ) Can be used.
본 발명의 상기의 목적을 달성하기 위하여, 본 발명에서는 FACS (magnetic magnetic cell sorting system)을 사용하여 표지인자 CD9를 띄는 개체를 분리해 내고 이러한 세포가 증식능, 부착능이 현저하게 증가됨을 확인하였다. 또한 이들 세포의 배양에 사용하는 자가혈청의 농도를 5 % (DMEM/IMDM)로 감소시켜도 세포증식능이 대조군 (DMEM/F12, 10 % HS)과 유사한 수준으로 유지되어 인체혈청사용을 최소화하며 생체 안전성을 확보한 배양방법을 확립하였다.In order to achieve the above object of the present invention, in the present invention, using a magnetic magnetic cell sorting system (FACS) to isolate the individual bearing the marker CD9 and confirmed that the proliferation ability, adhesion capacity of these cells significantly increased. In addition, even if the concentration of autologous serum used to culture these cells was reduced to 5% (DMEM / IMDM), cell proliferation was maintained at a level similar to that of the control group (DMEM / F12, 10% HS), minimizing human serum use and biosafety. A culture method was secured.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.
실시예Example 1. 골수 및 지방으로부터 1. From bone marrow and fat 중간엽Mesenchyme 줄기세포의 분리 Isolation of Stem Cells
부산대학병원에서 대퇴골치환술과 지방제거수술을 시행한 환자에서 얻은 지방과 골수로부터 기질 줄기세포를 분리하였다 (ADSC : 지방세포 유래 줄기세포(adipocyte derived stem cell), BMSC : 골수 유래 줄기세포 (Bone marrow stem cell)). 지방조직으로부터 중간엽줄기세포를 분리하기 위하여 성형외과에서 피하지방제거술을 시행한 환자에서 지방조직을 얻는다. 분리한 지방조직을 HBSS로 씻어준 후, 핀셋과 가위를 이용하여 지방조직을 얇게 자른 다음 0.075 % collagenase를 37 ℃에서 30분간 처리한다. 골수에서 기질 줄기세포를 분리하기 위하여 골수에 Ficoll을 첨가하고 3500rpm에서 30분간 원심분리를 해준 후 mononuclear cell layer인 상층액을 HBSS로 씻어준다. Stromal stem cells were isolated from fat and bone marrow obtained from patients undergoing femoral replacement and liposuction at Pusan National University Hospital (ADSC: adipocyte derived stem cells, BMSC: bone marrow derived stem cells) stem cell)). Adipose tissue is obtained from patients undergoing subcutaneous fat removal surgery in plastic surgery to separate mesenchymal stem cells from adipose tissue. After washing the separated adipose tissue with HBSS, cut the adipose tissue using tweezers and scissors and treat 0.075% collagenase at 37 ℃ for 30 minutes. Ficoll was added to bone marrow to separate stromal stem cells from bone marrow, centrifuged at 3500 rpm for 30 minutes, and the supernatant, a mononuclear cell layer, was washed with HBSS.
원심분리 후 pellet에 다음과 같은 3가지 조성의 배양용액을 각각 사용하여 기질 줄기세포를 배양하였다. fibronectin (10 ng/㎖)이 coating된 배양용기에 1x insulin-transferrin-selenium (Sigma), 10-8 M dexamethasone (Sigma), 100 U penicillin/1,000 U streptomycin (Sigma), 10 ng/ml FGF (R&D system) 50-60 % low-glucose DMEM (L-glutamine) (Life Technologies), ① 40~50 % F-12 Nutrient Mixture (Life Technologies) + 5 % 인체혈청으로 이루어진 배양용액, ②40~50 % F-12 Nutrient Mixture (Life Technologies) + 10 % 인체혈청으로 이루어진 배양용액, ③ 40~50 % Iscove's Modified Dulbecco's Medium (IMDM)(Life Technologies) + 5% 인체혈청으로 이루어진 배양용액에서 resuspension 시킨 후, 100 μm nylon mesh 에 통과시킨 후. 분리된 세포들을 dish에 plating해서 24시간 후 부착되지 않은 세포를 제거해준다.After centrifugation, the stromal stem cells were cultured using the following three culture solutions in pellets. 1x insulin-transferrin-selenium (Sigma), 10 -8 M dexamethasone (Sigma), 100 U penicillin / 1,000 U streptomycin (Sigma), 10 ng / ml FGF (R & D) system) 50-60% low-glucose DMEM (L-glutamine) (Life Technologies), ① 40 ~ 50% F-12 Nutrient Mixture (Life Technologies) + 5% human serum, ②40 ~ 50% F- 12 Nutrient Mixture (Life Technologies) + culture solution consisting of 10% human serum, ③ 40-50% Iscove's Modified Dulbecco's Medium (IMDM) (Life Technologies) + 100 μm nylon after resuspension in culture solution consisting of 5% human serum After passing through the mesh. The separated cells are plated in a dish to remove unattached cells after 24 hours.
세포가 70~80 %까지 자란 후 trypsin/EDTA를 처리하여 세포를 단일세포로 분리하고 이를 2000 cells/cm2의 밀도로 깔아 계대배양 하였다. 인체혈청분리를 위하여 채혈 후 항응고제 (heparin)을 처리한 용기에 넣어 혈액응고를 방지하고 이를 500 rpm으로 5분간 원심분리하고 혈청을 분리하였다. 0.2 μm Filter에서 혈청을 여과하여 멸균하고 세포배양에 사용하였다.Cells were grown to 70-80% and treated with trypsin / EDTA to separate the cells into single cells and subcultured at 2000 cells / cm 2 . In order to separate the human serum, blood was collected and placed in a container treated with anticoagulant (heparin) to prevent blood coagulation, which was centrifuged at 500 rpm for 5 minutes, and serum was separated. Serum was filtered and sterilized in a 0.2 μm filter and used for cell culture.
증식능의 측정을 위하여 세포를 3일 간격으로 계대배양하면서 각 계대배양시기마다 cell을 모아 현미경에서 haematometer를 이용하여 일정 용적 중의 세포수를 세는 방법으로 측정하였다. 그 결과, 도 1에서 보여지듯이, DMEM/IMDM 배지를 사용하였을 때는 자가혈청의 농도를 5 %로 감소시켜도 세포증식능이 대조군 (DMEM/F12, 10% HS) 과 유사한 수준으로 유지되는 것을 확인할 수 있었다 (도 1). 실험에 사용된 F-12 영양혼합물 (Nutrient Mixture)의 조성은 표 1에, IMDM 배지 조성은 표 2에 기재하였다.In order to measure the proliferation capacity, the cells were subcultured at three-day intervals, and the cells were collected at each subculture and counted in a certain volume using a haematometer under a microscope. As a result, as shown in Figure 1, using the DMEM / IMDM medium, even if the concentration of autologous serum was reduced to 5% it was confirmed that the cell proliferation is maintained at a level similar to the control (DMEM / F12, 10% HS) (FIG. 1). The composition of the F-12 nutrient mixture (Nutrient Mixture) used in the experiment is shown in Table 1, and the IMDM medium composition is shown in Table 2.
[표 1] F-12 Table 1 F-12 NutrientNutrient MixtureMixture ( ( LifeLife TechnologiesTechnologies )의 조성Composition
(L-glutamine, 25mM HEPES buffer는 포함하고, sodium bicarbonate, alpha-thioglycerol, 또는 beta-mercaptoethanol은 포함하지 않음)(Including L-glutamine, 25 mM HEPES buffer, no sodium bicarbonate, alpha-thioglycerol, or beta-mercaptoethanol)
[표 2] TABLE 2 Iscove'sIscove's ModifiedModified Dulbecco'sDulbecco's MediumMedium ( ( IMDMIMDM )의 조성Composition
(L-glutamine는 포함하고 sodium bicarbonate는 포함하지 않음) (Contains L-glutamine but no sodium bicarbonate)
실시예Example 2. 골수 및 지방유래 2. Bone marrow and fat derived 중간엽줄기세포에서In mesenchymal stem cells CD9CD9 의 발현확인Expression of
골수와 지방으로부터 분리한 중간엽줄기세포에 발현하는 CD9의 비율을 유세포분석을 통해 확인해 보았다. 유세포분석을 위하여 세포를 72시간 배양한 후 0.25 % trypsin/EDTA로 세포를 떼어낸 후 flow cytometry buffer (FCB; 1 x PBS, 2 % FBS, 0.05 % sodium azide)로 씻어주고 105 세포로 나눠 담은 뒤 CD9 항체 (BD biosciences)를 포함하는 flow cytometry buffer를 각각 처리하였다. 항체로 처리된 세포는 FACScan argon laser cytometer (Becton Dickson, San Jose, CA)를 이용하여 측정하였다 (도 2).The proportion of CD9 expressed in mesenchymal stem cells isolated from bone marrow and fat was confirmed by flow cytometry. For flow cytometry, cells were incubated for 72 hours, detached with 0.25% trypsin / EDTA, washed with flow cytometry buffer (FCB; 1 x PBS, 2% FBS, 0.05% sodium azide) and divided into 10 5 cells. Each was then treated with a flow cytometry buffer containing CD9 antibodies (BD biosciences). Cells treated with antibodies were measured using a FACScan argon laser cytometer (Becton Dickson, San Jose, Calif.) (FIG. 2).
실시예Example 3. 3. 유세포분석을Flow cytometry 통한 through 증식능에On proliferation 따른 개체의 Of objects according to CD9CD9 발현비율 확인 Expression rate confirmation
비교하여 유세포분석을 통하여 분석한 결과 표지인자 CD9의 발현비율이 현저한 차이를 보임을 확인하였다. In comparison, flow cytometric analysis showed that the expression rate of the marker CD9 showed a significant difference.
중간엽 줄기세포를 cm2 당 1개의 세포를 키워, 하나의 세포에서 자라난 세포군집 중 군집형성이 빠른 세포와 그렇지 못한 세포에서 표면항원의 발현이 변화하는지를 확인하기 위하여 유세포분석을 실시하였다. 유세포분석을 위하여 세포를 72시간 배양한 후 0.25 % trypsin/EDTA로 세포를 떼어낸 후 flow cytometry buffer (FCB; 1 x PBS, 2 % FBS, 0.05 % sodium azide)로 씻어주고 105 세포로 나눠 담은 뒤 CD9 항체 (BD biosciences)를 포함하는 flow cytometry buffer를 각각 처리하였다. 항체 로 처리된 세포는 FACScan argon laser cytometer (Becton Dickson, San Jose, CA)를 이용하여 측정하였다 (도 3a). 결과, CD9의 발현비율과 중간엽 줄기세포의 군집형성이 정의 상관관계를 가짐을 확인할 수 있었다 (도 3b).One cell per cm 2 of mesenchymal stem cells was grown, and flow cytometry was performed to determine whether the expression of surface antigens was changed in the fast-forming and non-population-grown cells. For flow cytometry, cells were incubated for 72 hours, detached with 0.25% trypsin / EDTA, washed with flow cytometry buffer (FCB; 1 x PBS, 2% FBS, 0.05% sodium azide) and divided into 10 5 cells. Each was then treated with a flow cytometry buffer containing CD9 antibodies (BD biosciences). Cells treated with antibodies were measured using a FACScan argon laser cytometer (Becton Dickson, San Jose, Calif.) (FIG. 3A). As a result, it was confirmed that the expression rate of CD9 and the colonization of mesenchymal stem cells had a positive correlation (FIG. 3b).
실시예Example 4. 4. FACSFACS 를 이용한 Using CD9CD9 항원을 가지는 세포의 분리 Isolation of Cells with Antigens
유세포분석을 위하여 중간엽줄기세포를 72시간 배양한 후 0.25 % trypsin/EDTA로 세포를 떼어낸 후 flow cytometry buffer (FCB; 1 x PBS, 2 % FBS, 0.05 % sodium azide)로 씻어주고 105 세포로 나눠 담은 뒤 CD9 항체 (BD biosciences)를 포함하는 flow cytometry buffer를 각각 처리하였다. 항체로 처리된 세포는 BD FACSCanto™ II Flow Cytometry System(BD biosciences)을 이용하여 세포를 분리한다. 그 결과, 분리 후 세포의 모양에는 커다란 차이를 보이지 않음을 확인할 수 있었다 (도 4).For flow cytometry, the cells were cultured with mesenchymal stem cells for 72 hours, detached with 0.25% trypsin / EDTA, washed with flow cytometry buffer (FCB; 1 x PBS, 2% FBS, 0.05% sodium azide) and 10 5 cells. After dividing into, each was treated with a flow cytometry buffer containing CD9 antibody (BD biosciences). Cells treated with antibodies are isolated using BD FACSCanto ™ II Flow Cytometry System (BD biosciences). As a result, it was confirmed that there was no significant difference in the shape of the cells after separation (FIG. 4).
이와 선택적으로, MAC를 이용하여 CD9 항원을 가지는 세포를 분리할 수 있다. 구체적으로는, 중간엽 줄기세포에 CD9-PE (BD biosciences) 항체를 넣고 4 ℃에서 30분 동안 암반응 후 PE-micro-bead (Miltenyi Biotec)를 붙여 MACS (magnetic magnetic cell sorting system)을 이용하여 세포를 분리한다. 그 결과, 분리 후 세포의 모양에는 커다란 차이를 보이지 않음을 확인할 수 있었다.Alternatively, MAC can be used to isolate cells with CD9 antigen. Specifically, CD9-PE (BD biosciences) antibody was added to mesenchymal stem cells, and after 30 minutes of cancer reaction at 4 ° C., PE-micro-bead (Miltenyi Biotec) was attached to the cells using a magnetic magnetic cell sorting system (MACS). Disconnect. As a result, it was confirmed that there was no significant difference in the shape of the cells after separation.
실시예Example 5. 5. CD9CD9 표지인자의Of cover factor 유무에 따른 With or without 중간엽Mesenchyme 줄기세포의 Stem cell 증식능Proliferative capacity 비교 compare
CD9의 유무에 따라 분리한 각각의 서로 다른 중간엽줄기세포 104개를 깔고 3일 후에 각각의 세포 수를 측정해 보았다. 결과, CD9를 띄는 세포의 수가 현저하게 증가되었음을 알 수 있었다 (도 5a).The number of cells was measured after 3 days with 10 4 different mesenchymal stem cells isolated according to the presence or absence of CD9. As a result, it was found that the number of cells bearing CD9 was significantly increased (FIG. 5A).
또한 각각 분리한 세포를 cm2 당 1개를 키워 40개의 세포 중 세포군집을 형성하는 수를 측정한 결과, CD9를 띄는 세포가 군집을 형성하는 비율이 높았으며 (도 5b), 각각의 군집당 세포수도 현저히 많음을 확인할 수 있었다 (도 5c).In addition, as a result of measuring the number of cells to form a cell population among 40 cells by growing one cell per cm 2 , the rate of forming a population of cells with CD9 was high (FIG. 5B). The number of cells was also remarkably high (FIG. 5c).
실시예Example 6. 6. CD9CD9 표지인자의Of cover factor 유무에 따른 With or without 중간엽Mesenchyme 줄기세포의 Stem cell 부착능Adhesion 비교 compare
CD9의 유무에 따라 분리한 각각의 서로 다른 중간엽 줄기세포 105를 깔고 90분 후에 부착된 세포의 수를 확인해 본 결과, CD9 표지인자를 띄는 세포의 수가 현저히 많은 부착능을 가진 것을 확인할 수 있었다 (도 6a, b).90 minutes after each different
실시예Example 7: 7: IMDMIMDM 배지에서 On the badge CD9CD9 발현의 증가Increase in expression
IMDM 배지가 CD9의 발현유무에 따라 세포의 증식능에 영향을 미치는 지를 확인해 보았다. CD9의 유무에 따라 각각 다른 중간엽 줄기세포 10000개를 깔고 1-4일 동안 trypsin/EDTA를 처리하여 단일세포로 분리한 후 현미경에서 haematometer를 이용하여 세포의 수를 측정하였다. 이로써 IMDM배지가 CD9의 발현을 증가시킴을 확인 할 수 있었다(도 7). We examined whether IMDM medium affects cell proliferation according to the expression of CD9. 10000 different mesenchymal stem cells were laid according to the presence or absence of CD9, and then treated with trypsin / EDTA for 1-4 days, and then separated into single cells. The number of cells was measured using a haematometer under a microscope. This confirmed that the IMDM medium increased the expression of CD9 (Fig. 7).
실시예Example 8: 8: CD9CD9 발현세포와 미발현세포의 Of expressing and unexpressed cells 생체생존능의Biological viability 비교 compare
CD9의 유무에 따라 분리한 각각의 서로 다른 중간엽 줄기세포를 CM-Dil red tracker(Molecular Probe)로 염색한 후 nude mouse의 좌측뇌에 CD9 발현세포를 우측뇌에 CD9 미발현세포를 각각 106개씩 주입하였다. 10일후 양쪽 뇌에 존재하는 세포를 비교해 본 결과, CD9 표지인자를 띄는 세포의 수가 현저히 높은 생체 생존 능을 가짐을 확인할 수 있었다(도 8).After staining the respective different mesenchymal stem cells separated by the presence or absence of CD9 as a CM-Dil red tracker (Molecular Probe ) for CD9 non-expressing cells in the brain right a CD9-expressing cells in the left brain of nude mouse, respectively 10 6 Dogs were injected one by one. As a result of comparing the cells present in both brains after 10 days, it was confirmed that the number of cells bearing CD9 markers had a significantly high biological viability (FIG. 8).
이상 설명한 바와 같이, 본 발명에 따르면 중간엽 줄기세포 한 개체가 이룬 세포군집 별로 표지인자를 확인해 본 결과 증식능이 뛰어난 군집일수록 CD9 발현율이 높음을 확인하고 표지인자 CD9를 발현하는 중간엽 줄기세포를 분리하고 이 인자가 중간엽 줄기세포의 증식능과 부착능 및 생체생존능을 증가시킴을 발견하였다. 따라서 이러한 CD9 표지인자를 발현하는 중간엽 줄기세포를 선별, 배양하는 기술은 줄기세포를 이용한 세포치료효과를 증진시킬 수 있는 방법으로 기대된다. 또한 이들 세포의 배양에 사용하는 자가혈청의 농도를 감소시킬 수 있는 DMEM/IMDM 배지를 사용함으로써, 인체혈청농도를 5%로 감소시켜도 CD9발현의 증가로 인하여 세포증식능이 대조군 (DMEM/F12, 10% HS)과 유사한 수준으로 유지되어 인체혈청사용을 최소화하고 생체 안전성을 확보할 수 있다.As described above, according to the present invention, as a result of confirming the markers for each cell population formed by one mesenchymal stem cell, it was confirmed that the higher the proliferation ability, the higher the expression rate of CD9 and the mesenchymal stem cells expressing the marker factor CD9. These factors were found to increase the proliferative capacity, adhesion and bio-viability of mesenchymal stem cells. Therefore, the technology for selecting and culturing mesenchymal stem cells expressing such CD9 markers is expected to be a way to enhance the cell therapeutic effect using stem cells. In addition, by using the DMEM / IMDM medium that can reduce the concentration of autologous serum used in the culture of these cells, even if the human serum concentration is reduced to 5% due to the increase of CD9 expression, cell proliferative capacity is increased (DMEM / F12, 10 % HS) can be maintained at a similar level to minimize the use of human serum and ensure biosafety.
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