KR20180072644A - Mesenchymal stem cell culture media for preventing or treating immune disease or inflammatory disease and a method for preparing the same - Google Patents
Mesenchymal stem cell culture media for preventing or treating immune disease or inflammatory disease and a method for preparing the same Download PDFInfo
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- KR20180072644A KR20180072644A KR1020180071300A KR20180071300A KR20180072644A KR 20180072644 A KR20180072644 A KR 20180072644A KR 1020180071300 A KR1020180071300 A KR 1020180071300A KR 20180071300 A KR20180071300 A KR 20180071300A KR 20180072644 A KR20180072644 A KR 20180072644A
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Abstract
본 발명은 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액 및 이의 제조방법에 관한 것으로, 본 발명의 방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 얻어지는 줄기세포 배양액은 종래의 방법에 따라 2차원 배양하여 얻어지는 줄기세포 배양액보다 면역조절인자인 PGE2가 고농도로 분비되고, 면역반응 억제 및 염증성 사이토카인 분비 억제 효과가 우수하므로, 면역 질환 또는 염증성 질환을 예방 또는 치료하는 조성물로 유용하게 사용될 수 있다.The present invention relates to a mesenchymal stem cell culture solution for the prevention or treatment of an immunological disease or an inflammatory disease and a method for producing the same. According to the method of the present invention, mesenchymal stem cells are cultured in a 3-dimensional The stem cell culture solution obtained by culturing the stem cell culture solution has a higher concentration of PGE2 which is an immunoregulatory factor than the stem cell culture solution obtained by two-dimensional culture according to the conventional method, and has excellent immunosuppressive and inflammatory cytokine secretion suppressing effect, Can be useful as a composition for preventing or treating diseases.
Description
본 발명은 3차원 배양방법에 의해 유의적인 면역억제 및 항염기능을 보유하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액 및 이의 제조방법에 관한 것이다.The present invention relates to a mesenchymal stem cell culture medium for preventing or treating an immunological disease or inflammatory disease which has significant immunosuppressive and anti-inflammatory functions by a three-dimensional culture method, and a method for producing the same.
인간의 골수에는 몇 가지 종류의 전구세포(progenitor)가 존재하는 것이 발견되었으며, 이들 중 다분화능을 나타내는 전구세포를 중간엽줄기세포라 칭한다. 중간엽줄기세포는 골수뿐 아니라 지방, 간, 근육 등 신체 대부분의 장기에 존재하는 것으로 알려져 있다. 중간엽줄기세포는 자가증식능이 있고 골아세포(osteoblasts), 연골세포(chondrocytes), 근세포(myocytes), 골수기질세포(marrow stromal cells), 건-인대 섬유아세포(tendonligamentfibroblasts), 지방세포(adipocytes) 등으로 분화할 수 있다.Several types of progenitors have been found in human bone marrow. Among them, precursor cells exhibiting multipotentiality are called mesenchymal stem cells. Mesenchymal stem cells are known to exist not only in bone marrow, but also in most organs of the body, including fat, liver, and muscle. The mesenchymal stem cells are self-renewing and have an ability to function as osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon ligament fibroblasts, adipocytes, etc. . ≪ / RTI >
또한, 중간엽줄기세포는 배양 과정에서 다양한 성장인자 및 생리활성물질을 분비하는 것으로 알려져 있다. 따라서, 줄기세포배양액은 의약품이나 화장품 조성물 등의 임상적 용도로 사용이 가능하다. In addition, mesenchymal stem cells are known to secrete various growth factors and physiologically active substances during the culturing process. Therefore, the stem cell culture solution can be used for clinical use such as pharmaceuticals and cosmetic compositions.
그러나, 중간엽줄기세포를 배양하기 위해서는 통상적으로 소혈청을 사용하는데, 소혈청이 함유된 배양액은 임상적 적용이 부적당하다. 즉, 세포 배양액의 임상적 적용을 위해서는 소혈청과 같은 동물 유래 단백질의 사용이 제한되므로, 줄기세포의 배양에는 소혈청을 배제하는 것이 바람직하다.However, in order to cultivate mesenchymal stem cells, bovine serum is usually used, but the culture medium containing bovine serum is inadequate for clinical application. That is, since the use of animal-derived proteins such as bovine serum is limited for clinical application of cell culture media, it is preferable to exclude bovine serum for culture of stem cells.
소혈청 단백질은 줄기세포의 증식 및 유지를 위해서 반드시 필요하므로, 무혈청 배지에서 중간엽줄기세포를 배양하면서도 고농도의 생리활성물질을 함유하는 배양액을 얻을 수 있는 방법이 요구된다.Bovine serum proteins are necessary for the proliferation and maintenance of stem cells. Therefore, a method of obtaining a culture solution containing a high concentration of physiologically active substance while culturing mesenchymal stem cells in a serum-free medium is required.
국내 특허공개 제10-2009-0001163호(2009. 01. 08)는 인간의 골수, 제대혈 또는 태반 조직으로부터 수득한 중간엽줄기세포를 혈청 배지에서 배양 후 무혈청 배지에서 계대 배양하고, 배양한 줄기세포에 저산소 배양의 물리적 자극을 가하여, 중간엽줄기세포로부터 bFGF 또는 TGF-β를 대량으로 생산하는 방법을 개시하고 있다.Korean Patent Laid-Open No. 10-2009-0001163 (2009. 01. 08) discloses that mesenchymal stem cells obtained from human bone marrow, umbilical cord blood or placenta tissue are cultured in serum medium, subcultured in serum-free medium, Discloses a method of mass-producing bFGF or TGF-? From mesenchymal stem cells by applying physical stimulation of hypoxic culture to the cells.
유사하게, 국제공개 WO 2007/086637호(2007. 08. 02)에서는 포유동물의 지방세포에서 추출한 성체 줄기세포를 혈청 배지에서 배양 후 무혈청 배지에서 계대배양하고, 배양한 줄기세포에 저산소 배양, 자외선 조사, 영양분 결핍, 또는 기계적 마찰과 같은 물리적 자극을 가하고, 비타민 A, B, C, 또는 D를 배양액에 첨가하여 중간엽줄기세포로부터 bFGF, VEGF 또는 TGF-β를 대량으로 생산하는 방법을 개시하고 있다. Similarly, in International Publication WO 2007/086637 (Aug. 02, 2007), adult stem cells extracted from adipocytes of mammals are cultured in serum medium, subcultured in serum-free medium, cultured in hypoxic culture, A method of mass-producing bFGF, VEGF or TGF-? From mesenchymal stem cells by adding physical stimulation such as ultraviolet irradiation, nutrient deficiency, or mechanical friction and adding vitamin A, B, C, .
즉, 종래의 방법에서는 줄기세포를 단순히 2차원 배양하거나, VEGF 등의 함량을 높이기 위해 저산소 조건에서 2차원 배양을 실시하고 있을 뿐으로, 무혈청 배지에서는 줄기세포가 안정적으로 유지되기 어렵다는 점을 고려할 때 줄기세포 배양액의 생리적 활성도가 저하되는 문제점이 있었다. That is, in the conventional method, only two-dimensional culture is carried out under hypoxic condition in order to increase the content of VEGF or the like in a simple two-dimensional culture of stem cells. Considering that the stem cells are not stably maintained in serum-free medium The physiological activity of the stem cell culture solution is lowered.
한편, 면역 질환은 포유류 면역계의 구성성분들이 포유류의 병리상태를 야기하거나, 매개하거나 또는 기타 공헌하는 질환으로서, 특히 염증성 질환은 전 세계적으로 주목을 하고 있는 질환으로 그 기저 질환을 치료할 수 있는 치료제가 시급한 질환이다. 염증은 일반적으로 외부 물질 또는 해로운 자극에 의한 숙주 침입에 대해 신체 조직의 국소화된 보호 반응이다. 염증의 원인은 박테리아, 바이러스 및 기생충과 같은 감염성 원인; 화상 또는 방사선 조사와 같은 물리적 원인; 독소, 약물 또는 산업적 제제와 같은 화학약품; 알레르기 및 자가면역 반응과 같은 면역반응, 또는 산화성 스트레스와 연관된 상태일 수 있다.On the other hand, immunological diseases are diseases in which components of the mammalian immune system cause, mediate, or contribute to mammalian pathological conditions, in particular, inflammatory diseases are globally noted diseases, and therapeutic agents capable of treating the underlying diseases It is an urgent disease. Inflammation is generally a localized protective response of the body tissue to host infiltration by foreign substances or by harmful stimuli. Causes of inflammation include infectious causes such as bacteria, viruses and parasites; Physical causes such as burns or irradiation; Chemicals such as toxins, drugs or industrial formulations; An immune response such as an allergic and autoimmune response, or a condition associated with oxidative stress.
염증은 통증, 적화현상, 부기, 열 및 감염된 영역의 궁극적인 기능 손실을 그 특징으로 한다. 이들 증상은 면역계의 세포 사이에서 일어나는 일련의 복잡한 상호작용의 결과이다. 세포의 반응으로 인해 결과적으로 여러 그룹의 염증 매개자의 상호작용 네트워크가 생성된다: 단백질(예를 들면, 사이토카인, 효소(예를 들면 프로테아제, 퍼옥시다제), 주요 염기성 단백질, 점착 분자(ICAM, VCAM), 지질 매개자(예를 들면, 에이코사노이드, 프로스타글란딘, 류코트라이엔, 혈소판 활성화 인자(PAF)), 반응성 산소 종(예를 들면, 하이드로퍼옥사이드, 슈퍼옥사이드 음이온 O2-, 산화질소(NO) 등). 그러나 염증의 이들 매개자 중 대부분은 또한 정상적인 세포 활성의 조절자이다. 따라서 염증 반응의 결핍으로 인해 숙주가 제어되지 않으면서 손상(즉, 감염)되고, 또는 만성 염증으로 인해 부분적으로는 상기 언급된 매개자 중 여럿이 과다 생성됨으로써 매개되는 염증성 질환이 야기된다.Inflammation is characterized by pain, numbness, swelling, heat and ultimate loss of function in the affected area. These symptoms are the result of a series of complex interactions that take place between cells of the immune system. The reaction of cells results in a network of interaction groups of inflammatory mediators as a result: proteins (eg, cytokines, enzymes (eg proteases, peroxidases), major basic proteins, (E.g., VCAM), lipid mediators (e.g., eicosanoids, prostaglandins, leukotrienes, platelet activating factors (PAF)), reactive oxygen species such as hydroperoxides, superoxide anions O2-, ), Etc. However, many of these mediators of inflammation are also regulators of normal cellular activity, and thus are either damaged (i.e., infected) without control of the host due to the deficiency of the inflammatory response, or partly due to chronic inflammation Inflammatory diseases mediated by the overproduction of several of the above-mentioned mediators.
또한, 면역 질환 중 하나인 자가면역 질환은 면역 체계가 그 자신의 기관을 공격하여 자발적인 반응을 일으키는 것을 특징으로 한다. 이러한 반응들은 T 림프구에 의한 자가항원(auto-antigen)의 인식에 기인하며, 이로 인하여 체액상(자가항체 생성) 및 세포상(림프구 및 대식세포 세포독성 활성 증가) 면역 반응이 유발된다. 자가면역 질환으로는 류마티스성 질환, 건선, 전신성 피부근염, 다발성 경화증, 홍반성 낭창, 또는 항원에 의한 면역반응 악화, 즉, 천식, 약물 또는 음식에 대한 알레르기 등이 있다. 이러한 질환들은 모두 제한성이고 만성인 질환들이며, 경우에 따라서는 치명적이고, 현재까지 상기 질환들을 치료할 수 있는 효과적인 치료 방법이 존재하지 않는 실정이다. 그러므로 당해 질환의 진행 중에 질환을 경감시키거나 완화시킬 수 있는 약물, 의약 또는 매체라면 환자의 건강을 위해서 중요한 해결 수단이 된다고 할 것이다.Also, an autoimmune disease, one of the immune diseases, is characterized by the immune system attacking its own organ and causing a spontaneous reaction. These reactions are caused by the recognition of auto-antigen by T lymphocytes, which leads to immune responses in the body fluid (autoantibody production) and in the cellular phase (increased lymphocyte and macrophage cytotoxic activity). Examples of autoimmune diseases include rheumatic diseases, psoriasis, systemic dermatomyositis, multiple sclerosis, lupus erythematosus, or aggravation of immune responses by antigens such as asthma, drugs or food allergies. These diseases are all limited and chronic diseases, and in some cases, they are fatal, and thus there is no effective treatment method for treating the diseases. Therefore, drugs, medicines or medicines that can alleviate or alleviate the disease during the course of the disease will be said to be an important solution for the health of the patient.
이에, 본 발명자들은 면역 질환 또는 염증성 질환 치료제 적용을 위한 임상 용도로 사용 가능한 줄기세포 배양액을 개발하기 위해 노력한 결과, 기존 2차원 배양에 따라 수득한 배양액을 비교하여 무혈청 배지에서 생체적합성 스캐폴드를 이용하여 3차원 배양에 따라 수득한 중간엽줄기세포 배양액이 면역반응을 유발하지 않고, 면역반응성을 증가시키는 역할을 하는 염증성 사이토카인인 TNF-α 및 IFN-γ의 분비량을 감소시키며, 면역조절인자인 PGE2를 고농도로 분비하는 것을 확인함으로써, 상기 방법에 따라 수득한 배양액을 면역 질환 또는 염증성 질환의 예방 또는 치료에 이용할 수 있음을 밝힘으로써, 본 발명을 완성하였다.Accordingly, the present inventors have made efforts to develop a stem cell culture solution which can be used for clinical use for the treatment of immunological diseases or inflammatory diseases. As a result, the inventors of the present invention found that biocompatible scaffolds The mesenchymal stem cell culture obtained by the three-dimensional culture does not induce an immune response and reduces the secretion amount of TNF-a and IFN-y, which are inflammatory cytokines that increase the immunoreactivity, And that the culture solution obtained according to the above method can be used for the prophylaxis or treatment of an immunological disease or an inflammatory disease by confirming that PGE2 is secreted at a high concentration.
본 발명의 목적은 면역 질환 또는 염증성 질환 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법을 제공하기 위한 것이다.It is an object of the present invention to provide a method for producing a mesenchymal stem cell culture solution for the prevention or treatment of an immune disease or an inflammatory disease.
본 발명의 다른 목적은 상기 제조방법으로 제조된 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액을 제공하기 위한 것이다.Another object of the present invention is to provide a mesenchymal stem cell culture solution for the prevention or treatment of an immunological disease or inflammatory disease produced by the above production method.
본 발명의 또 다른 목적은 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지를 유효성분으로 함유하는 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물을 제공하기 위한 것이다.Yet another object of the present invention is to provide a composition for preparing a mesenchymal stem cell culture liquid for the treatment of an immunological disease or inflammatory disease, which comprises a biocompatible scaffold and a serum-free medium containing any one or more of bEGF or EGF as an active ingredient will be.
본 발명의 목적을 달성하기 위하여 본 발명은In order to achieve the object of the present invention,
(a) 중간엽줄기세포를 수집하여 무혈청 배지에서 생체적합성 스캐폴드와 3차원 배양하는 단계; 및(a) collecting mesenchymal stem cells and culturing them in a serum-free medium in a three-dimensional culture with a biocompatible scaffold; And
(b) 배양 배지를 수집하는 단계를 포함하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법을 제공한다.(b) collecting the culture medium. The present invention also provides a method for producing a mesenchymal stem cell culture solution for preventing or treating an immunological disease or an inflammatory disease.
또한, 본 발명은 상기 방법으로 제조된 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액을 제공한다.In addition, the present invention provides a mesenchymal stem cell culture solution for preventing or treating an immunological disease or inflammatory disease produced by the above method.
또한, 본 발명은 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지를 유효성분으로 함유하는 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물을 제공한다.The present invention also provides a composition for the manufacture of a mesenchymal stem cell culture for the treatment of an immunological disease or inflammatory disease, which comprises a biocompatible scaffold and a serum-free medium containing any one or more of bEGF or EGF as an active ingredient.
본 발명의 방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 얻어지는 줄기세포 배양액은, 종래의 방법에 따라 2차원 배양하여 얻어지는 줄기세포 배양액보다 면역조절인자인 PGE2가 고농도로 분비되고, 면역반응 억제 및 염증성 사이토카인 분비 억제 효과가 우수하므로, 면역 질환 또는 염증성 질환을 예방 또는 치료하는 조성물로 유용하게 사용될 수 있다.According to the method of the present invention, a stem cell culture obtained by three-dimensionally culturing mesenchymal stem cells in a serum-free medium using a biocompatible scaffold can be used as an immunomodulator PGE2 is secreted at a high concentration and has excellent immunosuppressive and inflammatory cytokine secretion inhibiting effects, and thus can be effectively used as a composition for preventing or treating immunological diseases or inflammatory diseases.
도 1은 2차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(2D CM) 또는 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(3D CM)을 농도별로 처리하고 말초혈액단핵세포 증식 억제율을 나타낸 도이다.
도 2는 2차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(2D CM) 또는 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(3D CM)을 농도별로 처리하고 말초혈액단핵세포에서 분비되는 TNF-α 분비 억제율을 나타낸 도이다.
도 3은 2차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(2D CM) 또는 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(3D CM)을 농도별로 처리하고 말초혈액단핵세포에서 분비되는 IFN-γ 분비 억제율을 나타낸 도이다.
도 4는 2차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(2D CM) 또는 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액(3D CM)에서의 PGE2(prostaglandin E2)의 분비량을 나타낸 도이다.FIG. 1 is a graph showing the results of treatment of mesenchymal stem cell culture (2D CM) obtained by a two-dimensional culture method or mesenchymal stem cell culture (3D CM) obtained by a three-dimensional culture method by concentration and inhibition of peripheral blood mononuclear cell proliferation Fig.
FIG. 2 is a graph showing the results obtained by treating the mesenchymal stem cell culture medium (2D CM) obtained by the two-dimensional culture method or the mesenchymal stem cell culture medium (3D CM) obtained by the three- TNF-a < / RTI >
FIG. 3 is a graph showing the results obtained by treating the mesenchymal stem cell culture medium (2D CM) obtained by the two-dimensional culture method or the mesenchymal stem cell culture medium (3D CM) obtained by the three- 0.0 > IFN-y < / RTI > secretion inhibition rate.
4 is a graph showing the secretion amount of PGE2 (prostaglandin E2) in the mesenchymal stem cell culture solution (2D CM) obtained by the two-dimensional culture method or the mesenchymal stem cell culture solution (3D CM) obtained by the three- to be.
이하, 본 발명의 용어를 하기와 같이 정의한다.Hereinafter, terms of the present invention will be defined as follows.
본 명세서에서 "중간엽줄기세포(mesenchymal stem cells)"는 자기 증식이 가능하고 다분화능을 가지고 있으며, CD73+, CD90+, CD105+, CD14-, CD20-, CD34-, CD45-인 세포 표현형을 나타내는 세포를 의미하고, 골수, 지방조직, 제대혈 등에서 분리될 수 있으며, 이에 한정되지는 않는다."Mesenchymal stem cells (mesenchymal stem cells)" herein is self-propagation is possible, and it has a multipotent, CD73 +, CD90 +, CD105 +, CD14 - the cellular phenotype -, CD20 -, CD34 -, CD45 And may be isolated from bone marrow, adipose tissue, umbilical cord blood, etc., but is not limited thereto.
본 명세서에서 "생리활성물질"은 세포나 신체의 기능에 영향을 미칠 수 있는 사이토카인, 세포성장인자, 면역조절인자 등을 통칭하여 나타낸다. 생리활성물질의 예로는 VEGF(vascular endothelial growth factor), EGF(epidermal growth factor), HGF(hepatocyte growth factor), TGF-beta(Tumor growth factor-beta), IGF(Insulin growth factor)등을 포함하며, 이에 한정되지는 않는다.As used herein, the term " physiologically active substance " refers collectively to cytokines, cell growth factors, immunomodulatory factors, and the like that may affect the functions of cells or the body. Examples of physiologically active substances include VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), TGF-beta (Tumor growth factor-beta) But is not limited thereto.
본 명세서에서 "줄기세포 배양액"은 줄기세포를 배양한 세포배양 상등액을 지칭한다. 줄기세포 배양액은 줄기세포 배양 과정에서 세포로부터 분비되는 여러 가지 생리활성 물질을 함유하고 있다.As used herein, the term " stem cell culture fluid " refers to a cell culture supernatant in which stem cells have been cultured. Stem cell cultures contain various physiologically active substances secreted from cells during stem cell culture.
본 명세서에서 생체적합성 스캐폴드는 세포와 친화성을 가지며 이른바 세포 접착성인 표면을 지닌 재료로 만들어지며 세포를 3차원적으로 부착하고 배양시킬 수 있는 지지체를 의미한다. 천연 유래 지지체의 예로는 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산, 셀룰로오스 등이 있고 이에 한정되지는 않는다. 합성 고분자의 예를 들면 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 폴리안하이드라이드 등이 있으며 이에 한정되지는 않는다.In this specification, the biocompatible scaffold refers to a scaffold that has affinity with a cell and is made of a material having a so-called cell adhesion surface, and capable of adhering and culturing cells three-dimensionally. Examples of natural-derived supports include, but are not limited to, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, and the like. Examples of the synthetic polymer include poly (alpha-hydroxy acid) series, poly (vinyl alcohol), polyanhydride, and the like.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은The present invention
(a) 중간엽줄기세포를 수집하여 무혈청 배지에서 생체적합성 스캐폴드와 3차원 배양하는 단계; 및(a) collecting mesenchymal stem cells and culturing them in a serum-free medium in a three-dimensional culture with a biocompatible scaffold; And
(b) 배양 배지를 수집하는 단계를 포함하는 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법을 제공한다.(b) collecting the culture medium. The present invention also provides a method for producing a mesenchymal stem cell culture solution for preventing or treating an immunological disease or an inflammatory disease.
상기 단계 (a)에서 중간엽줄기세포는 지방, 골수, 간 및 근육으로 이루어진 군으로부터 선택되는 어느 하나로부터 분리된 것이 바람직하다.In the step (a), the mesenchymal stem cells are preferably isolated from any one selected from the group consisting of fat, bone marrow, liver and muscle.
상기 단계 (a)에서 중간엽줄기세포는 기질배지 및 증식배지에서 배양한 후 계대 배양한 것이 바람직하다.In step (a), the mesenchymal stem cells are preferably cultured in a medium and a proliferation medium, followed by subculture.
상기 단계 (a)에서 3차원 배양은 3일 간격으로 배지를 교환하면서 3회 이상 줄기세포 배양액을 얻는 것이 바람직하다.In the step (a), it is preferable to obtain a stem cell culture solution three times or more while exchanging the medium at 3-day intervals.
상기 단계 (a)에서 무혈청 배지에 bFGF(basic fibroblast growth factor) 또는 EGF(epidermal growth factor) 중 어느 하나 이상을 첨가하여 배양하는 것이 바람직하다.In step (a), it is preferable to add at least one of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) to the serum-free medium.
상기 단계 (b)에서 생체적합성 스캐폴드는 세포 접착성인 표면을 갖는 세포 지지체로서 천연 또는 합성 고분자인 것이 바람직하고, 예를 들어 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산, 셀룰로오스, 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 폴리안하이드라이드 등 일 수 있으나, 이에 한정되지 않는다.In the step (b), the biocompatible scaffold is preferably a natural or synthetic polymer as a cell support having a cell adhesion surface, and examples thereof include alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, Poly (vinyl alcohol), polyanhydride, and the like, but are not limited thereto.
상기 면역 질환은 베체트병, 다발성 근육염, 피부 근육염, 자가면역 혈구감소증, 자가면역 심근염, 아토피피부염, 알레르기, 천식, 일차성간경변, 피부근염, 굿파이처 증후군, 자가면역 뇌수막염, 쇼그렌 증후군, 전신 홍반성 루프스, 애디슨병, 원형 탈모증, 강직성 척수염, 자가면역성 간염, 자가면역성 이하선염, 크론병, 인슐린 의존성 당뇨병, 이영양성 수포성 표피박리증, 부고환염, 사구체 신염, 그레이브스병, 길랑바레 증후군, 하시모토병, 용혈성 빈혈, 다발성 경화증, 중증 근무력증, 심상천포창, 건선, 류마티스열, 류마티스 관절염, 유육종증, 피부 경화증, 척추관절증, 갑상선염, 혈관염, 백반증, 점액수종, 악성빈혈, 궤양성 대장염, 이식편대숙주질환 또는 비만일 수 있으나 이에 한정되지 않는다.The immunological diseases are selected from the group consisting of Behcet's disease, multiple myositis, skin myositis, autoimmune hemodilution, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary cirrhosis, dermatomyositis, Goodpie syndrome, autoimmune meningitis, Sjogren's syndrome, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient for the treatment and / or prophylaxis of diabetic nephropathy such as diabetes mellitus, Anemia, ulcerative colitis, graft-versus-host disease or obesity, anemia, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathies, thyroiditis, vasculitis, But is not limited thereto.
상기 염증성 질환은 상기 염증성 질환은 위염, 장염, 신장염, 간염, 만성 폐쇄성 폐질환(COPD), 폐섬유증, 과민성 대장 증후군, 염증성 통증, 편두통, 두통, 허리 통증, 섬유근육통, 근막 질환, 바이러스 감염, 박테리아 감염, 곰팡이 감염, 화상, 외과적 또는 치과적 수술에 의한 상처, PGE 과다 증후군, 아테롬성 동맥 경화증, 통풍, 호지킨병, 췌장염, 결막염, 홍채염, 공막염, 포도막염 또는 급성 및 만성 염증성 질환일 수 있으나 이에 한정되지 않는다.Wherein the inflammatory disease is selected from the group consisting of gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine, headache, back pain, fibromyalgia, It may be a bacterial infection, a fungal infection, a burn, a wound due to surgical or dental surgery, PGE hyperaemia, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis or acute and chronic inflammatory disease But is not limited thereto.
상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-? Or IFN-? By an immune response and an immune response, but is not limited thereto.
상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture solution preferably reduces the immunoreactivity and the inflammatory response upon xenotransplantation, but is not limited thereto.
상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것이 바람직하고, PGE2를 5000 pg/ml ~ 10000 pg/ml 포함하는 것이 보다 바람직하고, PGE2를 7900 pg/ml ~ 9800 pg/ml 포함하는 것이 보다 더 바람직하다.The mesenchymal stem cell culture solution preferably contains PGE2 (prostaglandin E2), more preferably 5000 pg / ml to 10000 pg / ml of PGE2, and more preferably 7900 pg / ml to 9800 pg / ml of PGE2 Is more preferable.
본 발명의 구체적인 실시예에서, 본 발명자들은 면역반응을 유발한 말초혈액단핵세포에 상기 3차원 배양방법에 따라 수득한 중간엽줄기세포 배양액을 처리한 결과, 상기 말초혈액단핵세포의 세포 증식이 억제되는 것을 확인하였다(도 1 참조).In a specific example of the present invention, the inventors treated the mesenchymal stem cell culture solution obtained by the above-mentioned three-dimensional culture method on the peripheral blood mononuclear cells that caused the immune response, and found that inhibition of cell proliferation of the peripheral blood mononuclear cells (See Fig. 1).
또한, 본 발명자들은 면역반응을 유발한 말초혈액단핵세포에 상기 3차원 배양방법에 따라 수득한 중간엽줄기세포 배양액을 처리한 결과, 상기 말초혈액단핵세포에서 TNF-α 및 IFN-γ 분비를 억제하는 것을 확인하였다(도 2 및 도 3 참조).Furthermore, the inventors of the present invention have found that treatment of mesenchymal stem cell culture obtained by the above-described three-dimensional culture method on peripheral blood mononuclear cells that caused an immune response inhibits TNF-α and IFN-γ secretion in the peripheral blood mononuclear cells (See Figs. 2 and 3).
또한, 본 발명자들은 상기 3차원 배양방법에 따라 수득한 중간엽줄기세포 배양액에서 면역조절인자인 PGE2가 2차원 배양방법에 따라 수득한 중간엽줄기세포 배양액보다 현저하게 증가하는 것을 확인하였다(도 4 참조).Further, the present inventors confirmed that PGE2, which is an immunoregulatory factor in the mesenchymal stem cell culture solution obtained according to the above three-dimensional culture method, was significantly increased as compared with the mesenchymal stem cell culture obtained by the two-dimensional culture method Reference).
따라서, 본 발명의 방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 줄기세포 배양액이 PGE2를 고농도 포함하고, 면역반응 및 염증성 사이토카인의 분비 억제 효과가 우수함을 확인함으로써, 본 발명은 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액의 제조방법에 유용하게 사용될 수 있다.Therefore, the mesenchymal stem cells were cultured three-dimensionally using biocompatible scaffolds in the serum-free medium according to the method of the present invention, and thus the stem cell culture medium contained a high concentration of PGE2 and excellent immunosuppressive and inflammatory cytokine secretion inhibitory effect The present invention can be usefully used in a method for producing a mesenchymal stem cell culture solution for the prevention or treatment of an immunological disease or an inflammatory disease.
본 발명에 따른 중간엽줄기세포의 배양은 다음 과정에 따라 실시될 수 있으며, 세포배양에 사용되는 배지는 이에 한정되지 않는다.The culture of the mesenchymal stem cells according to the present invention can be carried out according to the following procedure, but the culture medium used for cell culture is not limited thereto.
(1) 중간엽줄기세포의 배양(1) Culture of mesenchymal stem cells
해당 조직에서 얻은 중간엽줄기세포를 기질배지에 현탁시켜 10,000~40,000 cells/㎠의 농도로 배양용기에 접종한 후 배양한다. 기질배지는 10% 우혈청이 포함되어 있는 DMEM 또는 DMEM/F12(Dulbecco's Modified Eagle Medium/Ham's F-12 Nutrient Broth) 배지로, 약 24시간 배양한다.The mesenchymal stem cells obtained from the tissue are suspended in a medium and cultured in a culture vessel at a concentration of 10,000 to 40,000 cells /
(2) 증식배지(expansion media)에서의 배양(2) Culture in expansion media
기질배지를 제거한 후, 증식배지에서 배양하여 부착성 세포를 증식시킨다. 증식배지는 10% 우혈청, 0.1~100 ng/㎖ 농도의 EGF(epidermal growth factor) 및/또는 0.1~100 ng/㎖ 농도의 bFGF(basic fibroblast growth factor)를 포함하는 DMEM 또는 DMEM/F12로, 부착성인 중간엽줄기세포를 신속하게 증식시켜 세포 양을 단기간에 대량으로 증가시키는 작용을 한다.After the substrate medium is removed, the adherent cells are proliferated by culturing in a proliferation medium. The proliferation medium was DMEM or DMEM / F12 containing 10% fetal bovine serum, epidermal growth factor (EGF) at a concentration of 0.1 to 100 ng / ml and / or basic fibroblast growth factor (bFGF) at a concentration of 0.1 to 100 ng / Adherent mesenchymal stem cells are rapidly proliferating to increase the amount of cells in a short period of time.
(3) 계대 배양(3) Passage culture
세포가 배양용기 바닥의 80 내지 90%를 채우면 증식배지를 제거하고 트립신 처리를 통해 세포를 배양용기에서 떼어낸다. 계대배양을 위해서는 세포를 1:3~1:4로 희석하여 새로운 배양용기에서 증식배지와 함께 배양한다. 이와 같은 방법으로 추가적인 계대배양이 가능하다.When the cells fill 80 to 90% of the bottom of the culture vessel, the growth medium is removed and the cells are removed from the culture vessel by trypsinization. For subculture, cells are diluted 1: 3 ~ 1: 4 and incubated with the growth medium in a new culture vessel. Additional subculture is possible in this way.
(4) 생체적합성 스캐폴드와 함께 배양(4) incubation with a biocompatible scaffold
배양한 세포는 PBS로 3회 이상 세척하여 FBS를 제거해주고, 무혈청 배지에서 생체적합성 스캐폴드에 부착된 형태로 배양한다. 스캐폴드 내에서의 세포배양은 통상적인 세포배양 용기를 필요로 하지 않으므로, 무균 바틀 또는 culture bag에서 다량으로 배양이 가능하여 보다 낮은 비용으로 편리하게 배양할 수 있는 장점이 있다. The cultured cells are washed with PBS 3 times or more to remove FBS, and cultured in a serum-free medium in the form attached to a biocompatible scaffold. Cell culture in a scaffold does not require a conventional cell culture container, and thus it is possible to cultivate large quantities in an aseptic bottle or culture bag, thereby enabling convenient cultivation at a lower cost.
또한, 본 발명의 제조방법에 따라 제조된 면역 질환 또는 염증성 질환의 예방 또는 치료용 중간엽줄기세포 배양액을 함유하는 조성물을 제공한다.The present invention also provides a composition containing a mesenchymal stem cell culture solution for preventing or treating an immunological disease or inflammatory disease produced according to the production method of the present invention.
상기 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture medium preferably inhibits the secretion of TNF-? Or IFN-? By an immune response and an immune response, but is not limited thereto.
상기 중간엽줄기세포 배양액은 이종이식시 면역거부반응 및 염증반응을 감소시키는 것이 바람직하나 이에 한정되지 않는다.The mesenchymal stem cell culture solution preferably reduces the immunoreactivity and the inflammatory response upon xenotransplantation, but is not limited thereto.
상기 중간엽줄기세포 배양액은 PGE2(prostaglandin E2)를 포함하는 것이 바람직하고, PGE2를 5000 pg/ml ~ 10000 pg/ml 포함하는 것이 보다 바람직하고, PGE2를 7900 pg/ml ~ 9800 pg/ml 포함하는 것이 보다 더 바람직하다.The mesenchymal stem cell culture solution preferably contains PGE2 (prostaglandin E2), more preferably 5000 pg / ml to 10000 pg / ml of PGE2, and more preferably 7900 pg / ml to 9800 pg / ml of PGE2 Is more preferable.
본 발명의 제조방법에 따라 중간엽줄기세포를 무혈청 배지 중 생체적합성 스캐폴드를 이용하여 3차원 배양하여 줄기세포 배양액은 세포 배양액이 면역반응 및 염증성 사이토카인의 분비 억제 효과가 우수하므로, 면역 질환 또는 염증성 질환의 예방 또는 치료용 조성물로 유용하게 사용될 수 있다.According to the production method of the present invention, the mesenchymal stem cells are cultured in a three-dimensional manner using a biocompatible scaffold in a serum-free medium. Since the cell culture medium has excellent immunosuppressive and inflammatory cytokine secretion suppressing effects, Or as a composition for preventing or treating inflammatory diseases.
또한, 본 발명은 생체적합성 스캐폴드 및, bEGF 또는 EGF 중 어느 하나 이상을 포함하는 무혈청 배지를 유효성분으로 함유하는 면역 질환 또는 염증성 질환 치료용 중간엽줄기세포 배양액 제조용 조성물을 제공한다.The present invention also provides a composition for the manufacture of a mesenchymal stem cell culture for the treatment of an immunological disease or inflammatory disease, which comprises a biocompatible scaffold and a serum-free medium containing any one or more of bEGF or EGF as an active ingredient.
상기 생체적합성 스캐폴드는 세포 접착성인 표면을 갖는 세포 지지체로서 천연 또는 합성 고분자인 것이 바람직하고, 예를 들어 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산, 셀룰로오스, 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 폴리안하이드라이드 등 일 수 있으나, 이에 한정되지 않는다.The biocompatible scaffold is preferably a natural or synthetic polymer as a cell scaffold having a surface that is adhered to a cell. Examples of the scaffold include alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) But are not limited to, poly (vinyl alcohol), polyanhydride, and the like.
상기 무혈청 배지는 당업계에 알려진 기본 배지를 제한 없이 사용할 수 있으며, 다만 혈청이 첨가되지 않은 것을 사용한다. 기본 배지는 인위적으로 합성하여 제조할 수 있으며, 상업적으로 제조된 배지를 사용할 수도 있다. 상업적으로 제조되는 배지의 예를 들면, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium) 및 Isocove's Modified Dulbecco's Medium 등이 있으나, 이에 한정되는 것은 아니며, 바람직하게는 DMEM 또는 DMEM/F12 를 사용할 수 있다.The serum-free medium can be used without limitation in a basic medium known in the art, but serum-free medium is used. The basic medium may be prepared by artificial synthesis, or a commercially prepared medium may be used. Examples of commercially produced media include Dulbecco's Modified Eagle's Medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Essential Medium, G-MEM (Glasgow's Minimal Essential Medium), and Isocove's Modified Dulbecco's Medium, but are not limited thereto. DMEM or DMEM / F12 can be preferably used.
상기 무혈청 배지에 bFGF는 0.5 ~ 2 ng/㎖ 첨가된 것이 바람직하고, 0.7 ~ 1.5 ng/㎖ 첨가된 것이 보다 바람직하고, 1 ng/㎖ 첨가하는 것이 보다 더 바람직하다.It is preferable that bFGF is added to the serum-free medium in an amount of 0.5 to 2 ng / ml, more preferably 0.7 to 1.5 ng / ml, and even more preferably 1 ng / ml.
상기 무혈청 배지에 EGF는 3 ~ 7 ng/㎖ 첨가된 것이 바람직하고, 4 ~ 6 ng/㎖ 첨가된 것이 보다 바람직하고, 5 ng/㎖ 첨가된 것이 보다 더 바람직하다.It is preferable that 3 to 7 ng / ml of EGF is added to the serum-free medium, more preferably 4 to 6 ng / ml, and even more preferably 5 ng / ml.
이하 본 발명을 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 한정되는 것은 아니다.However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.
<실시예 1> 인간 지방유래 중간엽줄기세포의 배양 방법Example 1: Culture of human adipose-derived mesenchymal stem cells
지방 조직은 보통 지방 흡입술로 얻을 수 있지만, 이에 한정되지는 않는다. 지방 흡입에 의해 얻어진 지방 조직으로부터 다음과 같이 지방유래 중간엽줄기세포를 분리하였다: 혈액을 제거하기 위해 지방 조직을 같은 부피의 KRB 용액으로 3~4회 세척하였다. 그 다음, 지방 조직과 같은 부피의 콜라게나제 용액을 넣어 37℃ 수욕에서 반응시켰다. 이를 원심분리용 튜브에 옮겨 넣고 20, 1500 rpm에서 10분 동안 원심분리하였다. 원심분리 후, 상층액인 지방층을 제거하고 아래층인 콜라게나제 용액을 흔들리지 않도록 조심해서 분리하였다. 기질배지를 넣어 현탁시킨 후, 20℃, 1500 rpm에서 5분 동안 원심분리하였다. 이때, 아래에 가라앉는 것이 스트로마-혈관 분획으로, 상층액을 제거하였다. 스트로마-혈관 분획을 기질배지에 현탁시켜 배양용기에 접종하고, 37℃, 5% CO2 인큐베이터에서 24시간 동안 배양하였다. 배양액 제거 후 인산염 완충용액으로 세척하고, 기질배지(10% FBS가 함유된 DMEM/F12배지), 또는 기질배지에 염기성 섬유아세포 성장인자(bFGF)가 1 ng/㎖ 농도로 포함된 증식배지, 또는 기질배지에 표피세포 성장인자(EGF)가 5 ng/㎖ 농도로 포함된 배지를 이용하여 증식시켰다. 지방유래 중간엽줄기세포가 배양용기의 80~90% 정도로 자라면 트립신 처리하여 단일 세포로 분리하여 수득하였다. 얻어진 세포를 증식배지로 1:3~1:4로 희석하여 계대 배양을 실시하였다.Fat tissue is usually obtained by liposuction, but is not limited thereto. The adipose derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction as follows: The adipose tissue was washed 3-4 times with the same volume of KRB solution to remove blood. Then, a volume of collagenase solution such as adipose tissue was added and the mixture was reacted at 37 ° C in a water bath. This was transferred to a centrifuge tube and centrifuged at 20, 1500 rpm for 10 minutes . After centrifugation, the upper layer of fat layer was removed and the lower layer of collagenase solution was carefully removed to avoid shaking. After the substrate medium was suspended, it was centrifuged at 20 DEG C and 1500 rpm for 5 minutes. At this time, the lower layer was a stroma-vascular fraction, and the supernatant was removed. The stroma-vascular fraction was suspended in a substrate medium, inoculated into a culture vessel, and cultured in a 5% CO 2 incubator at 37 ° C for 24 hours. After removing the culture medium, the cells were washed with a phosphate buffer solution, and the medium (DMEM / F12 medium containing 10% FBS) or the growth medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng / The medium was grown in medium containing epidermal growth factor (EGF) at a concentration of 5 ng / ml. When the adipose-derived mesenchymal stem cells were grown to about 80 to 90% of the culture vessel, they were trypsinized and isolated into single cells. The obtained cells were diluted to 1: 3 to 1: 4 with a growth medium and subcultured.
<실시예 2> 인간 지방유래 중간엽줄기세포를 2차원 배양하는 방법<Example 2> Method for two-dimensional culture of human adipose-derived mesenchymal stem cells
트립신 처리하여 단일세포로 얻어진 줄기세포를 DMEM/F12 배지로 3회 세척하여 FBS를 제거하였다. 1.3 × 107개/㎖ 줄기세포에 DMEM/F12 용액을 첨가하여 세포를 현탁하여 접종하였다. 그 다음, 1 ng/㎖ 염기성 섬유아세포 성장인자 및 5 ng/㎖ 표피세포 성장인자가 포함된 DMEM/F12 용액을 세포 4.0 × 107개 당 500 ㎖가 되도록 첨가하였다. 72시간 후 세포 배양액을 회수하고 새 배지를 첨가하는 방법으로 2~11회 더 세포 배양액을 회수하여 냉장 보관하였다.The stem cells obtained by single-cell treatment with trypsin were washed three times with DMEM / F12 medium to remove FBS. DMEM / F12 solution was added to 1.3 x 10 7 cells / ml stem cells, and the cells were suspended and inoculated. Then, a DMEM / F12 solution containing 1 ng / ml basic fibroblast growth factor and 5 ng / ml epidermal growth factor was added to 500 ml of 4.0 x 107 cells. After 72 hours, the cell culture medium was recovered, and the cell culture medium was recovered 2 to 11 times by adding fresh medium, followed by refrigerated storage.
<실시예 3> 인간 지방유래 중간엽줄기세포를 피브린 글루와 3차원 배양하는 방법Example 3: Three-dimensional culture of human adipose-derived mesenchymal stem cells with fibrin glue
트립신 처리하여 단일세포로 얻어진 줄기세포를 DMEM/F12 배지로 3회 세척하여 FBS를 제거하였다. 1.3 × 107개/㎖ 줄기세포에 트롬빈 용액이 포함된 DMEM/F12 용액을 첨가하여 세포를 현탁하였다. 듀얼시린지를 이용하여 세포현탁액과 피브리노겐 희석액이 혼합되도록 한 후 바틀 또는 culture bag에서 3차원배양하였다. 세포배양배지는 1 ng/㎖ 염기성 섬유아세포 성장인자 및 5ng/㎖ 표피세포 성장인자가 포함된 DMEM/F12 용액을 사용하였다. 72시간 후 세포 배양액을 회수하고 새 배지를 첨가하는 방법으로 2~11회 더 세포 배양액을 회수하여 냉장 보관하였다.The stem cells obtained by single-cell treatment with trypsin were washed three times with DMEM / F12 medium to remove FBS. The cells were suspended by adding DMEM / F12 solution containing thrombin solution to 1.3 占07 / ml stem cells. The cell suspension and the fibrinogen dilution were mixed using a dual syringe and then cultured in a 3-dimensional culture in a bag or culture bag. The cell culture medium was a DMEM / F12 solution containing 1 ng / ml basic fibroblast growth factor and 5 ng / ml epidermal growth factor. After 72 hours, the cell culture medium was recovered, and the cell culture medium was recovered 2 to 11 times by adding fresh medium, followed by refrigerated storage.
<실험예 1> 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액의 면역반응Experimental Example 1 Immune response of the mesenchymal stem cell culture obtained by the three-dimensional culture method 억제 확인Check for inhibition
상기 <실시예 2> 및 <실시예 3>에서 획득한 중간엽줄기세포 배양액의 면역반응 억제능을 확인하기 위하여 EdU 세포 증식 분석법을 수행하였다.The EdU cell proliferation assay was performed to confirm the ability of the mesenchymal stem cell culture obtained in Example 2 and Example 3 to inhibit the immune response.
구체적으로, 조직 적합성 항원 (Human Leukocyte Antigen; HLA)이 다른 공여자에서 얻은 말초혈액단핵세포 (peripheral Blood Mononuclear Cell; PBMC) 5 × 105개를 24-웰 플레이트에 첨가하였다. 양성대조군으로는 말초혈액단핵세포에 마이토겐 (mitogen)인 PHA (phyto-hemagglutinin) 1.0 ㎍/㎖를 첨가하여 말초혈액단핵세포의 면역반응을 유발하였다. 그 다음, 상기 <실시예 2>의 2차원 배양방법으로 수득한 중간엽줄기세포 배양액 또는 <실시예 3>의 3차원 배양방법으로 수득한 중간엽줄기세포 배양액 두 세트(Lot#1 및 Lot#2)를 10% 또는 20%의 농도로 처리하여 반응 시작 후 3일째 상등액을 수집하여 말초혈액단핵세포의 증식능력을 FACS 기법으로 측정하였다.Specifically, 5 × 10 5 peripheral blood mononuclear cells (PBMC) obtained from other donors with human leukocyte antigen (HLA) were added to 24-well plates. As a positive control, 1.0 ㎍ / ㎖ of PHA (phyto-hemagglutinin), which is a mitogen, was added to peripheral blood mononuclear cells to induce immune response of peripheral blood mononuclear cells. Then, two sets (
그 결과, 도 1에 나타낸 바와 같이, 상기 <실시예 2>의 2차원 배양방법에 의해 수득한 중간엽줄기세포 배양액을 처리한 군에서는 세포 증식이 억제되지 않는 반면, 상기 <실시예 3>의 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액을 처리한 군에서는 세포 증식이 억제되므로, 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액이 면역반응을 유의적으로 억제함을 확인하였다(도 1).As a result, as shown in Fig. 1, cell proliferation was not inhibited in the group treated with the mesenchymal stem cell culture solution obtained by the two-dimensional culture method of Example 2, In the group treated with the mesenchymal stem cell culture obtained by the three-dimensional culture method, it was confirmed that the mesenchymal stem cell culture obtained by the three-dimensional culture method significantly inhibited the immune response since cell proliferation was inhibited 1).
<실험예 2> 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액의 염증성 사이토카인 분비 억제 확인Experimental Example 2 Confirmation of inhibition of inflammatory cytokine secretion of the mesenchymal stem cell culture obtained by the three-dimensional culture method
상기 <실시예 2> 및 <실시예 3>에서 획득한 중간엽줄기세포 배양액의 염증성 사이토카인 분비 억제 여부를 확인하기 위하여, ELISA 분석법을 수행하였다.In order to confirm the inhibition of inflammatory cytokine secretion of the mesenchymal stem cell culture obtained in Example 2 and Example 3, an ELISA assay was performed.
구체적으로, 상기 <실험예 1>과 같이 말초혈액단핵세포 5 × 105개를 24-웰 플레이트에 넣고 PHA로 활성화시켜 면역반응을 유발하였다. 그 다음, 상기 <실시예 2>의 2차원 배양방법으로 수득한 중간엽줄기세포 배양액 또는 <실시예 3>의 3차원 배양방법으로 수득한 중간엽줄기세포 배양액 두 세트(Lot#1 및 Lot#2)를 10% 또는 20%의 농도로 첨가하였다. 반응 3일째 상등액을 수집하여 TNF-α와 IFN-γ의 분비량을 ELISA 분석으로 측정하였다. Specifically, 5 × 10 5 peripheral blood mononuclear cells were put into a 24-well plate as described in <Experimental Example 1> and activated with PHA to induce an immune response. Then, two sets (
그 결과, 도 2 및 도 3에 나타낸 바와 같이, 상기 <실시예 3>의 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액을 처리한 군에서는 TNF-α와 IFN-γ의 분비량이 현저히 감소함을 확인하였다(도 2 및 도 3).As a result, as shown in Figs. 2 and 3, in the group treated with the mesenchymal stem cell culture obtained by the three-dimensional culture method of Example 3, the secretion amount of TNF-a and IFN- (Figs. 2 and 3).
따라서, 상기 <실험예 1> 및 <실험예 2>를 통해 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액은 면역반응을 유발하지 않으며, 면역반응성을 증가시키는 역할을 하는 염증성 사이토카인의 분비량을 감소시켜 면역 질환 또는 염증성 질환을 완화시켜 치유되도록 도울 수 있음을 확인하였다.Therefore, through the above <Experimental Example 1> and <Experimental Example 2>, the mesenchymal stem cell culture solution obtained by the three-dimensional culture method does not induce the immune response, and the secretion amount of the inflammatory cytokine To reduce the immune or inflammatory disease and to help to heal.
<실험예 3> 3차원 배양방법에 의해 수득된 중간엽줄기세포 배양액의 면역조절인자 확인<Experimental Example 3> Identification of immunoregulatory factors of the mesenchymal stem cell culture obtained by the three-dimensional culture method
상기 <실시예 2> 및 <실시예 3>에서 획득한 중간엽줄기세포 배양액에서 면역조절인자인 prostaglandin E2(PGE2)의 분비 정도를 확인하기 위하여, 상기 <실시예 2> 및 <실시예 3>에서 획득한 중간엽줄기세포액을 ELISA 분석을 수행하고, 450 nm에서 흡광도를 측정하였다.In order to confirm the secretion level of prostaglandin E2 (PGE2) which is an immunoregulatory factor in the mesenchymal stem cell culture fluid obtained in Example 2 and Example 3, in Example 2 and Example 3, Were analyzed by ELISA and the absorbance at 450 nm was measured.
그 결과, 도 4에 나타낸 바와 같이, 상기 <실시예 3>의 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액(Lot #1 및 Lot #2)에서는 PGE2의 분비량이 현저히 증가함을 확인하였다(도 4).As a result, as shown in Fig. 4, it was confirmed that the secretion amount of PGE2 was significantly increased in the mesenchymal stem cell culture solutions (
따라서, 상기 <실험예 1> 내지 <실험예 3>을 통해 3차원 배양방법에 의해 수득한 중간엽줄기세포 배양액은 면역반응을 유발하지 않으며, 면역반응성을 증가시키는 역할을 하는 염증성 사이토카인의 분비량을 감소시키고, 면역조절인자인 PGE2를 고농도로 분비하여 면역 반응성을 증가시켜 면역 질환 또는 염증성 질환을 완화시켜 치유되도록 도울 수 있음을 확인하였다.Therefore, through the above <Experimental Examples 1 to 3>, the mesenchymal stem cell culture solution obtained by the three-dimensional culture method does not induce the immune response, and the secretion amount of the inflammatory cytokine And PGE2, which is an immunoregulatory factor, is secreted at a high concentration to increase immunoreactivity, thereby alleviating immune or inflammatory diseases and thereby helping to be cured.
Claims (9)
(b) 배양 배지를 수집하는 단계를 거쳐, PGE2(prostaglandin E2)를 포함하는 중간엽줄기세포 배양액을 수득하는 단계를 포함하고,
여기서 중간엽줄기세포 배양액은 면역반응 및 면역반응에 의한 TNF-α 또는 IFN-γ 의 분비를 억제하는 면역 질환 또는 염증성 질환의 예방 또는 치료용인, 중간엽줄기세포 배양액의 제조방법.
(a) collecting mesenchymal stem cells and culturing them in a serum-free medium in a three-dimensional culture with a biocompatible scaffold; And
(b) collecting the culture medium to obtain a mesenchymal stem cell culture solution containing PGE 2 (prostaglandin E2)
Wherein the mesenchymal stem cell culture medium is for the prevention or treatment of immunological diseases or inflammatory diseases which inhibit the secretion of TNF-? Or IFN-? By the immune response and the immune response.
The method according to claim 1, wherein the mesenchymal stem cells are isolated from any one selected from the group consisting of fat, bone marrow, liver and muscle.
[2] The method according to claim 1, wherein the mesenchymal stem cells are cultured in a medium and a growth medium, followed by subculture.
[3] The method according to claim 1, wherein the three-dimensional culture in step (a) comprises culturing the stem cell culture medium three or more times while exchanging the medium at intervals of 3 days.
The method according to claim 1, wherein at least one of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added to the serum-free medium in step (a).
2. The method of claim 1, wherein the biocompatible scaffold is a natural or synthetic polymer as a cell support having a surface that is cell adhesive.
7. The composition of claim 6 wherein the biocompatible scaffold is selected from the group consisting of alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) Gt; and / or < / RTI >
The method of claim 1, wherein the immune disorder is selected from the group consisting of Behcet's disease, multiple myositis, skin myositis, autoimmune hemodilution, autoimmune myocarditis, atopic dermatitis, allergy, asthma, primary cirrhosis, dermatomyositis, , Sjogren's syndrome, systemic lupus erythematosus, Addison's disease, alopecia areata, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, Crohn's disease, insulin-dependent diabetes mellitus, eosinophilic epidermolysis, epididymitis, glomerulonephritis, Inflammatory bowel syndrome, hemorrhagic fever, multiple sclerosis, acute myasthenia gravis, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, mucoid, malignant anemia, ulcerative colitis, graft An autoimmune disease, an autoimmune disease, an autoimmune disease, an autoimmune disease, Way.
The method of claim 1, wherein the inflammatory disease is selected from the group consisting of gastritis, enteritis, nephritis, hepatitis, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, irritable bowel syndrome, inflammatory pain, migraine headache, back pain, fibromyalgia, PGE hyperaemia, atherosclerosis, gout, Hodgkin's disease, pancreatitis, conjunctivitis, iritis, scleritis, uveitis, and acute and chronic inflammatory diseases, including but not limited to infections, bacterial infections, fungal infections, burns, surgical or dental surgery wounds, ≪ / RTI >
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| CN111481572A (en) * | 2019-01-29 | 2020-08-04 | 安特罗根有限公司 | Autologous and homologous adipose-derived mesenchymal stem cell composition for curing tendon or ligament injury and preparation method thereof |
| WO2021100889A1 (en) * | 2019-11-18 | 2021-05-27 | 경희대학교 산학협력단 | Composition for treating spinal cord injury, comprising stem cells treated with novel compound |
| KR20220115289A (en) * | 2021-02-10 | 2022-08-17 | 사회복지법인 삼성생명공익재단 | Pharmaceutical composition for preventing or treating inflammatory bowel disease comprising Tumor necrosis factor alpha inhibitor and Prostaglandin E2 |
| WO2024161321A1 (en) * | 2023-01-31 | 2024-08-08 | Rhodes University | Corn endosperm-based biomaterial scaffold |
-
2018
- 2018-06-21 KR KR1020180071300A patent/KR20180072644A/en not_active Ceased
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111481572A (en) * | 2019-01-29 | 2020-08-04 | 安特罗根有限公司 | Autologous and homologous adipose-derived mesenchymal stem cell composition for curing tendon or ligament injury and preparation method thereof |
| WO2021100889A1 (en) * | 2019-11-18 | 2021-05-27 | 경희대학교 산학협력단 | Composition for treating spinal cord injury, comprising stem cells treated with novel compound |
| CN114760999A (en) * | 2019-11-18 | 2022-07-15 | 外治世拉普提斯株式会社 | Composition for treating spinal cord injury comprising stem cells treated with novel compound |
| KR20220115289A (en) * | 2021-02-10 | 2022-08-17 | 사회복지법인 삼성생명공익재단 | Pharmaceutical composition for preventing or treating inflammatory bowel disease comprising Tumor necrosis factor alpha inhibitor and Prostaglandin E2 |
| KR20230108251A (en) * | 2021-02-10 | 2023-07-18 | 사회복지법인 삼성생명공익재단 | Pharmaceutical composition for preventing or treating inflammatory bowel disease comprising Tumor necrosis factor alpha inhibitor and Prostaglandin E2 |
| WO2024161321A1 (en) * | 2023-01-31 | 2024-08-08 | Rhodes University | Corn endosperm-based biomaterial scaffold |
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