KR20210060922A - 3D Cultured Umbilical Cord-derived Mesenchymal Stem Cell and Uses thereof - Google Patents
3D Cultured Umbilical Cord-derived Mesenchymal Stem Cell and Uses thereof Download PDFInfo
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Abstract
Description
2D 배양조건 대비 3D 배양조건에서 특정 유전자와 사이토카인의 발현량이 변화하는 특성을 갖는 3차원 배양된 탯줄 유래 중간엽 줄기세포 및 이의 용도에 관련된 것이다.It relates to three-dimensional cultured umbilical cord-derived mesenchymal stem cells and uses thereof, which have the property of changing the expression levels of specific genes and cytokines in 3D culture conditions compared to 2D culture conditions.
일반적으로 사용되고 있는 단일층 세포배양 시스템(monolayer cell culture system)은 배양법이 비교적 용이하고 세포 생존 능력이 탁월하다는 장점을 가지고 있어 생물학 분야에 많이 이용되고 있다. 하지만 인체 조직과 같은 3차원적인 미세 환경 조건을 구현하기에는 한계가 있다. 실제, 세포가 생존하며 생활사를 유지하기 위해서는 세포 주변의 환경을 구성하고 있는 성장인자, 호르몬, 부착성분자(adhesion molecule), 세포외기질과 같은 인자들과의 상호작용이 유지되어야 하지만 이전까지 이루어진 특정 질환 세포의 생성 및 전이 그리고 이를 치료하기 위한 치료요법에 관한 연구에 있어서는 세포의 미세 환경 조건을 고려하지 않은 채 연구되어 왔다. 이러한 단일층 세포배양 시스템의 한계점을 보완할 수 있는 모델로서 3차원(3D) 세포배양이 주목을 받고 있다. 3D 세포배양 시스템의 이점은 형태학상 실제 특정 질환 세포와 매우 유사한 구조를 가지고 있다는 것이다. 세포의 스페로이드(spheroid) 구조는 단지 형태학적 의미뿐 아니라 그가 지니고 있는 기능과도 매우 연관성이 깊은 것으로 알려져 있기에 다양한 세포에서 형성될 수 있는 스페로이드의 구조가 세포 생활사에 미치는 영향을 고려할 수 있다. 또한 세포와 세포사이의 상호작용뿐 아니라 세포와 주변의 미세 환경과의 상호작용으로 인한 세포 내 신호전달이 가능하기에 특정 질환의 치료에서 나타나는 세포의 성장, 분화, 죽음을 조절하는 기전을 단일층에서 보다 정교하게 구현할 수 있다.A generally used monolayer cell culture system is widely used in the field of biology because it has the advantage of relatively easy culture method and excellent cell viability. However, there is a limit to realizing three-dimensional microenvironmental conditions such as human tissue. In fact, in order for cells to survive and maintain their life cycle, interactions with factors such as growth factors, hormones, adhesion molecules, and extracellular matrix that make up the environment around the cells must be maintained. In the study of the generation and metastasis of specific diseased cells and the treatment regimen for treating them, studies have been conducted without considering the microenvironmental conditions of the cells. Three-dimensional (3D) cell culture is attracting attention as a model that can compensate for the limitations of such a single layer cell culture system. The advantage of the 3D cell culture system is that it has a structure very similar to that of a specific disease cell in morphology. Since the spheroid structure of a cell is known to be highly related not only to its morphological meaning, but also to its functions, it is possible to consider the effect of the structure of spheroids that can be formed in various cells on the cell life cycle. In addition, since intracellular signaling is possible due to the interaction between cells and the surrounding microenvironment, as well as the interaction between cells and the surrounding microenvironment, the mechanism that regulates the growth, differentiation, and death of cells in the treatment of specific diseases is a single layer. It can be implemented more elaborately.
또한, 세포 치료제 상용화를 위해서 완제품의 수요가 대량으로 요구됨과 동시에 동일한 품질의 제품이 생산되어야 하나, 기존의 2D(2-Dimensional) 배양시스템 사용 시 세포치료제 생산이 매우 제한적이라는 문제점이 존재한다. 현재 2D 배양 생산으로 대량배양을 진행할 경우, 세포 및 배지의 양이 많아질수록 생산시간이 길어지게 되며, 또한 생산능력의 저하가 나타날 수 있다. 생산담당인원 및 보조 인력의 투입을 통해 일정부분 해결할 수 있지만, 이는 인건비 증가에 따라 생산 단가의 상승 원인이 되므로, 차후 상품 개발 후 가격 경쟁력이 낮아질 수 있다. 이에, 공정 효율성의 최적화할 수 있는 새로운 배양 시스템으로서, 3D(3-Dimensional) 바이오리액터(bioreactor)를 이용한 대량배양 시스템이 주목받고 있다.In addition, in order to commercialize cell therapy products, a large amount of demand for finished products is required and products of the same quality must be produced, but there is a problem that production of cell therapy products is very limited when using the existing 2-Dimensional (2D) culture system. When mass cultivation is carried out with the current 2D culture production, the production time becomes longer as the amount of cells and medium increases, and the production capacity may decrease. Although it can be partially solved through the input of production personnel and auxiliary personnel, this may cause an increase in production cost as the labor cost increases, and thus price competitiveness may decrease after product development in the future. Accordingly, as a new culture system capable of optimizing process efficiency, a mass culture system using a 3-Dimensional (3D) bioreactor is attracting attention.
2D 배양조건 대비 3D 배양조건에서 특정 유전자와 사이토카인의 발현량이 변화하는 특성을 갖는 3차원 배양된 탯줄 유래 중간엽 줄기세포 및 이의 용도를 제공하는 것을 목적으로 한다.It is an object of the present invention to provide a three-dimensional cultured umbilical cord-derived mesenchymal stem cell having a characteristic of varying the expression levels of specific genes and cytokines in 3D culture conditions compared to 2D culture conditions, and uses thereof.
일 양태로서, 하기 (a) 내지 (d)로 이루어진 군으로부터 선택된 어느 하나의 특성을 갖는 3차원 배양된 탯줄 유래 중간엽 줄기세포 집합체(stem cell cluster)를 제공한다:As an aspect, a three-dimensional cultured umbilical cord-derived mesenchymal stem cell cluster having any one characteristic selected from the group consisting of the following (a) to (d) is provided:
(a) IL-33, RAB27B, KDR 및 COL3A1 로 이루어진 군으로부터 선택되는 하나 이상이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것; (a) at least one selected from the group consisting of IL-33, RAB27B, KDR and COL3A1 is expressed more than two-dimensionally cultured umbilical cord-derived mesenchymal stem cells;
(b) ANKRD1, MYCT1, MFAP5, OLR1, SERPINB7, CEMIP 및 CD27로 이루어진 군으로부터 선택되는 하나 이상이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 적게 발현되는 것;(b) at least one selected from the group consisting of ANKRD1, MYCT1, MFAP5, OLR1, SERPINB7, CEMIP and CD27 is less expressed than that of two-dimensional cultured umbilical cord-derived mesenchymal stem cells;
(c) IGFBP1, KIT, IL-20, IL-3, HGF, PDFG-A, PDFG-B 및 VEGF-A로 이루어진 군으로부터 선택되는 하나 이상의 사이토카인이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것; 및 (c) IGFBP1, KIT, IL-20, IL-3, HGF, PDFG-A, PDFG-B, and at least one cytokine selected from the group consisting of VEGF-A in two-dimensional cultured umbilical cord-derived mesenchymal stem cells More expressed than; And
(d) IL-26, MMP 및 PF4로 이루어진 군으로부터 선택된 하나 이상의 사이토카인이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것.(d) IL-26, MMP, and at least one cytokine selected from the group consisting of PF4 is expressed more than two-dimensional cultured umbilical cord-derived mesenchymal stem cells.
상기 "탯줄(umbilical cord)"은 포유류의 태아가 태반에서 성장할 수 있도록 모체와 배를 연결해주는 줄을 의미할 수 있으며, 일반적으로 와튼 젤리로 둘러싸인 3개의 혈관, 즉, 2개의 배꼽 동맥과 1개의 배꼽 정맥으로 구성된 조직을 의미할 수 있다. 따라서, "탯줄 유래 중간엽 줄기세포(Umbilical Cord Mesenchymal Stem cells)"는 탯줄 또는 탯줄의 와튼 젤리(Wharton's Jelly) 조직으로부터 유래되고, 다양한 조직 세포로 분화할 수 있는 능력을 가지는 세포를 의미할 수 있다. The "umbilical cord" may mean a string that connects the mother and the abdomen so that the mammalian fetus can grow in the placenta, and is generally surrounded by three blood vessels, that is, two umbilical arteries and one It may mean a tissue composed of umbilical veins. Accordingly, "Umbilical Cord Mesenchymal Stem cells" is derived from the umbilical cord or Wharton's Jelly tissue of the umbilical cord, and may mean cells having the ability to differentiate into various tissue cells. .
상기 "세포 집합체(cell cluster)"는 2 이상의 세포가 밀집된 상태를 말하며, 조직 상태일 수도 있고, 단일 세포 상태일 수도 있다. 각각의 세포 집합체는 조직 자체 또는 일부, 또는 단일 세포의 집합체로 존재할 수 있으며, 3차원 배양된 탯줄 유래 중간엽 줄기세포로부터 분화된 세포 유사-조직체를 포함할 수 있다.The "cell cluster" refers to a state in which two or more cells are clustered, and may be a tissue state or a single cell state. Each cell aggregate may exist as a tissue itself or a part, or as an aggregate of single cells, and may include a cell-like-organism differentiated from three-dimensional cultured umbilical cord-derived mesenchymal stem cells.
상기 "3차원(three-dimension)"은 2차원이 아닌 기하학적인 3개의 파라미터(예를 들면, 깊이, 넓이, 높이 또는 X, Y, Z 축) 모델을 갖는 입체를 의미할 수 있으며, 따라서 일 구체예에 따른 3차원 배양된 탯줄 유래 중간엽 줄기세포로부터 분화된 세포집합체는 3차원 배양, 즉 배양 용기에서 탈착되어 부유상태로 배양되어 세포가 증식함에 따라 입체적으로 구형, 시트(sheet) 또는 그와 유사한 3차원의 형태(예를 들면, 유사 조직체)를 갖는 세포 집합체를 의미할 수 있다.The "three-dimension" may mean a three-dimensional model having three geometric parameters (eg, depth, area, height or X, Y, Z axis), not two-dimensional, and thus one The cell aggregate differentiated from the three-dimensional cultured umbilical cord-derived mesenchymal stem cells according to the embodiment is three-dimensionally cultured, that is, detached from the culture vessel and cultured in a suspended state, and as the cells proliferate, three-dimensionally spherical, sheet, or It may refer to an aggregate of cells having a three-dimensional shape similar to (eg, a similar tissue).
상기 "IL-33"은 치주(Dental) 유래 줄기세포의 골형성(osteogenesis) 유도 및 증식과 전분화능을 증가시키고, 조혈줄기세포(Hematopoietic stem and progenitor cells; HSPC)과 비-조혈줄기세포에서의 염증 과정의 모든 단계에 관여할 수 있다.The "IL-33" induces osteogenesis and increases proliferation and pluripotency of periodontal stem cells, and in hematopoietic stem and progenitor cells (HSPC) and non-hematopoietic stem cells. It can be involved in any stage of the inflammatory process.
상기 "RAB27B"는 소포성 융해와 트래피킹(Vesicular fusion and trafficking)에 관여하여 엑소좀 분비 경로를 제어할 수 있다.The "RAB27B" is involved in vesicular fusion and trafficking to control the exosome secretion pathway.
상기 "KDR"은 혈관 신생(angiogenesis), 림프관 신생(lymphangiogenesis), 혈관발달(vascular development), 혈관 투과성(vascular permeability), 그리고 배아에서의 조혈(embryonic hematopoiesis) 조절에 필수적인 역할을 수행하고, 내피 세포의 증식(proliferation), 생존(survival), 이동(migration) 및 분화(differentiation)를 촉진할 수 있다.The "KDR" plays an essential role in the regulation of angiogenesis, lymphangiogenesis, vascular development, vascular permeability, and embryonic hematopoiesis, and endothelial cells Can promote proliferation, survival, migration and differentiation.
상기 "COL3A1"은 초기 배아와 배아 발생 전반에 걸쳐 발현하며, 성인에서는 다양한 내부 장기 및 피부에 주요성분일 수 있다.The "COL3A1" is expressed throughout early embryonic and embryogenesis, and may be a major component in various internal organs and skin in adults.
상기 "HGF" 는 배아 기관 발달(embryonic organ development), 근육발달(myogenesis), 성인의 장기 기관 재생(organ regeneration) 및 상처 치유(wound healing)에 중요한 역할을 수행하고, 세포 성장(cell growth), 세포 운동성(cell motility), 형태 형성(morphogenesis)을 조절하며, 체세포 분열(mitogenesis)을 유도, 세포 운동성(cell motility), 및 혈관 신생(angiogenesis)과 조직재생(tissue regeneration)에 중요한 역할을 수행하고, 조혈 전구 세포(hematopoietic progenitor cells)의 성장을 자극할 수 있다.The "HGF" plays an important role in embryonic organ development, myogenesis, adult organ regeneration and wound healing, and cell growth, It regulates cell motility and morphogenesis, induces mitogenesis, plays an important role in cell motility, and angiogenesis and tissue regeneration. , Can stimulate the growth of hematopoietic progenitor cells.
상기 "ANKRD1"은 심장 유전자의 발현을 부정적으로 조절하는 핵 전사 인자(nuclear transcription factor)로 작용하고, 간세포에서 세포 사멸과 연관이 있을 수 있다.The "ANKRD1" acts as a nuclear transcription factor that negatively regulates the expression of heart genes, and may be associated with apoptosis in hepatocytes.
상기 "MYCT1"은 세포자연사(apoptosis) 촉진, 형태의 변경(alteration of morphology), 종양 형성 전환(tumorigenic conversion), 유전자 불안정성(genomic instability)의 촉진 및 조혈 분화(hematopoietic differentiation)의 억제 역할을 할 수 있다.The "MYCT1" can play a role in promoting apoptosis, alteration of morphology, tumorigenic conversion, promoting genomic instability, and inhibiting hematopoietic differentiation. have.
상기 "MFAP5"는 유방암(breast cancer)에서 ERK/MMP 신호 전달 경로를 조절함으로써 종양 진행(tumor progression) 및 뼈 전이(bone metastasis)를 촉진할 수 있다.The "MFAP5" may promote tumor progression and bone metastasis by regulating the ERK/MMP signaling pathway in breast cancer.
상기 "OLR1"은 '종양 유전자(oncogene)' 역할을 할 수 있음을 암시, 비정상적인 세포 증식(aberrant cellular proliferation), 이동(migration)에 관여할 수 있다.The "OLR1" implies that it can play a role of'oncogene', and may be involved in aberrant cellular proliferation and migration.
상기 "SERPINB7"은 췌관선암(pancreatic ductal adenocarcinoma)에 대한 최초의 예측 RNA 바이오 마커로 확인 및 위암 진행 관련 유전자일 수 있다.The "SERPINB7" is the first predictive RNA biomarker for pancreatic ductal adenocarcinoma and may be a gene related to gastric cancer progression.
상기 "CEMIP"는 암에서 발현이 증가, 세포 생존(regulator of cell survival), 성장 및 침습(growth and invasion)의 조절 인자, 류마티스 관절염(rheumatoid arthritis)에서 혈관 신생 마커(angiogenic marker)라고 불릴 수 있으며, 히알루론산 분해(hyaluronic acid degradation)에 참여할 수 있다.The "CEMIP" may be called an angiogenic marker in rheumatoid arthritis, which is an increased expression in cancer, a regulator of cell survival, a regulator of growth and invasion, and , Hyaluronic acid degradation (hyaluronic acid degradation) may participate.
상기 "CD274"는 상향 조절시 암이 숙주 면역계를 회피(evade the host immune system)하도록 허용할 수 있고, 자가 면역(autoimmunity)과 관련이 있을 수 있다.The “CD274” may allow cancer to evade the host immune system upon upregulation and may be related to autoimmunity.
상기 "IGFBP1"은 세포 이동(cell migration) 및 대사 조절에 중요하고, 진피유두세포 생존능력(dermal papilla cell viability)에 영향을 미치고 모발 유도(hair induction)와 관련된 다양한 단백질의 발현을 촉진할 수 있다.The "IGFBP1" is important for cell migration and metabolic regulation, affects dermal papilla cell viability, and can promote the expression of various proteins related to hair induction. .
상기 "KIT"은 세포 유형에 따라 세포 생존(cell survival), 이동(migration), 그리고 증식(proliferation)을 매개할 수 있고, 정상적인 조혈(혈구 형성, hematopoiesis), 색소 형성(pigmentation), 멜라닌 형성(melanogenesis), 생식(fertility), 정자 형성(spermatogenesis), 장 운동(gut movement) 및 신경계(nervous system) 조절에 중요할 수 있다.The "KIT" can mediate cell survival, migration, and proliferation depending on the cell type, and normal hematopoiesis (hematopoiesis), pigmentation (pigmentation), melanin formation ( melanogenesis, fertility, spermatogenesis, gut movement, and nervous system regulation.
상기 "IL20"은 피부와 관련된 염증에 각질 형성 세포(keratinocytes)의 증식(proliferation) 및 분화(differentiation)를 조절할 수 있고, 다능성 조혈 전구 세포(multipotential hematopoietic progenitor)의 세포 확장을 야기할 수 있다.The "IL20" can regulate the proliferation and differentiation of keratinocytes in skin-related inflammation, and can cause cell expansion of multipotential hematopoietic progenitors.
상기 "IL3"은 조혈 줄기 세포(hematopoietic stem cells)와 전구체(progenitors)의 생존(survival) 및 증식(proliferation), 이들의 성숙 세포(mature cells)로의 분화(differentiation)를 촉진함으로 혈액 세포 생산(blood-cell production)을 조절할 수 있다.The "IL3" is a blood cell production by promoting the survival and proliferation of hematopoietic stem cells and precursors, and their differentiation into mature cells. -cell production) can be adjusted.
상기 "HGF"는 배아 기관 발달(embryonic organ development), 근육발달(myogenesis), 성인의 장기 기관 재생(organ regeneration) 및 상처 치유(wound healing)에 중요한 역할할 수 있고, 세포 성장(cell growth), 세포 운동성(cell motility), 형태 형성(morphogenesis)을 조절할 수 있으며, 체세포 분열(mitogenesis)을 유도, 세포 운동성(cell motility), 및 혈관 신생(angiogenesis)과 조직재생(tissue regeneration)에 중요한 역할할 수 있고, 조혈 전구 세포(hematopoietic progenitor cells)의 성장을 자극할 수 있다.The "HGF" can play an important role in embryonic organ development, myogenesis, adult organ regeneration and wound healing, and cell growth, It can regulate cell motility and morphogenesis, and can play an important role in inducing mitogenesis, cell motility, and angiogenesis and tissue regeneration. And can stimulate the growth of hematopoietic progenitor cells.
상기 "PDGFA", "PDGFB"는 배아 발생(embryonal development) 동안 특정 세포 유형의 성장(growth) 및 생존(survival) 조절 및 성인의 조직 복구(tissue repair)에 중요한 역할을 할 수 있다.The “PDGFA” and “PDGFB” may play an important role in regulating the growth and survival of certain cell types during embryonic development and tissue repair in adults.
상기 "VEGFA"는 내피 세포에 특이적으로 작용하여 혈관 투과성(vascular permeability) 증가, 혈관 신생(angiogenesis), 혈관 형성(vasculogenesis) 및 내피 세포 성장(endothelial cell growth) 유도, 세포 이동(cell migration) 촉진 및 세포 자멸사 억제(inhibiting apoptosis) 등 다양한 효과를 나타낼 수 있다.The "VEGFA" specifically acts on endothelial cells to increase vascular permeability, angiogenesis, vasculogenesis and endothelial cell growth, and promote cell migration. And inhibiting apoptosis (inhibiting apoptosis).
상기 "IL26"은 건선(psoriasis)을 포함한 T 세포 매개 피부 염증(T cell-mediated skin inflammation)에서 유망한 치료 표적일 수 있다.The "IL26" may be a promising therapeutic target in T cell-mediated skin inflammation, including psoriasis.
상기 "MMP12"은 종양 세포가 침입하여 전이(invade and metastasize)되는 경로를 제거하여 종양 및 전이를 촉진하는 효소일 수 있다.The "MMP12" may be an enzyme that promotes tumor and metastasis by removing a pathway through which tumor cells invade and metastasize.
상기 "PF4"는 암 강화 내분비 신호(cancer-enhancing endocrine signal), 골수 거핵구형성(bone marrow megakaryopoiesis) 및 폐암 진행(lung cancer progression)을 자극할 수 있다.The “PF4” may stimulate cancer-enhancing endocrine signals, bone marrow megakaryopoiesis, and lung cancer progression.
상기 (a) 특성은 IL33 및 RAB27B 중 하나 이상이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 10배 이상 더 많이 발현되는 것일 수 있다. 이러한 경우, 개체에의 적용시에, IL-33의 높은 발현으로 인해 골형성(osteogenesis) 유도 및 증식과 전분화능을 증가시킬 수 있고, 염증 과정의 모든 단계에 관여하여 염증반응을 제어할 수 있으며, RAB27B의 높은 발현으로 인해 엑소좀 분비 경로를 제어할 수 있다는 장점이 존재한다.The (a) characteristic may be that at least one of IL33 and RAB27B is expressed 10 times or more compared to two-dimensional cultured umbilical cord-derived mesenchymal stem cells. In this case, when applied to an individual, due to the high expression of IL-33, it is possible to induce osteogenesis, increase proliferation and pluripotency, participate in all stages of the inflammatory process, and control the inflammatory response. , There is an advantage of being able to control the exosome secretion pathway due to the high expression of RAB27B.
상기 (b) 특성은 ANKRD1이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 60배 이상 더 적게 발현되는 것일 수 있다. 이러한 경우, 개체에의 적용시에, 심장 관련 유전자의 발현을 부정적으로 조절하는 핵 전사 인자의 발현을 현저히 낮출 수 있고, 간세포에서의 세포 사멸 역시 현저히 낮추어 관련 부작용을 억제할 수 있다는 장점이 존재한다.The (b) characteristic may be that ANKRD1 is expressed more than 60 times less than that of umbilical cord-derived mesenchymal stem cells cultured in two dimensions. In this case, when applied to an individual, the expression of nuclear transcription factors that negatively regulate the expression of heart-related genes can be significantly lowered, and apoptosis in hepatocytes can also be significantly lowered, thereby suppressing related side effects. .
일 양태로서, 상기 집합체, 이를 구성하는 개별세포 또는 이의 배양액을 포함하는 자가면역질환, 간질환, 산부인과적 질환 또는 근육 질환의 예방 또는 치료를 위한 약학적 조성물을 제공한다.In one aspect, there is provided a pharmaceutical composition for the prevention or treatment of autoimmune diseases, liver diseases, obstetric diseases or muscle diseases, including the aggregate, individual cells constituting the same, or a culture medium thereof.
상기 집합체는 상술한 바대로 3차원으로 배양되어 2차원으로 배양되는 경우 대비 차별적으로 인자들 발현(예를 들면, IL33, RAB27B, ANKRD1 등)함에 따라 다양한 질환의 예방 또는 치료에 이용될 수 있음은 당업계 통상의 기술자에 자명한 것이고, 구체적으로는 자가면역질환, 간질환, 산부인과적 질환 또는 근육 질환의 예방 또는 치료에 매우 효과적으로 이용될 수 있다.The aggregate can be used for the prevention or treatment of various diseases according to the differential expression of factors (eg, IL33, RAB27B, ANKRD1, etc.) compared to the case of being cultured in three dimensions and cultured in two dimensions as described above. It is obvious to those skilled in the art, and specifically, it can be used very effectively in the prevention or treatment of autoimmune diseases, liver diseases, obstetric diseases or muscle diseases.
상기 자가면역질환은 아토피성 피부염, 크론병, 궤양성 대장염, 베체트병, 루푸스, 쇼그렌증후군, 중증근무력증, 경피증, 결절성 다발동맥염, 갑상선기능저하증, 건선 및 류마티스성 관절염으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 반드시 이에 제한되는 것은 아니다.The autoimmune disease may be one or more selected from the group consisting of atopic dermatitis, Crohn's disease, ulcerative colitis, Behcet's disease, lupus, Sjogren's syndrome, myasthenia gravis, scleroderma, polyarteritis nodosa, hypothyroidism, psoriasis and rheumatoid arthritis. However, it is not necessarily limited thereto.
상기 간질환은 간기능 손상, 간 장애, 간염, 간독성, 담즙울체, 지방간, 간경변, 간허혈, 알코올성 간 질환, 간농양, 간성 혼수, 간위축증 및 간암으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 반드시 이에 제한되는 것은 아니다. The liver disease may be one or more selected from the group consisting of impaired liver function, liver disorder, hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, liver abscess, hepatic coma, hepatic atrophy, and liver cancer. It is not limited.
상기 산부인과적 질환은 난소 종양, 클라미디아, 다낭성 난포 증후군, 마이코플라즈마, 자궁 근종, 유리아플라즈마, 자궁 선근증, 자궁기형, 자궁 내막증, 매독 및 질염으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 반드시 이에 제한되는 것은 아니다.The obstetric diseases may be one or more selected from the group consisting of ovarian tumors, chlamydia, polycystic follicular syndrome, mycoplasma, uterine fibroids, free aplasma, adenomyosis, uterine anomalies, endometriosis, syphilis, and vaginitis, but are limited thereto. no.
상기 근육 질환은 긴장감퇴증, 근위축증, 근이영양증, 근무력증, 악액질, 경직성 척추 증후군, 근위축성 축삭경화증, 샤르코-마리-투스병 및 근육 감소증으로 이루어진 군으로부터 선택된 하나 이상일 수 있으나, 반드시 이에 제한되는 것은 아니다.The muscle disease may be one or more selected from the group consisting of hypotonia, muscular atrophy, muscular dystrophy, myasthenia, cachexia, stiff spine syndrome, amyotrophic axon sclerosis, Charco-Marie-tus disease, and muscle , but is not limited thereto. .
상기 약학적 조성물의 투여량은 탯줄 유래 중간엽 줄기세포를 기준으로 1.0 X 103 내지 1.0 X 1010 세포/kg(체중) 또는 개체, 또는 1.0 X 107 내지 1.0 X 108 세포/kg(체중) 또는 개체일 수 있다. 다만, 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성별, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있고, 당업자라면 이러한 요인들을 고려하여 투여량을 적절히 조절할 수 있다. 투여 횟수는 1회 또는 임상적으로 용인 가능한 부작용의 범위 내에서 2회 이상이 가능하고, 투여 부위에 대해서도 1개소 또는 2개소 이상에 투여할 수 있다. 인간 이외의 동물에 대해서도, kg당 또는 개체당 인간과 동일한 투여량으로 하거나, 또는 예를 들면 목적의 동물과 인간과의 기관(심장 등)의 용적비(예를 들면, 평균값) 등으로 상기의 투여량을 환산한 양을 투여할 수 있다. 일 구체예에 따른 치료의 대상동물로서는, 인간 및 그 밖의 목적으로 하는 포유동물을 예로 들 수 있고, 구체적으로는 인간, 원숭이, 마우스, 래트, 토끼, 양, 소, 개, 말, 돼지 등이 포함된다. The dosage of the pharmaceutical composition is based on the umbilical cord-derived mesenchymal stem cells, 1.0 X 10 3 to 1.0 X 10 10 cells/kg (body weight) or an individual, or 1.0 X 10 7 to 1.0 X 10 8 cells/kg (weight ) Or an individual. However, the dosage may be variously prescribed depending on factors such as the formulation method, the mode of administration, the patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity. In consideration of these factors, the dosage can be appropriately adjusted. The number of times of administration may be one time or two or more times within the range of clinically acceptable side effects, and the administration site may be administered to one or two or more sites. For animals other than humans, administration of the same dose per kg or per individual as humans, or, for example, the volume ratio (e.g., average value) of an organ (heart, etc.) between the target animal and human The amount converted to the amount can be administered. Examples of animals to be treated according to one embodiment include humans and mammals for other purposes, specifically humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, etc. Included.
상기 약학적 조성물은 유효성분으로서 상기 탯줄 유래 중간엽 줄기세포 집합체 또는 이의 배양액과, 약학적으로 허용가능한 담체 및/또는 첨가물을 포함할 수 있다. 예를 들어, 멸균수, 생리식염수, 관용의 완충제(인산, 구연산, 그 밖의 유기산 등), 안정제, 염, 산화방지제(아스코르브산 등), 계면활성제, 현탁제, 등장화제, 또는 보존제 등을 포함할 수 있다. 국소 투여를 위해, 생체고분자(biopolymer) 등의 유기물, 하이드록시아파타이트 등의 무기물, 구체적으로는 콜라겐 매트릭스, 폴리락트산 중합체 또는 공중합체, 폴리에틸렌글리콜 중합체 또는 공중합체 및 그의 화학적 유도체 등과 조합시키는 것도 바람직하다. 상기 약학적 조성물이 주사에 적당한 제형으로 조제되는 경우에는, 세포 집합체가 약학적으로 허용가능한 담체 중에 용해되어 있거나 또는 용해되어 있는 용액상태로 동결된 것일 수 있다. The pharmaceutical composition may include the umbilical cord-derived mesenchymal stem cell aggregate or a culture solution thereof, and a pharmaceutically acceptable carrier and/or additive as an active ingredient. For example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives, etc. can do. For topical administration, it is also preferable to combine organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. . When the pharmaceutical composition is prepared in a dosage form suitable for injection, the cell aggregate may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution state.
상기 탯줄 유래 중간엽 줄기세포 집합체는, 신체의 조직 또는 기관이 목적하는 세포 군집, 예를 들어 줄기세포 또는 유래세포군집의 생착, 이식 또는 주입에 의해 강화, 치료 또는 대체되는 다양한 종류의 치료 프로토콜에 사용될 수 있다. 상기 집합체는 존재하는 조직을 대체 또는 강화시켜, 새롭거나 변화된 조직이 되게 하거나 생물학적 조직 또는 구조와 결합시킬 수 있다. 또한, 전형적으로 탯줄 이외의 다른 조직 유래 줄기세포 집합체가 사용되는 치료 프로토콜에서 줄기세포가 상기 탯줄 유래 중간엽 줄기세포 집합체로 대체될 수 있다. The umbilical cord-derived mesenchymal stem cell aggregate is used in various types of treatment protocols that are strengthened, treated, or replaced by engraftment, transplantation, or injection of a target cell population, for example, stem cells or derived cell populations by a tissue or organ of the body. Can be used. The aggregates can replace or strengthen existing tissues, resulting in new or altered tissues, or can be associated with biological tissues or structures. In addition, stem cells may be replaced with the umbilical cord-derived mesenchymal stem cell aggregate in a treatment protocol in which a tissue-derived stem cell aggregate other than the umbilical cord is typically used.
상기 약학적 조성물은 그 투여방법이나 제형에 따라 필요한 경우, 현탁제, 용해보조제, 안정화제, 등장화제, 보존제, 흡착방지제, 계면활성화제, 희석제, 부형제, pH 조정제, 무통화제, 완충제, 환원제, 산화방지제 등을 적절히 포함할 수 있다. 상기에 예시된 것들을 비롯하여 본 발명에 적합한 약학적으로 허용되는 담체 및 제제는 문헌[Remington's Pharmaceutical Sciences, 19th ed., 1995]에 상세히 기재되어 있다.The pharmaceutical composition is a suspension agent, a solubilizer, a stabilizer, an isotonic agent, a preservative, an adsorption inhibitor, a surfactant, a diluent, an excipient, a pH adjuster, a painless agent, a buffering agent, a reducing agent, Antioxidants and the like may be appropriately included. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.
상기 약학적 조성물은 당해 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질 중의 용액, 현탁액 또는 유화액 형태이거나 분말, 과립, 정제 또는 캡슐 형태일 수 있다. 또한, 상기 약학적 조성물은 주사용 제형으로 제형화될 수 있다. 이 경우 제형화하기 위한 공지된 통상적 성분이 이용될 수 있고, 통상적인 방법으로 제형화될 수 있다.The pharmaceutical composition is prepared in a unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person having ordinary knowledge in the technical field to which the present invention belongs, or It can be manufactured by enclosing it in a multi-volume container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of powder, granules, tablets, or capsules. In addition, the pharmaceutical composition may be formulated as an injection formulation. In this case, known conventional ingredients for formulation may be used, and may be formulated in a conventional manner.
일 양태로서, 분리된 탯줄을 배양 용기에 부착하여 배양하는 단계; 상기 배양된 탯줄에 분리효소를 접촉시켜 탯줄 유래 중간엽 줄기세포를 분리하는 단계; 및 상기 분리된 탯줄 유래 중간엽 줄기세포를 겐타마이신을 포함하는 무혈청배지에서 3차원 배양하는 단계를 포함하는 탯줄 유래 중간엽 줄기세포 집합체를 제조하는 방법을 제공한다.In one aspect, culturing by attaching the separated umbilical cord to a culture vessel; Separating the umbilical cord-derived mesenchymal stem cells by contacting the cultured umbilical cord with a separating enzyme; And it provides a method for producing an umbilical cord-derived mesenchymal stem cell aggregate comprising the step of three-dimensional culturing the isolated umbilical cord-derived mesenchymal stem cells in a serum-free medium containing gentamicin.
상기 탯줄은 건강한 산모(예를 들면, HIV, HCV, HBV 음성인 산모)로부터 출산 후 분리된 태반을 사용할 수 있다. 즉, 상기 "분리된 탯줄"이라 함은 산모의 모체로부터 출산 후 분리되는 탯줄을 의미할 수 있다. 상기 분리된 탯줄은 분리된 후 신속하게 멸균된 용기 및 얼음에 담아 보관될 수 있다. The umbilical cord may use a placenta separated after childbirth from a healthy mother (eg, HIV, HCV, HBV negative mother). That is, the "separated umbilical cord" may mean an umbilical cord that is separated from the mother's mother after childbirth. The separated umbilical cord can be stored in a sterilized container and ice quickly after being separated.
상기 탯줄을 태반으로부터 분리하는 수득하는 방법은 예를 들면, 분리된 태반으로부터 탯줄을 분리하는 단계; 상기 분리된 탯줄의 외부의 혈액을 제거하는 단계; 상기 혈액이 제거된 탯줄의 동맥과 정맥을 제거하는 단계; 및/또는 상기 동맥과 정맥이 제거된 혈액을 일정한 크기(예를 들면, 1 내지 20 mm)로 세절하는 단계를 포함할 수 있다. 상기 혈액의 제거는 예를 들면, Ca/Mg free DPBS, 또는 겐타마이신 함유 Ca/Mg free DPBS를 사용할 수 있다. The method of obtaining the separation of the umbilical cord from the placenta may include, for example, separating the umbilical cord from the separated placenta; Removing blood from the outside of the separated umbilical cord; Removing the arteries and veins of the umbilical cord from which the blood has been removed; And/or the blood from which the arteries and veins have been removed may include the step of slicing the blood into a predetermined size (eg, 1 to 20 mm). For the removal of the blood, for example, Ca/Mg free DPBS or gentamicin-containing Ca/Mg free DPBS may be used.
다음으로, 상기 세절된 탯줄(예를 들면, 분리된 탯줄)로부터 줄기세포를 분리하는 단계가 수행될 수 있다. 상기 탯줄 유래 중간엽 줄기세포를 분리하는 단계는 상기 분리된 탯줄을 배양 용기에 부착하여 5 내지 20일, 예를 들면, 10 내지 20일, 예를 들면, 10 내지 15일 배양하는 단계; 상기 배양된 탯줄 조직에서 세포가 뻗어 나오는 것을 확인하는 단계; 및/또는 탯줄 조직에 분리효소를 처리하는 단계를 포함할 수 있다. Next, the step of separating stem cells from the shredded umbilical cord (eg, separated umbilical cord) may be performed. Separating the umbilical cord-derived mesenchymal stem cells may include attaching the separated umbilical cord to a culture vessel and culturing it for 5 to 20 days, for example, 10 to 20 days, for example, 10 to 15 days; Confirming that cells are protruding from the cultured umbilical cord tissue; And/or treating the umbilical cord tissue with a separating enzyme.
상기 분리효소는 콜라게나아제를 포함할 수 있다. 상기 콜라게나아제는 콜라겐의 펩티드 결합을 파괴하는 효소를 의미할 수 있으며, 콜라게나아제 타입 I, 타입 II, 타입 III, 타입 IV 또는 그들의 조합을 포함할 수 있다. 상기 분리효소는 5 내지 30 U/ml, 예를 들면, 5 내지 25 U/ml, 10 내지 25 U/ml, 또는 20 U/ml의 콜라게나아제를 포함할 수 있다. 또한, 상기 분리효소는 트립신(trypsin), 및/또는 디스파아제(Dispase)를 포함할 수 있으며, 또한, 상기 분리효소를 포함하는 용액은 콜라게나아제, 트립신, 및/또는 디스파아제를 포함하는 물, 염수(saline), 예를 들면, HBSS(Hank's Balanced Salt Solution)를 포함할 수 있다. 또한, 분리효소의 처리 시간은 예를 들면, 1시간 내지 20시간, 2시간 내지 10시간, 4시간 내지 9시간, 또는 5시간 내지 6시간일 수 있다. The separating enzyme may include collagenase. The collagenase may mean an enzyme that destroys the peptide bond of collagen, and may include collagenase type I, type II, type III, type IV, or a combination thereof. The separating enzyme may include 5 to 30 U/ml, for example, 5 to 25 U/ml, 10 to 25 U/ml, or 20 U/ml of collagenase. In addition, the separating enzyme may include trypsin, and/or dispase, and the solution containing the separating enzyme includes collagenase, trypsin, and/or dispase. It may include water, saline, for example, Hank's Balanced Salt Solution (HBSS). In addition, the treatment time of the separating enzyme may be, for example, 1 hour to 20 hours, 2 hours to 10 hours, 4 hours to 9 hours, or 5 hours to 6 hours.
일 구체예에 있어서, 상기 조직과 분리효소의 반응은 진탕을 수행하여 반응이 이루어질 수 있으며, 상기 진탕은 약 20 내지 40 ℃, 약 30 내지 40 ℃, 또는 35 내지 40 ℃, 예를 들면, 37 ℃에서 수행할 수 있고, 약 5 내지 60 분간 또는 10 내지 30분간 수행할 수 있으며, 또한 예를 들면 10 내지 30분간 2번을 수행할 수 있다. In one embodiment, the reaction between the tissue and the separating enzyme may be performed by shaking, the shaking is about 20 to 40 °C, about 30 to 40 °C, or 35 to 40 °C, for example, 37 It can be carried out at ℃, it can be carried out for about 5 to 60 minutes or 10 to 30 minutes, and for example, it can be carried out twice for 10 to 30 minutes.
추가적으로, 상기 조직과 분리 효소의 반응 후에, 분리 효소를 불활성화 하기 위한 과정을 추가로 수행할 수 있으며, 예를 들면, FBS를 첨가하여 상기 효소 반응을 정지시킬 수 있다. 또한, 효소 반응액으로부터 조직 세포, 예를 들면, 탯줄 유래 중간엽 줄기세포를 분리하는 방법은 통상의 당업계에 공지된 방법에 의해 수행할 수 있으며, 예를 들면 원심분리 한 후, 세포체(strainer)를 사용하여 세포를 분리할 수 있다. Additionally, after the reaction between the tissue and the separating enzyme, a process for inactivating the separating enzyme may be additionally performed. For example, the enzyme reaction may be stopped by adding FBS. In addition, the method of separating tissue cells, for example, umbilical cord-derived mesenchymal stem cells, from the enzyme reaction solution can be carried out by a method known in the art. For example, after centrifugation, the cell body (strainer ) Can be used to separate cells.
상기 방법은 상기 3차원 배양된 배지를 칼슘과 마그네슘이 제거된 인산염완충식염수로 세척한 후, EDTA(ethylenediaminetetraacetic acid)와 펙티나아제(pectinase)를 포함하는 수확용액(harvest solution)을 처리하여 3차원 배양된 탯줄 유래 중간엽 줄기세포 집합체를 분리하는 단계를 더 포함할 수 있다.The method includes washing the three-dimensional culture medium with phosphate-buffered saline from which calcium and magnesium have been removed, and then treating a harvest solution containing ethylenediaminetetraacetic acid (EDTA) and pectinase. It may further comprise the step of separating the cultured umbilical cord-derived mesenchymal stem cell aggregate.
상기 "탯줄 유래 중간엽 줄기세포 집합체의 분리"는 처리하지 않은 포유류 탯줄에서 줄기세포와 정상적으로 결부되어 있는 세포의 적어도 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% 또는 99%를 제거하는 것을 의미할 수 있다. 한 장기에서 얻은 줄기세포를 포함하는 세포군은 처리하지 않은 상태의 그 장기 속에서 그 줄기세포와 정상적으로 결부되어 있는 다른 세포가 전체 세포의 50% 미만일 때 "분리"되었다고 할 수 있다. The "Isolation of umbilical cord-derived mesenchymal stem cell aggregate" means at least 20%, 30%, 40%, 50%, 60%, 70%, 80% of cells normally associated with stem cells in an untreated mammalian umbilical cord, It can mean removing 90%, 95% or 99%. A cell group containing stem cells obtained from one organ can be said to be "separated" when the other cells normally associated with the stem cell in the untreated organ are less than 50% of the total cells.
상기 방법에 의해 제조된 탯줄 유래 중간엽 줄기세포 집합체는 하기 (a) 내지 (d)로 이루어진 군으로부터 선택된 어느 하나의 특성을 가질 수 있다:The umbilical cord-derived mesenchymal stem cell aggregate prepared by the above method may have any one characteristic selected from the group consisting of the following (a) to (d):
(a) IL-33, RAB27B, KDR 및 COL3A1으로 이루어진 군으로부터 선택되는 하나 이상이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것; (a) at least one selected from the group consisting of IL-33, RAB27B, KDR and COL3A1 is expressed more than that of two-dimensional cultured umbilical cord-derived mesenchymal stem cells;
(b) ANKRD1, MYCT1, MFAP5, OLR1, SERPINB7, CEMIP 및 CD27로 이루어진 군으로부터 선택되는 하나 이상이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 적게 발현되는 것;(b) at least one selected from the group consisting of ANKRD1, MYCT1, MFAP5, OLR1, SERPINB7, CEMIP and CD27 is less expressed than that of two-dimensional cultured umbilical cord-derived mesenchymal stem cells;
(c) IGFBP1, KIT, IL-20, IL-3, HGF, PDFG-A, PDFG-B 및 VEGF-A로 이루어진 군으로부터 선택되는 하나 이상의 사이토카인이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것; 및 (c) IGFBP1, KIT, IL-20, IL-3, HGF, PDFG-A, PDFG-B, and at least one cytokine selected from the group consisting of VEGF-A in two-dimensional cultured umbilical cord-derived mesenchymal stem cells More expressed than; And
(d) IL-26, MMP 및 PF4로 이루어진 군으로부터 선택된 하나 이상의 사이토카인이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것.(d) IL-26, MMP, and at least one cytokine selected from the group consisting of PF4 is expressed more than two-dimensional cultured umbilical cord-derived mesenchymal stem cells.
상기 3차원 배양, 탯줄 유래 중간엽 줄기세포, 차별적으로 발현되는 각 개별 인자들에 대한 구체적인 사항 등에 대한 설명은 상술한 바와 같다.The three-dimensional culture, umbilical cord-derived mesenchymal stem cells, and details of each individual factor differentially expressed are as described above.
일 양태로서 제공되는 탯줄 유래 중간엽 줄기세포 집합체는 3차원으로 배양되어 2차원으로 배양되는 경우 대비 차별적으로 인자들 발현함에 따라 다양한 질환의 예방 또는 치료에 이용될 수 있다.The umbilical cord-derived mesenchymal stem cell aggregate provided as an aspect can be used for the prevention or treatment of various diseases by differential expression of factors compared to the case of being cultured in three dimensions and cultured in two dimensions.
도 1은 일 구체예에 따른 탯줄 유래 중간엽 줄기세포 집합체의 분리에 있어서, 분리효소의 전, 후의 세포 형태를 나타내는 도면이다.1 is a diagram showing the cell morphology before and after the separating enzyme in the isolation of the umbilical cord-derived mesenchymal stem cell aggregate according to an embodiment.
이하, 보다 구체적인 설명을 위해 실시예를 들어 상세하게 설명하기로 한다. Hereinafter, for a more detailed description, examples will be described in detail.
실시예 1. 탯줄 유래 중간엽 줄기세포의 분리Example 1. Isolation of umbilical cord-derived mesenchymal stem cells
정상적으로 분만한 건강한 산모로부터 사전에 충분한 설명에 근거한 동의(informed consent)를 받고, 정상 태반 분만 시에 수집된 태반조직으로부터 탯줄을 분리하였다. 분리된 탯줄을 Ca/Mg free DPBS로 2 내지 5회 세척하여 혈액을 제거하였다. 이후, 외부 양막은 벗겨내지 않고, 동맥 2개와 정맥 1개를 제거한 뒤 1 내지 5 mm 정도 크기로 탯줄을 잘라내었다. 이후, 상기 탯줄을 배양용기에 부착하여 10 내지 15일 배양을 하였고, 상기 배양된 조직에서 세포가 뻗어나오는 것을 확인한 후, 5 내지 6시간 동안 200 U/ml의 콜라게나아제 I을 처리하여 탯줄 유래 중간엽 줄기세포를 분리하였다. 상기 콜라게나아제 I 처리 전, 후에 탯줄 부착된 조직에서 세포가 뻗어나오는 것을 확인하기 위하여 광학현미경하에서 40x, 100x 배율로 세포형태를 확인하였고, 그 결과를 도 1에 나타내었다. The umbilical cord was separated from the placental tissue collected at the time of normal placenta delivery after receiving informed consent from healthy mothers who delivered normally. The separated umbilical cord was washed 2 to 5 times with Ca/Mg free DPBS to remove blood. Thereafter, the external amniotic membrane was not peeled off, and two arteries and one vein were removed, and the umbilical cord was cut to a size of about 1 to 5 mm. Thereafter, the umbilical cord was attached to the culture vessel and cultured for 10 to 15 days, and after confirming that the cells were protruding from the cultured tissue, 200 U/ml of collagenase I was treated for 5 to 6 hours to derive the umbilical cord. Mesenchymal stem cells were isolated. Before and after the collagenase I treatment, the cell morphology was confirmed at 40x and 100x magnifications under an optical microscope in order to confirm that the cells are protruding from the tissues attached to the umbilical cord, and the results are shown in FIG. 1.
도 1은 일 구체예에 따른 탯줄 유래 중간엽 줄기세포 분리에 있어서, 분리효소의 전, 후의 세포 형태를 나타내는 도면이다. 1 is a diagram showing the cell morphology before and after the separating enzyme in isolation of umbilical cord-derived mesenchymal stem cells according to an embodiment.
도 1에서 나타낸 바와 같이, 분리효소인 콜라게나아제 I의 처리 후에 균질한(homogeneous) 세포의 형태를 나타냄을 알 수 있다.As shown in FIG. 1, it can be seen that a homogeneous cell morphology is shown after treatment with collagenase I, an enzyme that is isolated.
실시예 2. 탯줄 유래 중간엽 줄기세포의 배양Example 2. Culture of umbilical cord-derived mesenchymal stem cells
1. 2차원(2D) 배양1. Two-dimensional (2D) culture
15mL 코니컬튜브(conical tube)에 BM 8mL을 넣어 준비하고, 실시예 1에서 분리한 세포의 동결 바이알을 Heatblock을 사용하여 해동하였다. 이후, 기본배지(MEM alpha GlutaMAX(Gibco; 32561-102)에 겐타마이신(Gentamicin; Gibco; 15710-072; 50 μg/mL), 10% FBS 소분액(Gibco; 10099-141)을 혼합)가 담긴 15mL tube에 세포를 넣었다. 그 다음, 1,200 rpm에서 4분간 원심 분리 후, 상층액을 제거하고 펠렛에 완전배지(MEM alpha GlutaMAX에 헤파린 용액(중외제약; 0.2 U/mL), 겐타마이신(50 μg/mL), FBS 소분액 10%, FGF4(Peprotech; 100-31; 25 ng/mL)가 되도록 혼합)을 넣어 서스펜션(suspension)하였다. 세포부유액 10μL을 채취하여 트리판블루(trypan blue) 10μL와 섞은 후 핸드카운트를 사용하여 총 세포 수와 세포 생존율을 확인하였다. 이후, 35mL CCM을 넣은 T175 플라스크에 세포를 넣고 현미경으로 세포를 확인 후, 37°C, 5% CO2 로 설정된 인큐베이터에 넣고 3~4일간 배양하였다.Prepared by adding 8 mL of BM to a 15 mL conical tube, Freeze vials were thawed using a Heatblock. Thereafter, a basic medium (MEM alpha GlutaMAX (Gibco; 32561-102) mixed with gentamicin (Gentamicin; Gibco; 15710-072; 50 μg/mL), 10% FBS small aliquot (Gibco; 10099-141)) was added. The cells were placed in a 15 mL tube. Then, after centrifugation at 1,200 rpm for 4 minutes, the supernatant was removed, and a complete medium (mem alpha GlutaMAX heparin solution (Jungwey Pharmaceutical; 0.2 U/mL)), gentamicin (50 μg/mL), small aliquots of FBS 10%, FGF4 (Peprotech; 100-31; 25 ng/mL) was added thereto, followed by suspension. 10 μL of the cell suspension was collected, mixed with 10 μL of trypan blue, and then the total number of cells and cell viability were checked using a hand count. Thereafter, the cells were placed in a T175 flask containing 35 mL CCM, and the cells were checked under a microscope, and then placed in an incubator set at 37 °C and 5% CO 2 and cultured for 3 to 4 days.
광학현미경하에서 세포의 형태를 확인한 후 3D 배양 공정을 준비하였다. 마이크로캐리어(Microcarrier; Coming; 4897) 0.4g을 50mL 코니컬튜브에 담고 증류수 20mL을 넣어 부풀렸다(soaking). 이후, Ca/Mg free DPBS(Dulbecco's Phosphate-Buffered Saline; Gibco; 14190144) 20mL을 넣어 세척하였다. 3D 배양기(3D spinner flask)에 무혈청배지(Prime XV Serum Free Media(Irvine Scientific; 91135)에 겐타마이신(50 μg/mL)을 혼합)로 채운 후, 마이크로캐리어를 옮겨 담아 170mL이 되도록 하였다. 4시간 동안 37°C, pH 7.4, 0 rpm으로 인큐베이션하였다. 그 다음, T175 플라스크의 배지를 제거하고, Ca/Mg free DPBS 5mL을 넣어 2번 세척한 후 Ca/Mg free DPBS(1X)를 제거하였다. 이후, T175 플라스크에 TrypLE(Gibco; 12604-021)를 넣고, 37°C, 5% CO2로 설정된 인큐베이터에서 3분간 반응시켰다. 그 다음, TrypLE 반응 후 세포가 플라스크로부터 분리되었는지 광학현미경으로 확인하였다. 이후, 세포가 모두 분리된 것이 확인되면, 플라스크에 기본배지를 더하여 중화시킨 뒤 새로운 50mL 튜브에 옮겼다. 그 다음, 1,200 rpm에서 4분간 원심분리 후, 상층액을 제거하고 펠렛에 무혈청배지를 넣어 서스펜션하였다. 이후, 세포부유액 10μL를 채취하여 트리판블루 10μL와 섞은 후 핸드카운터를 사용하여 총 세포 수와 세포 생존율을 확인하였다.After confirming the morphology of the cells under an optical microscope, a 3D culture process was prepared. Microcarrier (Microcarrier; Coming; 4897) 0.4 g was placed in a 50 mL conical tube and 20 mL of distilled water was added to inflate (soaking). Thereafter, 20 mL of Ca/Mg free DPBS (Dulbecco's Phosphate-Buffered Saline; Gibco; 14190144) was added and washed. After filling a 3D incubator (3D spinner flask) with serum-free medium (Prime XV Serum Free Media (Irvine Scientific; 91135) mixed with gentamicin (50 μg/mL)), the microcarrier was transferred to make 170 mL. Incubated for 4 hours at 37 °C, pH 7.4, 0 rpm. Then, the medium of the T175 flask was removed, and 5 mL of Ca/Mg free DPBS was added and washed twice, and then Ca/Mg free DPBS (1X) was removed. Thereafter, TrypLE (Gibco; 12604-021) was added to a T175 flask, and reacted for 3 minutes in an incubator set at 37°C and 5% CO 2. Then, it was confirmed with an optical microscope whether the cells were separated from the flask after the TrypLE reaction. Thereafter, when it was confirmed that all the cells were isolated, the flask was neutralized by adding basic medium and transferred to a new 50 mL tube. Then, after centrifugation at 1,200 rpm for 4 minutes, the supernatant was removed, and a serum-free medium was added to the pellet to be suspended. Thereafter, 10 μL of the cell suspension was collected, mixed with 10 μL of trypan blue, and then the total number of cells and cell viability were checked using a hand counter.
2. 3차원(3D) 배양2. Three-dimensional (3D) culture
미리 준비한 3D 배양기(3D spinner flask)에 2D로 배양한 세포를 넣어 무혈청배지가 총 200 mL이 되게 한 후, 37°C, pH 7.2~7.4, 300~500 rpm으로 셋팅하여 5~8일 동안 배양하였다. 이후, 3~4일 후부터 수확하기 전까지, 배양액에서 200 μL를 채취하여 혈당측정기로 글루코오스 소모량을 측정하여 세포의 증식을 확인하며 글루코오스 양에 따라 50% 배지를 교환하였다.Put the cells cultured in 2D into a 3D incubator (3D spinner flask) prepared in advance to make a total of 200 mL of serum-free medium, then set at 37°C, pH 7.2~7.4, 300~500 rpm for 5~8 days. Cultured. Thereafter, from 3 to 4 days before harvesting, 200 μL of the culture medium was collected and the glucose consumption was measured with a blood glucose meter to check the proliferation of cells, and the 50% medium was exchanged according to the amount of glucose.
세포 수확을 위해 중력을 이용하여 세포 펠렛팅(cell pelleting)을 5분간 진행하였다. 이후, 3D 배양기(3D spinner flask)에 배지를 제거하고 Ca/Mg free DPBS(1X)를 100mL 넣어 2번 세척한 후 Ca/Mg free DPBS(1X)를 제거하였다. 그 다음, 3D 배양기(3D spinner flask)에 수확용액(harvest solution; TrypLE(5X)에 EDTA(ethylenediaminetetraacetic acid; Invitrogen; 15575-020; 10mM), 펙티나아제(pectinase; Sigma Aldrich; P2611; 100U/mL)을 혼합)을 60mL 넣고, 37°C, 5% CO2로 설정된 인큐베이터에서 15분간 반응시켰다. 이후, 수확용액에 반응 후 세포가 마이크로캐리어로부터 분리된 것을 육안으로 확인한 후 10mL짜리 혈청피펫(serological pipette)으로 완전히 풀어주었다. 그 다음, 세포가 모두 분리된 것이 확인되면, 3D 배양기(3D spinner flask)에 기본배지 100mL을 더하여 중화시킨 뒤 세포부유액을 40 μm 스트레이너(strainer; Falcon; Coming 352340)가 장착된 새로운 50mL 코니컬튜브에 옮겼다. 이후, 1,200 rpm에서 4분간 원심분리 후, 상층액을 제거하고 펠렛에 Ca/Mg free DPBS(1X)을 넣어 서스펜션하였다. 그 다음, 세포부유액 10 μL을 채취하여 트리판블루 10 μL와 섞은 후 핸드카운터를 사용하여 총 세포 수를 확인하고, LUNA 세포계수기를 통해 세포 사이즈와 생존율을 확인하였다.Cell pelleting was performed for 5 minutes using gravity for cell harvesting. Thereafter, the medium was removed in a 3D incubator (3D spinner flask), and 100 mL of Ca/Mg free DPBS (1X) was added and washed twice, and Ca/Mg free DPBS (1X) was removed. Then, harvest solution (ethylenediaminetetraacetic acid; Invitrogen; 15575-020; 10 mM), pectinase (Sigma Aldrich; P2611; 100U/mL) in a harvest solution (TrypLE (5X)) in a 3D incubator (3D spinner flask). ) Mixed) into 60 mL, and reacted for 15 minutes in an incubator set at 37°C and 5% CO 2. Thereafter, after reacting with the harvesting solution, the cells were visually confirmed to be separated from the microcarriers, and then completely released with a 10 mL serum pipette. Next, when it is confirmed that all cells are isolated, add 100 mL of basic medium to a 3D spinner flask to neutralize the cell suspension, and then add a new 50 mL conical tube equipped with a 40 μm strainer (Falcon; Coming 352340). Moved to. Thereafter, after centrifugation at 1,200 rpm for 4 minutes, the supernatant was removed, and Ca/Mg free DPBS (1X) was added to the pellet, followed by suspension. Then, 10 μL of the cell suspension was collected, mixed with 10 μL of trypan blue, and the total number of cells was checked using a hand counter, and the cell size and viability were confirmed through a LUNA cell counter.
실시예 3. 배양된 탯줄 유래 중간엽 줄기세포의 유전자 발현량 및 사이토카인 분비량 분석Example 3. Analysis of gene expression and cytokine secretion of cultured umbilical cord-derived mesenchymal stem cells
1. 분석방법1. Analysis method
2D 및 3D 배양 세포로 RNA prep(RNeasy® Mini Kit, Cat#74106)을 진행한다. 이후, RNA Prep은 키트 프로토콜에 제시된 대로 진행하였고, 추출한 RNA로 마크로젠에 마이크로어레이(GeneChip?? Human Gene 2.0 ST Array, Cat# 902112)를 의뢰하여 2D 배양 대비 3D 배양에 따라 발현이 증가 및 감소하는 유전자를 확인하였다. 그 다음, 2D 및 3D 배양으로 얻어진 배양액(conditioned media)을 모으고 초원심분리기로 다운시켜주었다. 상층액만 따서 이바오젠에 사이토카인 어레이(Human L507 Array, cat# AAH-BLG-1-4)를 의뢰하여 2D 배양 대비 3D 배양에 따라 발현이 증가 및 감소하는 사이토카인(cytokine)을 확인하였다. 유전자 및 단백질 발현 특성 비교를 통해 3D 배양세포에서 분비하는 우수인자를 발굴하였다.RNA prep (RNeasy® Mini Kit, Cat#74106) is performed with 2D and 3D cultured cells. Thereafter, RNA Prep proceeded as indicated in the kit protocol, and the extracted RNA was requested to macrogen microarray (GeneChip?? Human Gene 2.0 ST Array, Cat# 902112), and the expression increased and decreased according to 3D culture compared to 2D culture. The gene was identified. Then, the conditioned media obtained by 2D and 3D culture were collected and down with an ultracentrifuge. By requesting a cytokine array (Human L507 Array, cat# AAH-BLG-1-4) to Ibaogen by picking only the supernatant, it was confirmed that cytokines whose expression increased and decreased according to 3D culture compared to 2D culture. Excellent factors secreted from 3D cultured cells were identified through comparison of gene and protein expression characteristics.
2. 분석결과2. Analysis result
발현량의 차이가 가장 많이 나는 유전자를 선정하여 하기 표 1에 나타냈다. 이들 중 IL33, RAB27B, KDR, COL3A1, 및 HGF가 2D 배양에 비해 3D 배양에서 발현량이 증가한 유전자이다. 또한, ANKRD1, MYCT1, MFAP5, OLR1, SERPINB7, CEMIP 및 CD274가 2D 배양에 비해 3D 배양에서 발현량이 감소한 유전자이다.Genes having the most difference in expression levels were selected and shown in Table 1 below. Among these, IL33, RAB27B, KDR, COL3A1, and HGF are genes whose expression levels are increased in 3D culture compared to 2D culture. In addition, ANKRD1, MYCT1, MFAP5, OLR1, SERPINB7, CEMIP and CD274 are genes whose expression levels are reduced in 3D culture compared to 2D culture.
발현량의 차이가 가장 많이 나는 사이토카인을 선정하여 하기 표 2에 나타냈다. 이들 중 IGFBP1, KIT, IL20, IL3, HGF, PDGFA, PDGFB가 2D 배양에 비해 3D 배양에서 발현량이 증가한 사이토카인이다. 또한, IL26, MMP12, PF4가 2D 배양에 비해 3D 배양에서 발현량이 감소한 사이토카인이다.The cytokines having the most difference in expression levels were selected and shown in Table 2 below. Among these, IGFBP1, KIT, IL20, IL3, HGF, PDGFA, and PDGFB are cytokines whose expression levels are increased in 3D culture compared to 2D culture. In addition, IL26, MMP12, and PF4 are cytokines whose expression levels are reduced in 3D culture compared to 2D culture.
Claims (14)
(a) IL-33, RAB27B, KDR 및 COL3A1으로 이루어진 군으로부터 선택되는 하나 이상이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것;
(b) ANKRD1, MYCT1, MFAP5, OLR1, SERPINB7, CEMIP 및 CD27로 이루어진 군으로부터 선택되는 하나 이상이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 적게 발현되는 것;
(c) IGFBP1, KIT, IL-20, IL-3, HGF, PDFG-A, PDFG-B 및 VEGF-A로 이루어진 군으로부터 선택되는 하나 이상의 사이토카인이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것; 및
(d) IL-26, MMP 및 PF4로 이루어진 군으로부터 선택된 하나 이상의 사이토카인이 2차원 배양된 탯줄 유래 중간엽 줄기세포에 비하여 더 많이 발현되는 것.
A three-dimensional cultured umbilical cord-derived mesenchymal stem cell cluster having any one characteristic selected from the group consisting of the following (a) to (d):
(a) at least one selected from the group consisting of IL-33, RAB27B, KDR and COL3A1 is expressed more than that of two-dimensional cultured umbilical cord-derived mesenchymal stem cells;
(b) at least one selected from the group consisting of ANKRD1, MYCT1, MFAP5, OLR1, SERPINB7, CEMIP, and CD27 is less expressed than two-dimensional cultured umbilical cord-derived mesenchymal stem cells;
(c) IGFBP1, KIT, IL-20, IL-3, HGF, PDFG-A, PDFG-B, and at least one cytokine selected from the group consisting of VEGF-A in two-dimensional cultured umbilical cord-derived mesenchymal stem cells More expressed than; And
(d) IL-26, MMP, and at least one cytokine selected from the group consisting of PF4 is expressed more than two-dimensional cultured umbilical cord-derived mesenchymal stem cells.
The aggregate of claim 1, wherein the (a) characteristic is that at least one of IL33 and RAB27B is expressed 10 times or more compared to two-dimensional cultured umbilical cord-derived mesenchymal stem cells.
The aggregate of claim 1, wherein the (b) characteristic is that ANKRD1 is expressed more than 60 times less than that of two-dimensionally cultured umbilical cord-derived mesenchymal stem cells.
A pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising the aggregate of any one of claims 1 to 3, individual cells constituting the same, or a culture medium thereof.
The group consisting of atopic dermatitis, Crohn's disease, ulcerative colitis, Behcet's disease, lupus, Sjogren's syndrome, myasthenia gravis, scleroderma, polyarteritis nodosa, hypothyroidism, psoriasis and rheumatoid arthritis. One or more compositions selected from.
A pharmaceutical composition for the prevention or treatment of liver disease, comprising the aggregate of any one of claims 1 to 3, individual cells constituting the same, or a culture medium thereof.
The method according to claim 6, wherein the liver disease is at least one selected from the group consisting of impaired liver function, liver disorder, hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, liver abscess, hepatic coma, hepatic atrophy, and liver cancer. Composition.
A pharmaceutical composition for the prevention or treatment of obstetric diseases comprising the aggregate of any one of claims 1 to 3, individual cells constituting the same, or a culture medium thereof.
The composition of claim 8, wherein the obstetric disease is at least one selected from the group consisting of ovarian tumors, chlamydia, polycystic follicular syndrome, mycoplasma, fibroids, free plasma, adenomyosis, uterine anomalies, endometriosis, syphilis and vaginitis.
A pharmaceutical composition for the prevention or treatment of muscle diseases comprising the aggregate of any one of claims 1 to 3, individual cells constituting the same, or a culture medium thereof.
The composition of claim 10, wherein the muscle disease is at least one selected from the group consisting of hypotonia, muscular dystrophy, muscular dystrophy, myasthenia, cachexia, spastic spine syndrome, amyotrophic axonal sclerosis, Charco-Marie-tus disease and sarcopenia.
상기 배양된 탯줄에 분리효소를 접촉시켜 탯줄 유래 중간엽 줄기세포를 분리하는 단계; 및
상기 분리된 탯줄 유래 중간엽 줄기세포를 겐타마이신을 포함하는 무혈청배지에서 3차원 배양하는 단계를 포함하는 탯줄 유래 중간엽 줄기세포 집합체를 제조하는 방법.
Culturing by attaching the separated umbilical cord to a culture vessel;
Separating umbilical cord-derived mesenchymal stem cells by contacting the cultured umbilical cord with a separating enzyme; And
Method for producing an umbilical cord-derived mesenchymal stem cell assembly comprising the step of three-dimensional culturing the isolated umbilical cord-derived mesenchymal stem cells in a serum-free medium containing gentamicin.
The method of claim 12, wherein the isolating enzyme is a collagenase.
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| CN115572709A (en) * | 2022-10-18 | 2023-01-06 | 中晶生物技术股份有限公司 | Application of three-dimensional culture mesenchymal stem cell microspheres in preparation of alopecia repairing and regenerating medicine or preparation |
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| CN115572709A (en) * | 2022-10-18 | 2023-01-06 | 中晶生物技术股份有限公司 | Application of three-dimensional culture mesenchymal stem cell microspheres in preparation of alopecia repairing and regenerating medicine or preparation |
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