KR102047768B1 - Media including mesenchymal stem cells derived high purity and high concentration exosome and the producing method thereof - Google Patents
Media including mesenchymal stem cells derived high purity and high concentration exosome and the producing method thereof Download PDFInfo
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Abstract
본 발명은 중간엽줄기세포 유래 고순도, 고농도 엑소좀을 포함하는 배양액 및 이의 제조방법에 관한 것으로, 구체적으로 본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 엑소좀을 추출할 경우, 고순도의 엑소좀을 보다 높은 수율로 수득할 수 있음을 확인하였으므로, 본 발명 따른 방법을 고순도, 고농도 엑소좀 추출방법으로 유용하게 이용할 수 있다.The present invention relates to a culture medium containing high purity, high concentration exosomes derived from mesenchymal stem cells, and a method for preparing the same, specifically, when extracting exosomes from mesenchymal stem cells cultured three-dimensionally according to the present invention, exo of high purity Since it was confirmed that the moth can be obtained in a higher yield, the method according to the present invention can be usefully used as a high purity, high concentration exosome extraction method.
Description
본 발명은 중간엽줄기세포 유래 고순도, 고농도 엑소좀을 포함하는 배양액 및 이의 제조방법에 관한 것으로, 구체적으로 3차원 배양방법으로 배양한 중간엽줄기세포 유래 고순도, 고농도 엑소좀을 포함하는 배양액에서 고순도, 고농도 엑소좀을 추출하는 방법 및 3차원 배양방법으로 배양한 중간엽줄기세포로부터 추출된 고순도, 고농도 엑소좀에 관한 것이다.
The present invention relates to a culture medium containing high purity, high concentration exosomes derived from mesenchymal stem cells, and a method for preparing the same, specifically, a high purity in a culture medium containing high purity, high concentration exosomes derived from mesenchymal stem cells cultured by a three-dimensional culture method It relates to a high-purity, high-concentration exosomes extracted from mesenchymal stem cells cultured by a high-concentration exosomes and a three-dimensional culture method.
인간의 골수에는 몇 가지 종류의 전구세포(progenitor)가 존재하는 것이 발견되었으며, 이들 중 다분화능을 나타내는 전구세포를 중간엽줄기세포(Mesenchymal Stem Cells; MSC)라 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 kinds of progenitors have been found in the human bone marrow, and among these, progenitor cells showing pluripotency are called mesenchymal stem cells (MSCs). Mesenchymal stem cells are known to exist in most organs of the body, including bone marrow, fat, liver, and muscle. Mesenchymal stem cells have self-proliferative capacity, osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon- ligament fibroblasts, and adipocytes Etc. can be differentiated.
이러한 중간엽줄기세포는 중간엽줄기세포 자체의 분화 기능보다는 중간엽줄기세포가 분비하는 여러 가지 인자들(paracrine/secretory factors), 예를 들어 케모카인(chemokine), 사이토카인(cytokine), 성장인자(growth factors) 등이 줄기세포의 효과를 대변하는 것으로 주목받고 있다. 또한, 중간엽줄기세포는 이러한 인자들뿐만 아니라 세포외 소포체(extracellular vesicle; EV)를 분비하는 것으로 알려져 있으며 세포외 소포체가 세포간의 신호전달을 통해 세포의 운명, 기능, 분화 등 다방면으로 영향을 미치는 것으로 알려져 있다.
These mesenchymal stem cells are not the differentiation function of mesenchymal stem cells themselves, but paracrine / secretory factors secreted by mesenchymal stem cells, for example, chemokine (chemokine), cytokines (cytokine), growth factors ( growth factors, etc., are drawing attention as representing the effects of stem cells. In addition, mesenchymal stem cells are known to secrete extracellular vesicles (EVs) as well as these factors, and extracellular vesicles affect various aspects of cell fate, function, and differentiation through signaling between cells. It is known.
한편, 세포외 소포체는 세포가 세포외로 분비하는 막 구조의 소포체의 총칭으로 마이크로소포체(microvesicle), 엑소좀(exosome), 엑토좀(ectosome), 사멸체(apoptotic body) 등으로 구분된다. 이중 엑소좀은 약 40 ~ 150 nm의 직경과 1.09 ~ 1.18 g/ml의 밀도를 가지는 세포외 소포체로 정의되는데 다양한 종류의 세포 활성 기능이 있다. 엑소좀은 다양한 종류의 체액, 예를 들어 침, 소변, 혈장, 혈청, 양수로부터 분리할 수 있고 여러 종류의 세포배양 상층액에서도 분리가능하다.On the other hand, the extracellular vesicles are a general term of the vesicles of the membrane structure that the cells secrete extracellularly and are classified into microvesicles, exosomes, ectosomes, and apoptotic bodies. The double exosomes are defined as extracellular vesicles having a diameter of about 40 to 150 nm and a density of 1.09 to 1.18 g / ml. Exosomes can be separated from various types of body fluids, such as saliva, urine, plasma, serum, amniotic fluid, and also from various cell culture supernatants.
아울러, 순수하게 분리한 엑소좀은 in vitro와 in vivo에서 임상적 치료효능이 있는 것으로 밝혀지고 있어 그동안 중간엽줄기세포를 이용하여 시도되던 여러 질환들에 대해 엑소좀이 새로운 대안으로 떠오르고 있다. 또한 엑소좀은 면역조절, 세포간 신호전달 역할을 하며 세포의 기능적 변화를 유도해 세포재생 프로그램을 활성화시키며, 무세포계(cell free system)이므로 종양 형성의 위험이 없고 동결보존제 없이 영하 20℃에서 6개월간 생물학적 활성이 유지되는 상태로 보존되며 캡슐화(encapsulation) 되어 있어 엑소좀 내 물질(cytokine, growth factor, mRNA, miRNA 등)이 분해되지 않는 장점이 있다.In addition, purely isolated exosomes have been shown to have clinical therapeutic efficacy in vitro and in vivo , and exosomes have emerged as a new alternative to various diseases that have been attempted using mesenchymal stem cells. In addition, exosomes play a role in immune regulation, intercellular signal transduction, activate cell regeneration programs by inducing functional changes in cells, and are a cell free system. The biological activity is preserved for a period of time and encapsulation (encapsulation) has the advantage that the material in the exosomes (cytokine, growth factor, mRNA, miRNA, etc.) is not degraded.
다만, 엑소좀의 이러한 우수한 효과에도 불구하고 중간엽줄기세포 배양액에서 고순도의 엑소좀을 고농도로 분리하는 기술적 어려움이 존재하므로, 고순도의 엑소좀만을 고농도로 분리하는 기술이 요구되고 있다.
However, despite these excellent effects of exosomes, there is a technical difficulty in separating high-purity exosomes from mesenchymal stem cell cultures at high concentrations. Therefore, a technique for separating only high-purity exosomes at high concentrations is required.
또한, 엑소좀 분리방법 중 가장 보편적으로 쓰이는 초원심분리 (Ultracetrifugation)는 한 번에 다량의 엑소좀을 분리할 수 있는 장점이 있지만, 고가의 장비가 필요하고 분리하는데 많은 시간이 소요되며 강한 원심분리로 인해 엑소좀에 물리적으로 손상이 발생할 수 있고, 특히 분리된 엑소좀의 순도가 떨어지는 등의 단점이 있다. 이러한 단점을 개선하기 위한 방법 중, 엑소좀의 막 (membrane)에 존재하는 단백질인 포스파티딜세린(phosphatidylserine, PS)에 특이적으로 결합하는 물질을 이용함으로써 분리되는 엑소좀의 순도를 높인 PS 친화성 방법 (PS affinity method)이 있다. 이는 초원심분리 방법에 비해 높은 순도의 엑소좀을 분리할 수 있지만 수율이 낮다는 단점이 있다.
In addition, ultratrace separation (Ultracetrifugation), which is the most widely used exosome separation method, has the advantage of separating a large amount of exosomes at one time, but it requires expensive equipment, takes a long time to separate, and strong centrifugation. Due to this physical damage to the exosomes can occur, in particular, there is a disadvantage such as falling purity of the separated exosomes. Among the methods to improve this disadvantage, the PS-affinity method to increase the purity of the exosomes separated by using a substance that specifically binds to phosphatidylserine (PS), a protein present in the membrane of the exosomes (membrane) (PS affinity method). This can separate high purity exosomes compared to ultracentrifugation method, but has a disadvantage of low yield.
이에, 본 발명자들은 중간엽줄기세포 배양액으로부터 고순도의 엑소좀을 높은 수율로 추출하기 위해 노력한 결과, 본 발명에 따라 3차원 배양방법으로 배양하여 중간엽줄기세포 배양액을 수득하고, PS 친화성 방법을 이용하여 고순도 및 고농도의 엑소좀을 추출할 수 있음을 확인함으로써, 본 발명에 따른 방법을 중간엽줄기세포로부터 고순도 및 고농도의 엑소좀 분리방법으로 이용할 수 있음을 밝힘으로써, 본 발명을 완성하였다.
Thus, the present inventors endeavor to extract high-purity exosomes from the mesenchymal stem cell culture medium in high yield, and obtained by culturing in a three-dimensional culture method according to the present invention to obtain a mesenchymal stem cell culture solution, PS affinity method By confirming that high purity and high concentration of exosomes can be extracted using the method, the present invention was completed by revealing that the method according to the present invention can be used as a method for separating high purity and high concentration of exosomes from mesenchymal stem cells.
본 발명의 목적은 종래 중간엽줄기세포 배양액에서 엑소좀을 고순도로 분리하기 어렵고, 종래 엑소좀 분리방법으로 분리한 엑소좀의 순도 또는 수율이 낮다는 단점을 해결함으로써, 중간엽줄기세포로부터 고순도 및 고농도의 엑소좀을 추출하는 방법을 제공하기 위한 것이다.
An object of the present invention is difficult to separate the exosomes with high purity in the conventional mesenchymal stem cell culture medium, and by solving the disadvantage that the purity or yield of the exosomes separated by the conventional exosome separation method, high purity from mesenchymal stem cells It is to provide a method for extracting high concentration of exosomes.
본 발명의 목적을 달성하기 위하여, 본 발명은In order to achieve the object of the present invention, the present invention
1) 중간엽줄기세포를 무혈청 배지에서 생체적합성 스캐폴드와 함께 3차원 배양하고 배양액을 수집하는 단계; 및1) three-dimensional culture of mesenchymal stem cells with a biocompatible scaffold in serum-free medium and collecting the culture solution; And
2) 상기 단계 1)에서 수집한 배양액에서 엑소좀을 추출하는 단계를 포함하는, 3차원 배양한 중간엽줄기세포로부터 고순도 및 고농도의 엑소좀 추출방법을 제공한다.
2) provides a method for extracting exosomes of high purity and high concentration from mesenchymal stem cells cultured in three dimensions, comprising the step of extracting exosomes from the culture solution collected in step 1).
본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 엑소좀을 추출할 경우, 종래 2차원 배양한 중간엽줄기세포로부터 엑소좀을 추출하는 경우보다 고순도의 엑소좀을 보다 높은 수율로 수득할 수 있음을 확인하였으므로, 본 발명에 따른 방법을 중간엽줄기세포로부터 고순도, 고농도의 엑소좀 추출방법으로 유용하게 이용할 수 있다.When exosomes are extracted from mesenchymal stem cells cultured in three dimensions according to the present invention, high-purity exosomes can be obtained in higher yield than exosomes extracted from conventional two-dimensional cultured mesenchymal stem cells. Since it was confirmed, the method of the present invention can be usefully used as a method for extracting high purity and high concentration of exosomes from mesenchymal stem cells.
도 1은 본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 추출된 엑소좀(3D) 및 2차원 배양한 중간엽줄기세포로부터 추출된 엑소좀(2D)의 크기별 분포데이터를 나타낸 도이다.
도 2는 본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 추출된 엑소좀(3D) 및 2차원 배양한 중간엽줄기세포로부터 추출된 엑소좀(2D)의 농도를 나타낸 도이다.1 is a diagram showing the distribution data for each size of exosomes (3D) extracted from mesenchymal stem cells cultured in three-dimensional culture and exosomes (2D) extracted from mesenchymal stem cells cultured in two dimensions according to the present invention.
Figure 2 is a view showing the concentration of exosomes (3D) extracted from mesenchymal stem cells cultured in three-dimensional culture in accordance with the present invention and exosomes (2D) extracted from mesenchymal stem cells in two-dimensional culture.
이하, 본 발명을 보다 상세히 설명한다.
Hereinafter, the present invention will be described in more detail.
본 발명은The present invention
1) 중간엽줄기세포를 무혈청 배지에서 생체적합성 스캐폴드와 함께 3차원 배양하고 배양액을 수집하는 단계; 및1) three-dimensional culture of mesenchymal stem cells with a biocompatible scaffold in serum-free medium and collecting the culture solution; And
2) 상기 단계 1)에서 수집한 배양액에서 엑소좀을 추출하는 단계를 포함하는, 3차원 배양한 중간엽줄기세포로부터 고순도 및 고농도의 엑소좀 추출방법을 제공한다.2) provides a method for extracting exosomes of high purity and high concentration from mesenchymal stem cells cultured in three dimensions, comprising the step of extracting exosomes from the culture solution collected in step 1).
본 발명에서, 상기 "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, 간 또는 근육 등에서 분리될 수 있으며, 보다 구체적으로 지방조직에서 분리될 수 있으나, 이에 한정되지는 않는다. 특히 지방유래 줄기세포는 비교적 접근이 수월하고 채취가 간단하며 한 개체로부터 다량을 얻을 수 있다는 장점이 있다.In the present invention, the "mesenchymal stem cells (mesenchymal stem cells)" are self-propagation is possible, and it has a multipotent, CD73 +, CD90 +, CD105 +, CD14 -, CD20 -, CD34 -, CD45 - the cell Means a cell exhibiting a phenotype, can be isolated from bone marrow, adipose tissue, umbilical cord blood, liver or muscle, and more specifically, can be isolated from adipose tissue, but is not limited thereto. In particular, fat-derived stem cells have the advantage of being relatively easy to access, simple to harvest, and obtain large quantities from one individual.
본 발명에서, 상기 "배양액"은 중간엽줄기세포 배양액으로 중간엽줄기세포를 배양한 세포배양 상등액을 지칭한다. 중간엽줄기세포 배양액은 중간엽줄기세포의 배양 과정에서 세포로부터 분비되는 여러가지 생리활성 물질을 함유하고 있다. 또한 상기 "생리활성 물질"은 세포나 신체의 기능에 영향을 미칠 수 있는 사이토카인, 세포성장인자, 면역조절인자 등을 통칭하는 것으로, 생리활성 물질의 예로는 VEGF(vascular endothelial growth factor), EGF(epidermal growth factor), HGF(hepatocyte growth factor), TGF-beta(Tumor growth factor-beta), IGF(Insulin growth factor) 등이 있으나, 이에 한정되지는 않는다.In the present invention, the "culture medium" refers to a cell culture supernatant cultured with mesenchymal stem cells as a mesenchymal stem cell culture. Mesenchymal stem cell culture medium contains various bioactive substances secreted from cells during the culture of mesenchymal stem cells. In addition, the "physiologically active substance" is a generic name for cytokines, cell growth factors, immunomodulators, etc. that may affect the function of cells or the body, examples of physiologically active substances are VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), TGF-beta (Tumor growth factor-beta), IGF (Insulin growth factor) and the like, but is not limited thereto.
본 발명에서, 상기 "엑소좀(exosome)"은 여러 종류의 세포들로부터 분비되는 막 구조의 소포체(vesicle)로, 다른 세포 및 조직에 결합하여 막 구성요소, 단백질, RNA를 전달하는 등 다양한 역할을 하는 것으로 알려져 있다. 구체적으로 상기 엑소좀은 본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 분비되는 막 구조의 소낭체로, 40 내지 180 nm의 지름을 가지는 것이 바람직하고, 45 내지 140 nm의 지름을 가지는 것이 보다 바람직하며, 90 내지 140 nm의 지름을 가지는 것이 보다 더 바람직하나, 이에 한정되지는 않는다.In the present invention, the "exosome" is a vesicle of membrane structure secreted from various kinds of cells, and binds to other cells and tissues to transfer membrane components, proteins, RNA, etc. It is known to do. Specifically, the exosomes are membrane vesicles secreted from mesenchymal stem cells cultured in three dimensions according to the present invention, preferably 40 to 180 nm in diameter, and more preferably 45 to 140 nm in diameter. It is more preferable to have a diameter of 90 to 140 nm, but is not limited thereto.
본 발명에 따른 방법에 있어서, 상기 단계 1)에서 중간엽줄기세포를 무혈청 배지에서 생체적합성 스캐폴드와 함께 3차원 배양을 실시함으로써, 세포배양을 위해 일반적으로 사용하는 플레이트를 사용하지 않고 간단한 형태의 바틀에서 배양이 가능하며, 장기간 동안 세포를 안정하게 유지할 수 있어 다량의 세포 배양액을 생산할 수 있다. 또한, 종래의 세포배양방법인 2차원 배양으로 생산된 배양액에 비해 고순도 엑소좀의 함량을 월등히 높일 수 있다. In the method according to the present invention, the three-dimensional culture of the mesenchymal stem cells with the biocompatible scaffold in the serum-free medium in step 1), a simple form without using a plate commonly used for cell culture It is possible to culture in the bottle of, and to keep the cells stable for a long time, it is possible to produce a large amount of cell culture. In addition, it is possible to significantly increase the content of high-purity exosomes compared to the culture solution produced by the two-dimensional culture of the conventional cell culture method.
상기 단계 1)에서 중간엽줄기세포는 다음 단계로 배양한 것이 바람직하다:The mesenchymal stem cells in step 1) is preferably cultured in the following steps:
a) 중간엽줄기세포를 기질배지에서 배양한 후 증식배지에서 배양하는 단계; 및a) culturing the mesenchymal stem cells in a matrix medium and then in a growth medium; And
b) 상기 세포를 증식배지에서 계대 배양하는 단계.b) passaging the cells in a growth medium.
상기 배양방법에 있어서, 상기 기질배지는 무혈청기질배지 또는 혈청기질배지일 수 있고, 보다 구체적으로 혈청기질배지일 수 있다. 또한, 상기 증식배지는 무혈청기질배지 또는 혈청기질배지 일 수 있고, 보다 구체적으로 혈청증식배지일 수 있다.In the culture method, the substrate medium may be serum-free medium or serum substrate medium, more specifically, serum substrate medium. In addition, the proliferation medium may be serum-free medium or serum substrate medium, and more specifically, may be a serum growth medium.
상기 배양방법에 있어서, 단계 b)에서 세포를 증식배지에서 1 계대 이상 배양하는 것이 바람직하고, 2계대 이상 배양하는 것이 보다 바람직하나, 이에 한정되지는 않는다. 또한, 상기 증식배지는 기질배지에 bFGF(basic fibroblast growth factor) 또는 EGF(epidermal growth factor) 중 어느 하나 이상을 첨가하는 것이 바람직하고, 구체적으로 상기 bFGF는 0.1 ~ 100 ng/㎖, 상기 EGF는 0.1 ~ 100 ng/㎖ 첨가하는 것이 바람직하고, 상기 bFGF는 0.3 ~ 10 ng/㎖, 상기 EGF는 1 ~ 10 ng/㎖ 첨가하는 것이 보다 바람직하며, 상기 bFGF는 0.5 ~ 5 ng/㎖, 상기 EGF는 3 ~ 7 ng/㎖ 첨가하는 것이 보다 더 바람직하나, 이에 한정되지는 않는다.In the above culturing method, it is preferable to culture cells at least one passage in the growth medium in step b), and more preferably at least two passages, but is not limited thereto. In addition, the growth medium is preferably added at least one of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) to the substrate medium, specifically, the bFGF is 0.1 ~ 100 ng / ㎖, the EGF is 0.1 It is preferable to add ~ 100 ng / ㎖, the bFGF is 0.3 ~ 10 ng / ㎖, the EGF is more preferably added 1 ~ 10 ng / ㎖, the bFGF is 0.5 ~ 5 ng / ㎖, the EGF is It is more preferable to add 3 to 7 ng / ml, but is not limited thereto.
상기 단계 1)에서 무혈청 배지에 bFGF 또는 EGF 중 어느 하나 이상을 첨가하는 것이 바람직하고, 구체적으로 상기 bFGF는 0.1 ~ 100 ng/㎖, 상기 EGF는 0.1 ~ 100 ng/㎖ 첨가하는 것이 바람직하고, 상기 bFGF는 0.3 ~ 10 ng/㎖, 상기 EGF는 1 ~ 10 ng/㎖ 첨가하는 것이 보다 바람직하며, 상기 bFGF는 0.5 ~ 5 ng/㎖, 상기 EGF는 3 ~ 7 ng/㎖ 첨가하는 것이 보다 더 바람직하나, 이에 한정되지는 않는다.Preferably, in step 1), at least one of bFGF or EGF is added to the serum-free medium, specifically, the bFGF is added in an amount of 0.1 to 100 ng / ml, and the EGF is preferably added in an amount of 0.1 to 100 ng / ml, More preferably, the bFGF is added in an amount of 0.3-10 ng / ml, the EGF is added in an amount of 1-10 ng / ml, the bFGF is added in an amount of 0.5-5 ng / ml, and the EGF is added in an amount of 3-7 ng / ml. Preferred, but not limited to.
상기 단계 1)에서 생체적합성 스캐폴드는 세포 접착성인 표면을 갖는 세포지지체로서, 구체적으로 세포와 친화성을 가지며 세포 접착성인 표면을 지닌 재료, 예컨대 천연 또는 합성 고분자로 만들어지며, 세포를 3차원적으로 부착하고 배양시킬 수 있는 지지체를 의미한다. 상기 천연 고분자는 예를 들어 알지네이트, 단백질, 콜라젠, 피브린, 히알루론산 또는 셀룰로오스일 수 있고, 상기 합성 고분자는 예를 들어 폴리(알파-하이드록시산) 계열, 폴리(비닐 알콜), 또는 폴리안하이드라이드일 수 있으나, 이에 한정되지는 않는다.In step 1), the biocompatible scaffold is a cell support having a cell-adhesive surface, specifically made of a material having a surface compatible with the cell and having a cell-adhesive surface, such as a natural or synthetic polymer, and making the cell three-dimensional. Means a support that can be attached and cultured. The natural polymer may be, for example, alginate, protein, collagen, fibrin, hyaluronic acid or cellulose, and the synthetic polymer may be, for example, poly (alpha-hydroxy acid) based, poly (vinyl alcohol), or polyanhydr. It may be a ride, but is not limited thereto.
상기 단계 1)에서 3차원 배양은 3회 이상 배양액을 얻는 것이 바람직하고, 구체적으로 2일 내지 4일 간격으로, 보다 구체적으로 3일 간격으로 배지를 교환하면서 3회 이상 배양액을 얻는 것이 바람직하다. 특히, 한정된 수의 세포에서 다량의 줄기세포 배양액을 얻고 배양액 중에 포함되어 있는 엑소좀의 함량을 높이기 위해서는 3회 이상 배양액을 얻는 것이 바람직하다.In the step 1), the three-dimensional culture is preferably obtained at least three times the culture medium, and in particular, it is preferable to obtain the culture medium at least three times while replacing the medium at intervals of two to four days, more specifically three days. In particular, in order to obtain a large amount of stem cell culture from a limited number of cells and increase the content of exosomes contained in the culture, it is preferable to obtain a culture solution three or more times.
본 발명에 따른 방법에 있어서, 상기 단계 2)에서 엑소좀은 다음 단계로 추출될 수 있으나, 이에 한정되지는 않는다:In the method according to the invention, the exosome in step 2) may be extracted to the following steps, but is not limited thereto:
i) 상기 단계 1)에서 수집한 배양액을 여과하는 단계; i) filtering the culture solution collected in step 1);
ii) 여과액을 필터가 장착된 원심분리 튜브에 첨가하고 원심분리하여 상등액을 획득하는 단계; 및ii) adding the filtrate to a filter-mounted centrifuge tube and centrifuging to obtain a supernatant; And
iii) 상등액에서 엑소좀을 추출하는 단계.iii) extracting the exosomes from the supernatant.
본 발명에 따른 방법에 있어서, 상기 단계 i)에서 배양액을 0.10 내지 0.30 ㎛ 메쉬 필터로 여과하는 것이 바람직하고, 0.15 내지 0.25 ㎛ 메쉬 필터로 여과하는 것이 바람직하나, 이에 한정되지는 않는다. 상기 필터가 0.15 ㎛ 메쉬 미만일 경우 여과액 내에 엑소좀이 포함되지 않을 수 있고, 상기 필터가 0.30 ㎛ 메쉬를 초과할 경우 엑소좀 이외 세포외 소포체들이 여과되어 엑소좀을 고순도로 분리하는 것이 어려울 수 있다.In the method according to the invention, it is preferable to filter the culture in step i) with a 0.10 to 0.30 ㎛ mesh filter, it is preferred, but not limited to filtering with a 0.15 to 0.25 ㎛ mesh filter. Exosome may not be included in the filtrate when the filter is less than 0.15 ㎛ mesh, extracellular vesicles other than the exo bit may be difficult to separate the exosome with high purity if the filter exceeds 0.30 ㎛ mesh .
본 발명에 따른 방법에 있어서, 상기 단계 ii)에서 여과액을 필터가 장착된 원심분리 튜브에 첨가하고 원심분리함으로써, 여과액을 농축할 수 있고 이를 통해 고순도의 엑소좀을 고농도로 얻을 수 있다.In the method according to the present invention, by adding the filtrate to the filter-mounted centrifuge tube in step ii) and centrifugation, the filtrate can be concentrated to thereby obtain a high purity exosomes.
상기 단계 ii)에서 필터가 장착된 원심분리 튜브는 80 내지 120 kDa MWCO(molecular weight cut off)를 갖는 필터가 장착된 원심분리 튜브인 것이 바람직하고, 90 내지 110 KDa MWCO를 갖는 필터가 장착된 원심분리 튜브인 것이 보다 바람직하나, 이에 한정되지는 않는다. 또한, 상기 단계 ii)에서 원심분리는 2,000 내지 4,000×g에서 45 내지 75분간 수행하는 것이 바람직하고, 2,500 내지 3,500×g에서 50 내지 70분간 수행하는 것이 보다 바람직하나, 이에 한정되지는 않는다.The centrifuge tube equipped with the filter in step ii) is preferably a centrifuge tube equipped with a filter having a molecular weight cut off (80-120 kDa MWCO), and a centrifuge tube equipped with a filter having a 90-110 KDa MWCO. More preferably, the separation tube is not limited thereto. In addition, the centrifugation in step ii) is preferably performed at 2,000 to 4,000 × g for 45 to 75 minutes, and more preferably at 2,500 to 3,500 × g for 50 to 70 minutes, but is not limited thereto.
상기 단계 iii)에서 상등액에 대해 크기-배제 크로마토그래피(size exclusion chromatography), 밀도 구배 원심분리(density gradient centrifugation), 분별 원심분리(differential centrifugation), 초원심분리(ultrafilteration), 면역흡착포획(immunoabsorbent capture), 친화성 방법(affinity method), 예를 들어, 친화성 포획(affinity capture), 친화성 정제(affinity purification), 면역분석법(immunoassay), 미세유체 분리(microfluidic separation) 또는 이들의 조합을 수행하여 엑소좀을 추출할 수 있고, 구체적으로 친화성 방법을 수행하여 엑소좀을 추출할 수 있으며, 보다 구체적으로 포스파티딜세린(phosphatidylserin; PS) 친화성 방법을 수행하여 엑소좀을 추출할 수 있으나, 이에 한정되지는 않는다. 상기 엑소좀 추출 방법은 당업계에 잘 알려져 있으며, 예컨대 David W.Greening et al., 2015 및 Wataru Nakai et al., 2016 논문에 개시되어 있다.Size exclusion chromatography, density gradient centrifugation, differential centrifugation, ultrafilteration, immunoabsorbent capture of the supernatant in step iii). ), An affinity method such as, for example, affinity capture, affinity purification, immunoassay, microfluidic separation, or a combination thereof. Exosome can be extracted, and specifically, exosomes can be extracted by performing an affinity method, and more specifically, exosomes can be extracted by performing a phosphatidylserin (PS) affinity method, but is not limited thereto. It doesn't work. Such exosome extraction methods are well known in the art and are described, for example, in David W. Greening et al., 2015 and Wataru Nakai et al., 2016.
본 발명에 따른 방법으로 종래 2차원 배양한 중간엽줄기세포로부터 엑소좀을 추출하는 것보다 고순도 및 고농도의 엑소좀을 추출할 수 있다. 구체적으로, 본 발명에 따른 방법으로 40 내지 180 nm의 지름을 가지는 고순도의 엑소좀을 추출할 수 있고, 보다 구체적으로 45 내지 140 nm의 지름을 가지는 고순도의 엑소좀을 추출할 수 있으며, 보다 더 구체적으로 90 내지 140 nm의 지름을 가지는 고순도의 엑소좀을 추출할 수 있으나, 이에 한정되지는 않는다. 또한, 본 발명에 따른 방법으로 고농도의 엑소좀을 0.5×107 particles/ml 이상 추출할 수 있고, 보다 구체적으로 1×107 particles/ml 이상 추출할 수 있으나, 이에 한정되지는 않는다.
The method according to the present invention can extract exosomes of high purity and high concentration than extracting exosomes from mesenchymal stem cells conventionally cultured in two dimensions. Specifically, the method according to the present invention can extract high purity exosomes having a diameter of 40 to 180 nm, more specifically can extract high purity exosomes having a diameter of 45 to 140 nm, more Specifically, a high purity exosome having a diameter of 90 to 140 nm may be extracted, but is not limited thereto. In addition, by the method according to the present invention can extract a high concentration of exosomes 0.5 × 10 7 particles / ml or more, more specifically 1 × 10 7 particles / ml or more can be extracted, but is not limited thereto.
본 발명의 일 실시예에서 본 발명에 따른 중간엽줄기세포의 3차원 배양은 다음 과정에 따라 실시될 수 있으며, 세포배양에 사용되는 배지는 이에 한정되지 않는다.In one embodiment of the present invention, the three-dimensional culture of mesenchymal stem cells according to the present invention can be carried out according to the following procedure, the medium used for cell culture is not limited thereto.
(1) (One) 중간엽줄기세포의Mesenchymal stem cells 배양 culture
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.Mesenchymal stem cells obtained from the tissues are suspended in the substrate medium and inoculated in a culture vessel at a concentration of 10,000-40,000 cells / ㎠ and then cultured. The substrate medium is incubated in DMEM or DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth) medium containing 10% bovine serum for about 24 hours.
(2) (2) 증식배지(expansion media)에서의In expansion media 배양 culture
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 removing the substrate medium, the cells are grown in the growth medium to proliferate adherent cells. The growth medium is DMEM or DMEM / F12 containing 10% bovine serum, an epidermal growth factor (EGF) at 0.1-100 ng / ml and / or a basic fibroblast growth factor (bFGF) at 0.1-100 ng / ml, Rapidly proliferates the adherent mesenchymal stem cells to increase the amount of cells in a short period of time.
(3) 계대 배양(3) subculture
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 cells fill 80 to 90% of the bottom of the culture vessel, the growth medium is removed and cells are removed from the culture vessel by trypsin treatment. For subculture, cells are diluted 1: 3 to 1: 4 and incubated with proliferation medium in a new culture vessel. In this way, additional subcultures are possible.
(4) 생체적합성 (4) biocompatibility 스캐폴드와With scaffold 함께 배양 Incubated together
배양한 세포는 PBS로 3회 이상 세척하여 FBS를 제거해주고, 무혈청 배지(페놀레드 미포함)에서 생체적합성 스캐폴드에 부착된 형태로 배양한다. 생체적합성 스캐폴드에 부착된 형태로 배양하기 위해서는 배양한 세포의 현탁액과 생체적합성 스캐폴드 재료 현탁액을 혼합한 후, 상기 혼합 현탁액을 무균 바틀 또는 culture bag에 뿌려 3차원 구조의 응괴를 형성한다. 응괴가 완전히 경화하면, 0.1~100 ng/㎖ 농도의 EGF 및/또는 0.1~100 ng/㎖ 농도의 bFGF를 포함하는 무혈청 배지(페놀레드 미포함)로 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 three or more times with PBS to remove FBS, and cultured in a serum-free medium (without phenol red) attached to a biocompatible scaffold. For culturing in a form attached to a biocompatible scaffold, a suspension of the cultured cells and a suspension of the biocompatible scaffold material are mixed, and then the mixed suspension is sprayed into a sterile bottle or culture bag to form a three-dimensional structure. Once the clot has fully cured, it is incubated in serum-free medium (without phenol red) containing EGF at a concentration of 0.1-100 ng / ml and / or bFGF at a concentration of 0.1-100 ng / ml. Cell culture in the scaffold does not require a conventional cell culture container, there is an advantage that can be cultured in a large amount in a sterile bottle or culture bag can be conveniently cultured at a lower cost.
또한, 본 발명의 일 실시예에서 본 발명자들은 인간 지방유래 중간엽줄기세포를 상기와 같은 방법으로 3차원 배양한 후, 0.22 ㎛ 메쉬 크기 필터를 이용하여 여과하고, 여과 후 회수한 여과액을 100 kD MWCO를 갖는 필터가 장착된 원심분리 튜브를 이용해 원심분리하여 농축하였다. 농축 후 수득한 상등액에서 엑소좀을 추출하였다. In addition, in one embodiment of the present invention, the present inventors three-dimensional culture of human adipose-derived mesenchymal stem cells in the same manner as described above, and then filtered using a 0.22 ㎛ mesh size filter, the filtrate recovered after filtration 100 It was concentrated by centrifugation using a centrifuge tube equipped with a filter with kD MWCO. Exosome was extracted from the supernatant obtained after concentration.
삭제delete
또한, 본 발명자들은 본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 엑소좀을 추출할 경우, 2차원 배양한 중간엽줄기세포로부터 엑소좀을 추출하는 경우보다 고순도의 엑소좀을 보다 높은 수율로 수득할 수 있음을 확인하였으므로, 본 발명에 따른 방법은 엑소좀을 고순도 및 고농도로 추출하는 데 있어 유용하게 이용될 수 있다.In addition, the inventors of the present invention, when extracting the exosomes from the mesenchymal stem cells cultured in three-dimensional culture according to the present invention, the exosomes of high purity in higher yield than when extracting the exosomes from the mesenchymal stem cells cultured in two-dimensional culture Since it can be obtained, the method according to the present invention can be usefully used to extract exosomes in high purity and high concentration.
이하 본 발명을 실시예, 비교예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by Examples, Comparative Examples and Experimental Examples.
단, 하기 실시예, 비교예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예, 비교예 및 실험예에 한정되는 것은 아니다.
However, the following Examples, Comparative Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Comparative Examples and Experimental Examples.
<< 실시예Example 1> 인간 지방유래 1> derived from human fat 중간엽줄기세포의Mesenchymal stem cells 배양 방법 Culture method
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℃, 1,200 rpm에서 10분 동안 원심분리하였다. 원심분리 후, 상층액인 지방층을 제거하고 아래층인 콜라게나제 용액을 흔들리지 않도록 조심해서 분리하였다. 기질배지를 넣어 현탁한 후, 20℃, 1,200 rpm에서 5분 동안 원심분리하였다. 이때, 아래에 가라앉는 것이 스트로마-혈관 분획으로, 상층액을 제거하였다. Adipose tissue is usually obtained by liposuction, but is not limited thereto. Adipose-derived mesenchymal stem cells were isolated from adipose tissue obtained by liposuction as follows: Adipose tissue was washed 3-4 times with the same volume of KRB solution to remove blood. Then, the same volume of collagenase solution as adipose tissue was added and reacted in a 37 ° C. water bath. This was transferred to a centrifuge tube and centrifuged at 20 ° C. and 1,200 rpm for 10 minutes . After centrifugation, the fat layer as a supernatant was removed and the collagenase solution as a lower layer was separated carefully so as not to shake. After the substrate medium was suspended and centrifuged for 5 minutes at 20 ℃, 1,200 rpm. At this time, the subsidence was the stromal-vascular fraction, and the supernatant was removed.
상기 스트로마-혈관 분획을 기질배지로 현탁하여 배양용기에 접종하고, 37℃, 5% CO2 인큐베이터에서 24시간 동안 배양하였다. 배양액 제거 후 인산염 완충용액으로 세척하고, 기질배지(10% FBS가 함유된 DMEM/F12 배지), 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 트립신을 처리하여 단일 세포로 분리하여 수득하였다. 얻어진 세포를 증식배지로 1:3~1:4로 희석하여 계대 배양을 실시하였다.
The stromal-vascular fractions were suspended in a substrate medium and inoculated in a culture vessel and incubated in 37 ° C., 5% CO 2 incubator for 24 hours. After removal of the culture solution, the solution was washed with phosphate buffer, and the substrate medium (DMEM / F12 medium containing 10% FBS), or the growth medium containing basic fibroblast growth factor (bFGF) at a concentration of 1 ng / ml in the substrate medium, or Epithelial growth factor (EGF) in the substrate medium was grown using a medium containing a concentration of 5 ng / ㎖. When adipose-derived mesenchymal stem cells grow to about 80-90% of the culture vessel, trypsin treatment was obtained to separate them into single cells. The obtained cells were diluted 1: 3 to 1: 4 with proliferation medium to carry out passage culture.
<<
실시예Example
2> 인간 지방유래 2> derived from human fat
중간엽줄기세포를Mesenchymal stem
인간 지방유래 중간엽줄기세포를 피브린 글루와 3차원 배양하였다.Human adipose derived mesenchymal stem cells were cultured three-dimensionally with fibrin glue.
구체적으로, 상기 <실시예 1>에서 계대 배양한 중간엽줄기세포를 트립신 처리하여 단일세포로 얻은 후, DMEM/F12 배지로 3회 세척하여 FBS를 제거하였다. 그 다음, 1.6 × 107 개/㎖ 줄기세포에 트롬빈 용액이 20% 포함된 DMEM/F12 배지(페놀레드(phenol red) 미포함)를 첨가하여 세포를 현탁하였다. 피브리노겐은 DMEM/F12 배지(페놀레드 미포함)으로 1:2의 비율로 희석하였다. 듀얼시린지를 이용하여 상기 세포현탁액과 피브리노겐 희석액이 혼합되도록 한 후 바틀 또는 culture bag에 뿌려 피브린 응괴를 만들었다. 25분 후 피브린 응괴가 완전히 굳으면 1 ng/ml 염기성 섬유아세포 성장인자(bFGF) 및 5 ng/ml 표피세포 성장인자(EGF)가 포함된 DMEM/F12 배지(페놀레드 미포함)를 세포 4 × 107 개 당 500 ㎖가 되도록 첨가하였다. 72시간 후 세포 배양액을 회수하고 새 배지를 첨가하는 방법으로 2회 더 세포 배양액을 회수하여 냉장 보관하였다.
Specifically, the mesenchymal stem cells passaged in <Example 1> were trypsinized to obtain single cells, and then washed three times with DMEM / F12 medium to remove FBS. Then, the cells were suspended by adding DMEM / F12 medium (without phenol red) containing 20% of thrombin solution to 1.6 × 10 7 stem cells / ml. Fibrinogen was diluted in DMEM / F12 medium (without phenol red) at a ratio of 1: 2. Fibrin clots were made by dual syringes and the cell suspension and fibrinogen dilutions were mixed and then sprayed into bottles or culture bags. After 25 minutes, the fibrin clot was completely solidified. DMEM / F12 medium containing 1 ng / ml basic fibroblast growth factor (bFGF) and 5 ng / ml epidermal growth factor (EGF) was added to cells 4 × 10. 500 ml per 7 was added. After 72 hours, the cell culture was recovered and refrigerated two more times by adding fresh medium.
<< 실시예Example 3> 3차원 배양방법으로 배양한 3> Cultured by 3D culture method 중간엽줄기세포로부터From mesenchymal stem cells 엑소좀( Exosomes exosomeexosome ) 추출) extraction
3차원 배양방법으로 배양한 중간엽줄기세포로부터 엑소좀을 추출하였다.Exosomes were extracted from mesenchymal stem cells cultured by three-dimensional culture.
구체적으로, 상기 <실시예 2>에서 회수한 중간엽줄기세포 배양액을 0.22 ㎛ 메쉬를 갖는 필터를 이용하여 여과하였다. 여과 후 여과액을 회수하고, 이를 100 kD MWCO(molecular weight cut off)를 갖는 필터가 장착된 원심분리 튜브를 이용하여 3,000×g에서 60분간 원심분리하여 150배 농축하였다. 농축 후 수득한 상등액을 PS 친화성(PS affinity) 엑소좀 분리 키트(WAKO cat no. 293-77601)를 이용하여 엑소좀을 추출하였다.
Specifically, the mesenchymal stem cell culture recovered in Example 2 was filtered using a filter having a 0.22 μm mesh. The filtrate was recovered after filtration and concentrated 150 times by centrifugation at 3,000 × g for 60 minutes using a centrifuge tube equipped with a filter having a 100 kD MWCO (molecular weight cut off). The supernatant obtained after concentration was extracted using the PS affinity exosome separation kit (WAKO cat no. 293-77601).
<< 비교예Comparative example 1> 2차원 배양방법으로 배양한 1> Cultured by 2D culture method 중간엽줄기세포로부터From mesenchymal stem cells 엑소좀Exosomes 추출 extraction
종래 2차원 배양방법으로 배양한 중간엽줄기세포로부터 추출된 엑소좀과 본 발명에 따른 3차원 배양방법으로 배양한 중간엽줄기세포로부터 추출된 엑소좀을 비교하기 위하여, 종래 2차원 배양방법으로 인간 지방유래 중간엽줄기세포를 배양하고, 엑소좀을 추출하였다.In order to compare exosomes extracted from mesenchymal stem cells cultured by a conventional two-dimensional culture method and exosomes extracted from mesenchymal stem cells cultured by a three-dimensional culture method according to the present invention, a human by a conventional two-dimensional culture method Adipose-derived mesenchymal stem cells were cultured and exosomes were extracted.
구체적으로, 상기 <실시예 1>에서 계대 배양한 중간엽줄기세포를 트립신 처리하여 단일세포로 얻은 후, DMEM/F12 배지로 3회 세척하여 FBS를 제거하였다. 1.6 × 107개/㎖ 줄기세포에 DMEM/F12 배지을 첨가하여 세포를 현탁하여 접종하였다. 그 다음, 1 ng/㎖ 염기성 섬유아세포 성장인자(bFGF) 및 5 ng/㎖ 표피세포 성장인자(EGF)가 포함된 DMEM/F12 배지를 세포 4.0 × 107개 당 500 ㎖가 되도록 첨가하였다. 72시간 후 세포 배양액을 회수하고 새 배지를 첨가하는 방법으로 2회 더 세포 배양액을 회수하여 냉장 보관하였다.Specifically, the mesenchymal stem cells passaged in <Example 1> were trypsinized to obtain single cells, and then washed three times with DMEM / F12 medium to remove FBS. Cells were inoculated by adding DMEM / F12 medium to 1.6 × 10 7 stem cells / ml. Next, DMEM / F12 medium containing 1 ng / ml basic fibroblast growth factor (bFGF) and 5 ng / ml epidermal growth factor (EGF) was added to 500 ml per 4.0 × 10 7 cells. After 72 hours, the cell culture was recovered and refrigerated two more times by adding fresh medium.
그 다음, 상기 회수한 세포 배양액을 상기 <실시예 3>에 기재된 방법과 동일한 방법으로 여과하고 농축한 후 엑소좀을 추출하였다.
Then, the recovered cell culture was filtered and concentrated in the same manner as described in <Example 3>, and then exosomes were extracted.
<< 실험예Experimental Example 1> 3차원 배양방법으로 배양한 1> Cultured by 3D culture method 중간엽줄기세포로부터From mesenchymal stem cells 추출된 Extracted 엑소좀의Exosome 특성 분석 Characterization
종래 2차원 배양방법으로 배양한 중간엽줄기세포로부터 추출된 엑소좀과 본 발명에 따라 3차원 배양방법으로 배양한 중간엽줄기세포로부터 추출된 엑소좀의 크기 및 농도를 확인하였다.Exosomes extracted from mesenchymal stem cells cultured by a conventional two-dimensional culture method and exosomes extracted from mesenchymal stem cells cultured by a three-dimensional culture method according to the present invention was confirmed.
구체적으로, 상기 <실시예 3>의 3차원 배양방법으로 배양한 중간엽줄기세포로부터 추출된 엑소좀(3D) 및 상기 <비교예 1>의 2차원 배양방법으로 배양한 중간엽줄기세포로부터 추출된 엑소좀(2D)을 나노입자추적분석기(nanoparticle tracking analysis)를 사용하여 크기 및 농도를 측정하였다.Specifically, exosomes (3D) extracted from the mesenchymal stem cells cultured by the three-dimensional culture method of <Example 3> and mesenchymal stem cells cultured by the two-dimensional culture method of the <Comparative Example 1> Exosomes (2D) were measured in size and concentration using a nanoparticle tracking analysis (nanoparticle tracking analysis).
그 결과, 도 1 및 도 2에 나타낸 바와 같이, 종래 2차원 배양한 중간엽줄기세포로부터 추출된 엑소좀의 크기는 40~600 nm로 나타났고, 농도는 5×106 particles/mL로 나타났다. 이를 통해 종래 2차원 배양한 중간엽줄기세포로부터 엑소좀을 추출하는 경우 엑소좀 이외 다양한 크기의 세포외 소포(extracellular vesicle)가 많이 혼재되어 있음을 확인하였다. As a result, as shown in Figures 1 and 2, the size of the exosomes extracted from the mesenchymal stem cells in the conventional two-dimensional culture appeared to be 40 ~ 600 nm, the concentration was 5 × 10 6 particles / mL. Through this, when extracting exosomes from the mesenchymal stem cells in the conventional two-dimensional culture, it was confirmed that a lot of extracellular vesicles (extracellular vesicle) of various sizes other than the exosomes are mixed.
반면, 본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 추출된 엑소좀의 크기는 40~290 nm로 나타났고, 대부분의 엑소좀이 100~140 nm의 크기로 나타났으며, 농도는 1×107 particles/mL로 나타났다. 이를 통해 본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 엑소좀을 추출하는 경우 100~140 nm 크기의 엑소좀을 수득할 수 있음을 확인하였다(도 1 및 도 2).On the other hand, the size of the exosomes extracted from the mesenchymal stem cells cultured in three dimensions according to the present invention was found to be 40 ~ 290 nm, most exosomes appeared in the size of 100 ~ 140 nm, the concentration is 1 × 10 7 particles / mL. Through this, it was confirmed that when the exosomes were extracted from the mesenchymal stem cells cultured in three dimensions according to the present invention, exosomes having a size of 100 to 140 nm could be obtained (FIGS. 1 and 2).
상기와 같은 결과를 통해 본 발명에 따라 3차원 배양한 중간엽줄기세포로부터 엑소좀을 추출할 경우 종래 2차원 배양한 중간엽줄기세포로부터 엑소좀을 추출하는 경우보다 고순도 엑소좀의 수득율을 높일 수 있음을 확인하였다.When the exosomes are extracted from the mesenchymal stem cells cultured in three dimensions according to the present invention through the above results, the yield of high-purity exosomes can be increased compared to the case of extracting the exosomes from the conventional two-dimensional cultured mesenchymal stem cells. It was confirmed that there is.
Claims (13)
2) 상기 단계 1)에서 수집한 배양액을 여과하는 단계;
3) 상기 단계 2)의 여과액을 필터가 장착된 원심분리 튜브에 첨가하고 원심분리하여 상등액을 획득하는 단계; 및
4)) 상기 단계 3)의 상등액에 대해 PS 친화성 방법(phosphatidylserin affinity method)을 수행하여 상등액에서 엑소좀을 추출하는 단계를 포함하는, 3차원 배양한 중간엽줄기세포로부터 고순도 및 고농도의 엑소좀 추출방법.
1) culturing the mesenchymal stem cells together with the biocompatible scaffold in a serum-free medium to which any one or more of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) are added and collecting the culture solution;
2) filtering the culture solution collected in step 1);
3) adding the filtrate of step 2) to a filter-mounted centrifuge tube and centrifuging to obtain a supernatant; And
4)) extracting exosomes from the supernatant by performing a PS affinity method (phosphatidylserin affinity method) on the supernatant of step 3), high purity and high concentration of exosomes from mesenchymal stem cells cultured in three dimensions Extraction method.
a) 중간엽줄기세포를 기질배지에서 배양한 후 증식배지에서 배양하는 단계; 및
b) 상기 세포를 증식배지에서 계대 배양하는 단계.
According to claim 1, wherein the mesenchymal stem cells in step 1) characterized in that the mesenchymal stem cells cultured in the next step, high-purity and high concentration exosomes extraction method from three-dimensional cultured mesenchymal stem cells:
a) culturing the mesenchymal stem cells in a matrix medium and then in a growth medium; And
b) passaging the cells in a growth medium.
The method of claim 2, wherein the growth medium is a high-purity and three-dimensional cultured mesenchymal stem cells, characterized in that any one or more of the basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) added to the substrate medium High concentration exosome extraction method.
According to claim 1 or 3, wherein the bFGF is 0.1 ~ 100 ng / ㎖, EGF is characterized in that the addition of 0.1 ~ 100 ng / ㎖, high purity and high concentration of exo from three-dimensional cultured mesenchymal stem cells How to extract.
According to claim 1, wherein the biocompatible scaffold in step 1) is a cell support having a cell-adhesive surface, characterized in that the natural or synthetic polymer, high purity and high concentration of exosomes from mesenchymal stem cells Extraction method.
According to claim 1, wherein the three-dimensional culture in step 1) characterized in that to obtain a stem cell culture medium at least three times while replacing the medium at intervals of 2 to 4 days, high purity and high concentration from the three-dimensional cultured mesenchymal stem cells Exosomes extraction method.
The method for extracting high purity and high concentration of exosomes from three-dimensionally cultured mesenchymal stem cells according to claim 1, wherein the culture solution is filtered with a 0.10 to 0.30 µm mesh filter in step 2).
The method of claim 1, wherein the centrifuge tube is equipped with a filter in step 3) is characterized in that the centrifuge tube is equipped with a filter having a molecular weight cut off (80 to 120 kD MWCO), three-dimensional culture medium Method for extracting high purity and high concentration of exosomes from leaf stem cells.
The method for extracting high purity and high concentration of exosomes from mesenchymal stem cells cultured in three dimensions, wherein the exosomes have a diameter of 40 to 180 nm.
The method for extracting high purity and high concentration of exosomes from three-dimensional cultured mesenchymal stem cells according to claim 1, wherein the exosomes have higher purity and higher concentration than exosomes extracted from two-dimensional cultured mesenchymal stem cells.
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