WO2022114724A1 - Medium composition for producing exosome with high efficacy and high purity - Google Patents
Medium composition for producing exosome with high efficacy and high purity Download PDFInfo
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- WO2022114724A1 WO2022114724A1 PCT/KR2021/017257 KR2021017257W WO2022114724A1 WO 2022114724 A1 WO2022114724 A1 WO 2022114724A1 KR 2021017257 W KR2021017257 W KR 2021017257W WO 2022114724 A1 WO2022114724 A1 WO 2022114724A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present invention relates to a medium additive for producing high-efficiency and high-purity exosomes comprising albumin and lipid as active ingredients and a composition comprising the same, and more specifically, to a high-efficiency, high-purity medium for producing exosomes comprising albumin and lipid as active ingredients. It relates to an additive, a stem cell culture medium composition comprising the medium additive, and a method for producing high-efficiency, high-purity exosomes.
- stem cell therapy is highly anticipated in various diseases, the results of clinical trials in which stem cells are administered show only a slight improvement in the damaged tissue or the problem that the effect does not last, and long-term follow-up Results It is reported that the long-term survival rate in vivo after stem cell transplantation is extremely low. The cause of this problem is that, as with most cell therapy drugs, the therapeutic effect of stem cells appears when they survive and adapt to the environment at the site where they are administered, but the basic viability is lowered, that is, these cells cannot be maintained after transplantation. This is because it is destroyed after being injected into the human body, and it is considered to be an insufficient therapeutic effect.
- exosomes are nanovesicles with a diameter of 30 to 200 nm that are naturally secreted and contain RNA and proteins of cells, and it is known that they can act as a medium for transporting various substances. Exosomes contain cell-specific components that reflect the unique biological function of derived cells, and transmembrane proteins such as CD9 and CD81 are well known as marker proteins.
- an object of the present invention is to provide a medium additive for producing high-efficiency and high-purity exosomes, including albumin and lipid as active ingredients.
- Another object of the present invention is to provide a medium composition comprising the medium additive and the basal medium.
- the present invention provides a medium additive for producing high-efficiency and high-purity exosomes, including albumin and lipids.
- the albumin may be included in a concentration of 0.01 - 1 mg/ml in the medium additive.
- the lipid may be included in 0.5 - 50 ug/ml in the medium additive.
- the albumin may be recombinant albumin.
- the lipid may include phosphatidic acid or phosphatidylcholine.
- the present invention provides a medium composition comprising the medium additive and the basal medium.
- the basal medium is DMEM (Dulbecco's Modified Eagle's Medium); Minimal Essential Medium (MEM); BME (Basal Medium Eagle); RPMI 1640; DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12); a-MEM (a-Minimal essential Medium); Glasgow's Minimal Essential Medium (G-MEM); IMDM (Isocove's Modified Dulbecco's Medium); and KnockOut DMEM (GIBCO, USA) may be any one selected from the group consisting of.
- the medium composition may be a serum-free medium.
- the present invention provides a method for producing high-efficiency and high-purity exosomes, including the step of culturing stem cells in the medium composition.
- the stem cells may be human adipose-derived mesenchymal stem cells.
- the present inventors confirmed an increase in the production of stem cell-derived exosomes and isolation of high-purity exosomes. It is expected that it can be used as an effective means in the field of developing cosmetic raw materials and therapeutics using this.
- 1 is a culture medium of stem cells cultured in each of the albumin and lipid raw material addition culture medium (medium A) and albumin and lipid-free culture medium (medium B) of the present invention as exosome marker markers CD9, CD81 antibody Western blotting results.
- Figure 2 is the last step of the separation process of the exosomes from the stem cell culture medium cultured in each of the culture medium (medium A) and the non-added culture medium (medium B) of the present invention albumin and lipids are analyzed by ion chromatography is a result
- Figure 2a is the result of confirming that the exosomes isolated from the culture medium of medium A have high purity by removing total protein and RNA.
- Figure 2b confirms the result of Figure 2a through SDS-PAGE.
- Figure 2c confirms the result of Figure 2a by Western blotting with the exosome marker CD9, CD81 antibody.
- the present invention provides a medium additive for producing high-efficiency and high-purity exosomes, including albumin and lipid, and a medium composition comprising the medium additive and a basal medium, to separate exosomes of high purity from stem cells.
- the albumin may be included in the medium additive at a concentration of 0.01 - 1 mg/ml, and may be recombinant albumin, and the lipid may be included in the medium additive at a concentration of 0.5 - 50 ug/ml, and phosphatidic acid. Or it may include phosphatidylcholine (phosphatidylcholine), but is not limited thereto.
- phosphatidylcholine phosphatidylcholine
- the term "basic medium” means that the basal medium is DMEM (Dulbecco's Modified Eagle's Medium); Minimal Essential Medium (MEM); BME (Basal Medium Eagle); RPMI 1640; DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12); a-MEM (a-Minimal essential Medium); Glasgow's Minimal Essential Medium (G-MEM); IMDM (Isocove's Modified Dulbecco's Medium); or KnockOut DMEM (GIBCO, USA), but is not limited thereto.
- DMEM Dulbecco's Modified Eagle's Medium
- MEM Minimal Essential Medium
- BME Base Medium Eagle
- RPMI 1640 DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM
- stem cells refers to the ability to differentiate into cells of various tissues by specific differentiation-inducing stimuli as well as self-renewal capacity in an undifferentiated state (multi-differentiation potency). means cells.
- mesenchymal stem cells refers to the ability to differentiate into cells of various tissues by specific differentiation-inducing stimuli as well as self-renewal capacity in an undifferentiated state (multi-differentiation potency).
- cells that have The mesenchymal stem cells of the present invention may be any cell having differentiation and proliferative capacity, and may be human, monkey, pigs, horses, cows, sheep, or dogs. It can be of any animal origin, including dogs, cats, mice, and rabbits.
- adipose-derived mesenchymal stem cells are undifferentiated adult stem cells isolated from adipose tissue, and are abbreviated herein as “adipose-derived adult stem cells,” “adipose stem cells,” or “adipose-derived stem cells.” It is also referred to as " It can be obtained through a conventional method known in the art, the separation method may be, for example, as follows.
- Adipose-derived mesenchymal stem cells can be isolated through recovery or other methods.
- exosome or exosome is a vesicle of a certain size derived from a cell. Exosomes mean that the vesicles in the maturation process, the multivesicular body, fuse with the cell membrane and are discharged. The discharged exosomes contain various proteins, nucleic acids, lipids, etc., and can be fused with other cells to deliver the contents. Exosomes have been found to perform important functions in processes such as blood coagulation, intercellular communication, and waste disposal. It includes all body fluids such as blood and urine of multicellular eukaryotic organisms, and all that can be observed in cultures of eukaryotic cells. Preferably, it may be an extracellular vesicle.
- high-purity exosomes refers to exosomes having fewer impurities or components other than exosomes in the experimental group to which a specific medium composition is applied compared to the control group.
- culture media refers to a culture medium that can support the growth and survival of stem cells in in vitro culture conditions, and a conventional medium used in the art suitable for culturing stem cells. includes all In addition, the medium and culture conditions can be selected according to the type of cell.
- the term "separation” refers to an operation to increase purity by removing impurities mixed in a substance, recrystallization or fractional crystallization using the difference in solubility as a method, distillation or fractional distillation based on vapor pressure, and distributional adsorption ⁇ Refers to several types of methods such as ion exchange chromatography, electrophoresis, and gel filtration.
- the agent When it is for measuring the expression level of a high-purity exosome by the medium composition of the present invention, the agent may be an antibody that specifically binds to a protein expressed in the exosome.
- the term “antibody” refers to an immunoglobulin that specifically binds to an antigenic site.
- the antibody can be prepared by cloning the gene into an expression vector to obtain a protein encoded by the gene, and from the obtained protein according to a conventional method in the art.
- a protein-specific antibody can also be prepared using the fragment of the gene protein containing the antigenic site.
- the form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody or a monoclonal antibody. In addition, as long as it has antigen-antibody binding properties, a part of the entire antibody is also included in the antibody of the present invention, and all types of immunoglobulin antibodies that specifically bind to the gene are included.
- a functional fragment of an antibody molecule as well as a complete antibody having two full-length light chains and two full-length heavy chains, i.e. Fab, F(ab'), F(ab') with antigen-binding function. 2 and Fv, and the like.
- the antibodies of the present invention include special antibodies such as humanized antibodies and chimeric antibodies, and recombinant antibodies as long as they can specifically bind to CD9 or CD81 proteins.
- the medium additive may be a serum-free medium that does not contain animal serum, and by using a serum-free medium, immunogenicity may be low and safety and stability may be improved.
- the present inventors confirmed the high efficiency and high purity exosome production ability of the medium composition according to the present invention through Examples.
- the composition of the present invention does not contain albumin and lipids. It was confirmed that the total protein and RNA of the isolated exosomes were removed compared to the control, indicating high purity (see Example 4).
- the present invention provides a method for producing exosomes with high efficiency and high purity, comprising the step of culturing stem cells in the medium composition to produce exosomes.
- the stem cells may be isolated from human adipose tissue, bone marrow, peripheral blood, or umbilical cord blood, preferably human-derived mesenchymal stem cells, and more preferably human adipose-derived mesenchymal stem cells.
- Albumin was prepared at a concentration of 0.01 - 1 mg/ml, and a culture medium containing lipids at a concentration of 0.5 - 50 ug/ml.
- the present inventors purchased a primary cultured mesenchymal stem cell line (ThermoFisher scientific, USA) obtained from adipose tissue (lipoaspirate) of normal people for a fast and efficient study of mesenchymal stem cells (MSC) existing in adipose. was used.
- mesenchymal stem cells For subculture of mesenchymal stem cells for a certain period of time, they were cultured using the serum-free stem cell culture medium prepared in Example 1. The culture conditions were maintained at 37°C and humidity of 95% or higher in a 5% carbon dioxide incubator. After 3 days of incubation, when the cells were 80% confluent, subcultivation was performed by treatment with Accutase (1x accutase enzyme). .
- An exosome sample was obtained from the stem cells from the cultured stem cells in the same manner as above, the exosomes were separated from the sample, and the expression level was measured.
- Example 3 Confirmation of expression level of stem cell-derived exosome marker marker cultured in a medium composition comprising albumin and lipid
- the medium cultured in the medium prepared in Example 1 was referred to as medium A, and the medium containing the remaining components except for albumin and lipid in the medium of Example 1 was referred to as medium B.
- the expression level of the marker was confirmed by Western blotting of the culture medium of stem cells cultured in each medium with CD9 and CD81 antibodies, which are exosome marker markers.
- Example 4 Confirmation of purity of stem cell-derived exosomes cultured in a medium composition comprising albumin and lipid
Abstract
The present invention relates to a medium additive comprising albumin and a lipid as active ingredients for producing exosomes at high efficiency and high purity and a medium composition comprising same and, more specifically, to a medium additive comprising albumin and a lipid as active ingredients for producing exosomes at high efficiency and high purity, a stem cell culture medium composition containing the medium additive, and a method for producing exosomes at high efficiency and high purity. As the present inventors identified the expansion of an output of stem cell-derived exosomes and the separation of highly pure exosomes as a result of culturing cells in a medium containing albumin and a lipid through concrete experiments, it is possible for the medium to produce exosomes at high efficiency and high purity, relative to conventional media. The composition is expected to be utilized as an effective means in the field of developing cosmetic raw materials and therapeutic agents utilizing same.
Description
본 발명은 알부민 및 리피드를 유효성분으로 포함하는 고효율 및 고순도 엑소좀 생산용 배지첨가제 및 이를 포함하는 조성물에 관한 것으로, 보다 구체적으로 알부민 및 리피드를 유효성분으로 포함하는 고효율, 고순도 엑소좀 생산용 배지 첨가제, 상기 배지 첨가제를 포함하는 줄기세포 배양배지 조성물, 및 고효율, 고순도 엑소좀 생산 방법에 관한 것이다.The present invention relates to a medium additive for producing high-efficiency and high-purity exosomes comprising albumin and lipid as active ingredients and a composition comprising the same, and more specifically, to a high-efficiency, high-purity medium for producing exosomes comprising albumin and lipid as active ingredients. It relates to an additive, a stem cell culture medium composition comprising the medium additive, and a method for producing high-efficiency, high-purity exosomes.
다양한 질환에서 줄기세포 치료요법이 큰 기대를 모으고 있으나, 줄기세포를 투여한 임상 시험의 결과에서 손상된 조직의 미미한 개선만이 이루어지거나, 효과도 지속되지 못한다는 문제점이 확인되고 있으며, 장기 추적 관찰의 결과 줄기세포 이식 후 생체 내에서 장기간 생존하는 비율이 극히 낮았다고 보고되고 있다. 이러한 문제점의 원인으로는 대부분의 세포치료제들이 그러하듯 줄기세포 또한 투여된 부위에서의 환경에 적응하고 살아남았을 때 줄기세포의 치료효능이 나타나지만 기본적인 생존능의 저하 즉 이들 세포들이 이식된 후 유지되지 못하거나 인체에 투입 후에 사멸되기 때문이고 그에 따른 불충분한 치료효능이라고 생각되어지고 있다.Although stem cell therapy is highly anticipated in various diseases, the results of clinical trials in which stem cells are administered show only a slight improvement in the damaged tissue or the problem that the effect does not last, and long-term follow-up Results It is reported that the long-term survival rate in vivo after stem cell transplantation is extremely low. The cause of this problem is that, as with most cell therapy drugs, the therapeutic effect of stem cells appears when they survive and adapt to the environment at the site where they are administered, but the basic viability is lowered, that is, these cells cannot be maintained after transplantation. This is because it is destroyed after being injected into the human body, and it is considered to be an insufficient therapeutic effect.
한편, 엑소좀은 세포의 RNA 및 단백질 등을 포함하며 자연적으로 분비되는 30 내지 200 nm 직경의 나노소낭으로, 다양한 물질을 운반하는 매개체로 작용할 수 있음이 알려져 있다. 엑소좀은 유래 세포 특유의 생물학적 기능을 반영하는 세포특이적 구성 성분을 함유하며, 마커 단백질로는 막관통 단백질인 CD9, CD81 등이 잘 알려져 있다.On the other hand, exosomes are nanovesicles with a diameter of 30 to 200 nm that are naturally secreted and contain RNA and proteins of cells, and it is known that they can act as a medium for transporting various substances. Exosomes contain cell-specific components that reflect the unique biological function of derived cells, and transmembrane proteins such as CD9 and CD81 are well known as marker proteins.
최근에는 세포치료제로서 중간엽 줄기세포 자체를 사용하지 않고, 중간엽 줄기세포가 분비하는 엑소좀을 이용하여 다양한 질환의 치료 효과에 대한 연구가 활발하게 진행 중이며(대한민국 공개특허 10-2020-0112092호), 학계 및 산업계에서는 이를 통해 기존의 줄기세포 치료법의 한계를 극복할 수 있는 새로운 대안이 될 수 있을 것으로 예상한다.Recently, instead of using mesenchymal stem cells themselves as cell therapy agents, studies on the therapeutic effects of various diseases using exosomes secreted by mesenchymal stem cells are being actively conducted (Korean Patent Application Laid-Open No. 10-2020-0112092). ), academia and industry expect it to be a new alternative that can overcome the limitations of existing stem cell therapy.
이러한 엑소좀을 상업적으로 이용하기 위해서는 다량의 고순도 엑소좀을 효율적으로 생산, 및 분리하는 방법이 필요하다. 그러나 현재 줄기세포로부터 얻을 수 있는 엑소좀의 양은 매우 소량에 불과하고, 줄기세포 유래 엑소좀의 생산량을 증가시킬 수 있는 물질에 대한 개발도 아직까지 미비한 실정이다.In order to commercially use these exosomes, a method for efficiently producing and separating a large amount of high-purity exosomes is required. However, the amount of exosomes currently obtainable from stem cells is only a very small amount, and the development of substances capable of increasing the production of stem cell-derived exosomes is still insufficient.
이에 따라 줄기세포 유래 엑소좀의 생산을 촉진하는 신규한 조성물 개발에 대한 필요성이 제기되고 있다.Accordingly, the need for the development of a novel composition that promotes the production of stem cell-derived exosomes has been raised.
이에, 본 발명자들은 중간엽 줄기세포로부터 보다 순도 높은 엑소좀을 분리하고자 노력한 결과, 중간엽 줄기세포로부터 엑소좀을 획득하는 과정에 있어서 특정 조성물이 고순도의 엑소좀을 획득하는 결과에 영향을 준다는 것을 확인하였고, 따라서 이러한 배지 조성물을 고순도의 엑소좀을 생산하는데 사용할 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, as a result of the present inventors' efforts to isolate higher-purity exosomes from mesenchymal stem cells, in the process of obtaining exosomes from mesenchymal stem cells, a specific composition affects the results of obtaining high-purity exosomes. It was confirmed, and thus the present invention was completed by confirming that this medium composition can be used to produce exosomes of high purity.
이에, 본 발명은 알부민 및 리피드를 유효성분으로 포함하는, 고효율 및 고순도 엑소좀 생산용 배지 첨가제를 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a medium additive for producing high-efficiency and high-purity exosomes, including albumin and lipid as active ingredients.
또한, 본 발명은 상기 배지 첨가제 및 기본 배지를 포함하는 배지 조성물을 제공하는 것을 다른 목적으로 한다.Another object of the present invention is to provide a medium composition comprising the medium additive and the basal medium.
또한, 본 발명은 상기 배지 조성물에 줄기세포를 배양하는 단계를 포함하는, 고효율 및 고순도 엑소좀 생산방법을 제공하는 것을 또 다른 목적으로 한다.In addition, it is another object of the present invention to provide a method for producing high-efficiency and high-purity exosomes, including the step of culturing stem cells in the medium composition.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.
상기 목적을 달성하기 위하여, 본 발명은 알부민 및 리피드를 포함하는, 고효율 및 고순도 엑소좀 생산용 배지 첨가제를 제공한다.In order to achieve the above object, the present invention provides a medium additive for producing high-efficiency and high-purity exosomes, including albumin and lipids.
본 발명의 일구현예로, 상기 알부민은 배지첨가제에 0.01 - 1 mg/ml 농도로 포함되는 것일 수 있다.In one embodiment of the present invention, the albumin may be included in a concentration of 0.01 - 1 mg/ml in the medium additive.
본 발명의 다른 구현예로, 상기 리피드는 배지첨가제에 0.5 - 50 ug/ml 포함되는 것일 수 있다.In another embodiment of the present invention, the lipid may be included in 0.5 - 50 ug/ml in the medium additive.
본 발명의 또 다른 구현예로, 상기 알부민은 재조합 알부민일 수 있다.In another embodiment of the present invention, the albumin may be recombinant albumin.
본 발명의 또 다른 구현예로, 상기 리피드는 포스파티딘산(phosphatidic acid) 또는 포스파티딜콜린(phosphatidylcholine)을 포함할 수 있다.In another embodiment of the present invention, the lipid may include phosphatidic acid or phosphatidylcholine.
또한, 본 발명은 상기 배지 첨가제 및 기본 배지를 포함하는, 배지 조성물을 제공한다.In addition, the present invention provides a medium composition comprising the medium additive and the basal medium.
본 발명의 일구현예로, 상기 기본 배지는 DMEM (Dulbecco's Modified Eagle's Medium); MEM (Minimal Essential Medium); BME (Basal Medium Eagle); RPMI 1640; DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12); a-MEM(a-Minimal essential Medium); G-MEM(Glasgow's Minimal Essential Medium); IMDM (Isocove's Modified Dulbecco's Medium); 및 KnockOut DMEM (GIBCO, USA)으로 이루어진 군에서 선택되는 어느 하나일 수 있다.In one embodiment of the present invention, the basal medium is DMEM (Dulbecco's Modified Eagle's Medium); Minimal Essential Medium (MEM); BME (Basal Medium Eagle); RPMI 1640; DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12); a-MEM (a-Minimal essential Medium); Glasgow's Minimal Essential Medium (G-MEM); IMDM (Isocove's Modified Dulbecco's Medium); and KnockOut DMEM (GIBCO, USA) may be any one selected from the group consisting of.
본 발명의 다른 구현예로 상기 배지 조성물은 무혈청 배지일 수 있다.In another embodiment of the present invention, the medium composition may be a serum-free medium.
또한, 본 발명은 상기 배지 조성물에 줄기세포를 배양하는 단계를 포함하는, 고효율 및 고순도 엑소좀 생산방법을 제공한다.In addition, the present invention provides a method for producing high-efficiency and high-purity exosomes, including the step of culturing stem cells in the medium composition.
본 발명의 일구현예로, 상기 줄기세포는 인간 지방유래 중간엽 줄기세포일 수 있다.In one embodiment of the present invention, the stem cells may be human adipose-derived mesenchymal stem cells.
본 발명자들은 구체적인 실험을 통해 알부민 및 리피드가 포함된 배지에 세포를 배양한 결과 줄기세포 유래 엑소좀의 생산량 증대와 고순도 엑소좀 분리를 확인하였는 바, 기존 배지 대비 고효율 고순도의 엑소좀 생산이 가능하므로 이를 활용한 화장품 원료 및 치료제 개발 분야에 효율적인 수단으로 활용될 수 있을 것으로 기대된다.As a result of culturing the cells in a medium containing albumin and lipids through a specific experiment, the present inventors confirmed an increase in the production of stem cell-derived exosomes and isolation of high-purity exosomes. It is expected that it can be used as an effective means in the field of developing cosmetic raw materials and therapeutics using this.
도 1은 본 발명의 알부민 및 리피드 원료 첨가 배양 배지(배지 A) 및 알부민 및 리피드가 첨가되지 않은 배양 배지(배지 B) 각각에서 배양된 줄기세포의 배양액을 엑소좀 표지 마커인 CD9, CD81 항체로 웨스턴 블랏팅한 결과이다. 1 is a culture medium of stem cells cultured in each of the albumin and lipid raw material addition culture medium (medium A) and albumin and lipid-free culture medium (medium B) of the present invention as exosome marker markers CD9, CD81 antibody Western blotting results.
도 2는 본 발명의 알부민 및 리피드가 첨가된 배양 배지(배지 A) 및 첨가되지 않은 배양 배지(배지 B) 각각에서 배양된 줄기세포 배양액으로부터 엑소좀을 분리과정의 마지막 단계인 이온 크로마토그래피를 분석한 결과이다.Figure 2 is the last step of the separation process of the exosomes from the stem cell culture medium cultured in each of the culture medium (medium A) and the non-added culture medium (medium B) of the present invention albumin and lipids are analyzed by ion chromatography is a result
도 2a는 배지 A의 배양액으로부터 분리된 엑소좀이 총단백질 및 RNA가 제거되어 높은 순도를 가지는 것을 확인한 결과이다. Figure 2a is the result of confirming that the exosomes isolated from the culture medium of medium A have high purity by removing total protein and RNA.
도 2b는 도 2a의 결과를 SDS-PAGE를 통해 확인한 것이다. Figure 2b confirms the result of Figure 2a through SDS-PAGE.
도 2c는 도 2a의 결과를 엑소좀 표지 마커인 CD9, CD81 항체로 웨스턴 블랏팅하여 확인한 것이다.Figure 2c confirms the result of Figure 2a by Western blotting with the exosome marker CD9, CD81 antibody.
상기한 목적을 달성하기 위하여, 알부민 및 리피드를 포함하는, 고효율 및 고순도 엑소좀 생산용 배지 첨가제에 의해 달성된다.In order to achieve the above object, it is achieved by a medium additive for producing high efficiency and high purity exosomes, including albumin and lipids.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 중간엽 줄기세포로부터 보다 순도 높은 엑소좀을 분리하고자 노력한 결과, 중간엽 줄기세포로부터 엑소좀을 획득하는 과정에 있어서 특정 조성물이 고순도의 엑소좀을 획득하는 결과에 영향을 준다는 것을 확인하였고, 따라서 이러한 배지 조성물을 고순도의 엑소좀을 생산하는데 사용할 수 있음을 확인함으로써 본 발명을 완성하였다.As a result of the present inventors' efforts to isolate higher-purity exosomes from mesenchymal stem cells, it was confirmed that a specific composition affects the results of obtaining high-purity exosomes in the process of obtaining exosomes from mesenchymal stem cells. , thus completing the present invention by confirming that this medium composition can be used to produce exosomes of high purity.
이에, 본 발명은 줄기세포로부터 고순도의 엑소좀을 분리하기 위해 알부민 및 리피드를 포함하는, 고효율 및 고순도 엑소좀 생산용 배지 첨가제 및 상기 배지 첨가제와 기본배지를 포함하는 배지 조성물을 제공한다.Accordingly, the present invention provides a medium additive for producing high-efficiency and high-purity exosomes, including albumin and lipid, and a medium composition comprising the medium additive and a basal medium, to separate exosomes of high purity from stem cells.
본 발명에서 상기 알부민은 배지첨가제에 0.01 - 1 mg/ml 농도로 포함될 수 있으며 재조합 알부민일 수 있고, 상기 리피드는 배지첨가제에 0.5 - 50 ug/ml 농도로 포함될 수 있으며 포스파티딘산(phosphatidic acid) 또는 포스파티딜콜린(phosphatidylcholine)을 포함할 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the albumin may be included in the medium additive at a concentration of 0.01 - 1 mg/ml, and may be recombinant albumin, and the lipid may be included in the medium additive at a concentration of 0.5 - 50 ug/ml, and phosphatidic acid. Or it may include phosphatidylcholine (phosphatidylcholine), but is not limited thereto.
본 발명에서 사용되는 용어, "기본배지"는 상기 기본 배지는 DMEM (Dulbecco's Modified Eagle's Medium); MEM (Minimal Essential Medium); BME (Basal Medium Eagle); RPMI 1640; DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12); a-MEM(a-Minimal essential Medium); G-MEM(Glasgow's Minimal Essential Medium); IMDM (Isocove's Modified Dulbecco's Medium); 또는 KnockOut DMEM (GIBCO, USA)일 수 있으나, 이에 제한되는 것은 아니다.As used herein, the term "basic medium" means that the basal medium is DMEM (Dulbecco's Modified Eagle's Medium); Minimal Essential Medium (MEM); BME (Basal Medium Eagle); RPMI 1640; DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12); a-MEM (a-Minimal essential Medium); Glasgow's Minimal Essential Medium (G-MEM); IMDM (Isocove's Modified Dulbecco's Medium); or KnockOut DMEM (GIBCO, USA), but is not limited thereto.
본 발명에서 사용되는 용어, "줄기세포"는 미분화 상태에서 무한 증식능력(self-renewal capacity)과 더불어 특정한 분화유도 자극에 의해 다양한 조직의 세포로 분화될 수 있는 능력(multi-differentiation potency)을 가진 세포를 의미한다.As used herein, the term "stem cells" refers to the ability to differentiate into cells of various tissues by specific differentiation-inducing stimuli as well as self-renewal capacity in an undifferentiated state (multi-differentiation potency). means cells.
본 발명에서 사용되는 용어, "중간엽 줄기세포"는 미분화 상태에서 무한 증식능력(self-renewal capacity)과 더불어 특정한 분화유도 자극에 의해 다양한 조직의 세포로 분화될 수 있는 능력(multi-differentiation potency)을 가진 세포를 말한다. 본 발명의 중간엽줄기세포는 분화능 및 증식능을 갖는 모든 세포일 수 있으며, 인간(human), 원숭이(monkeys), 돼지(pigs), 말(horses), 소(cows), 양(sheep), 개(dogs), 고양이(cats), 생쥐(mice), 토끼(rabbits) 등의 모든 동물로부터 유래할 수 있다.As used herein, the term "mesenchymal stem cells" refers to the ability to differentiate into cells of various tissues by specific differentiation-inducing stimuli as well as self-renewal capacity in an undifferentiated state (multi-differentiation potency). cells that have The mesenchymal stem cells of the present invention may be any cell having differentiation and proliferative capacity, and may be human, monkey, pigs, horses, cows, sheep, or dogs. It can be of any animal origin, including dogs, cats, mice, and rabbits.
본 발명에서, "지방유래 중간엽 줄기세포"란 지방조직에서 분리해 낸 미분화된 성체 줄기세포로서, 본 명세서에 축약하여 "지방 유래 성체 줄기세포", "지방 줄기세포" 또는 "지방 유래 줄기세포"라고 지칭하기도 한다. 이는 당업계에 공지된 통상의 방법을 통하여 수득할 수 있는데, 그 분리 방법은 예를 들어 다음과 같을 수 있다. 즉, 지방흡입술로부터 얻어지는 생리 식염수에 부유된 지방 함유 suspension을 배양한 다음, 플라스크 등 배양용기에 부착된 줄기세포 층을 트립신으로 처리한 다음 회수하거나, 스크래퍼로 긁어서 소량의 생리식염수에 부유되는 것을 직접 회수하거나 하는 방법 등을 통해 지방 유래 중간엽 줄기세포를 분리할 수 있다.In the present invention, "adipose-derived mesenchymal stem cells" are undifferentiated adult stem cells isolated from adipose tissue, and are abbreviated herein as "adipose-derived adult stem cells," "adipose stem cells," or "adipose-derived stem cells." It is also referred to as " It can be obtained through a conventional method known in the art, the separation method may be, for example, as follows. That is, after culturing a suspension containing fat in physiological saline obtained from liposuction, and then treating the stem cell layer attached to a culture vessel such as a flask with trypsin, and then recovering it, or scraping it with a scraper and floating in a small amount of physiological saline directly. Adipose-derived mesenchymal stem cells can be isolated through recovery or other methods.
본 발명에서 사용되는 용어, "엑소좀 또는 엑소솜은(exosome)"은 세포에서 유래한 일정 크기의 소포(vesicle)이다. 엑소좀은 성숙과정 중의 소포인 다중소포체(multivesicular body)가 세포막과 융합하여 배출되는 것을 의미한다. 배출된 엑소솜은 다양한 단백질, 핵산, 지질 등이 포함되어 있으며, 다른 세포와 융합하여 내용물을 전달할 수 있다. 엑소솜은 혈액 응고, 세포 간 통신, 노폐물처리 등의 과정에서 중요한 기능을 수행하는 것으로 밝혀져 있다. 다세포 진핵 생명체의 혈액, 오줌 등 체액과 진핵세포를 배양한 배양액에서 관찰할 수 있는 모두를 포함한다. 바람직하게는 세포 외 소포체 (Extracellular vesicle)일 수 있다.As used herein, the term "exosome or exosome" is a vesicle of a certain size derived from a cell. Exosomes mean that the vesicles in the maturation process, the multivesicular body, fuse with the cell membrane and are discharged. The discharged exosomes contain various proteins, nucleic acids, lipids, etc., and can be fused with other cells to deliver the contents. Exosomes have been found to perform important functions in processes such as blood coagulation, intercellular communication, and waste disposal. It includes all body fluids such as blood and urine of multicellular eukaryotic organisms, and all that can be observed in cultures of eukaryotic cells. Preferably, it may be an extracellular vesicle.
본 발명에서 사용되는 용어, "고순도의 엑소좀"은 대조군과 비교하였을 때 특정 배지 조성물을 적용한 실험군에서 불순물 또는 엑소좀을 제외한 성분이 적은 엑소좀을 의미한다.As used in the present invention, the term "high-purity exosomes" refers to exosomes having fewer impurities or components other than exosomes in the experimental group to which a specific medium composition is applied compared to the control group.
본 발명에서 사용되는 용어, "배지 (culture media)"는 체외배양 조건에서 줄기세포의 성장 및 생존을 지지할 수 있게 하는 배양액을 의미하고, 줄기세포의 배양에 적절한 당 분야에서 사용되는 통상의 배지를 모두 포함한다. 또한, 세포의 종류에 따라 배지와 배양 조건을 선택할 수 있다.As used herein, the term "culture media" refers to a culture medium that can support the growth and survival of stem cells in in vitro culture conditions, and a conventional medium used in the art suitable for culturing stem cells. includes all In addition, the medium and culture conditions can be selected according to the type of cell.
본 발명에서 사용된 용어, "분리"는 어떤 물질로부터 혼재해 있는 불순물을 제거하여, 순도를 높이는 조작, 그 방법으로 용해도의 차이를 이용한 재결정이나 분별결정, 증기압에 근거한 증류나 분별증류 및 분배흡착·이온교환 크로마토그래피법, 전기영동법, 겔여과법 등 여러 종류의 방법을 의미한다.As used in the present invention, the term "separation" refers to an operation to increase purity by removing impurities mixed in a substance, recrystallization or fractional crystallization using the difference in solubility as a method, distillation or fractional distillation based on vapor pressure, and distributional adsorption · Refers to several types of methods such as ion exchange chromatography, electrophoresis, and gel filtration.
본 발명의 배지 조성물에 의한 고순도의 엑소좀 발현수준을 측정하기 위한 것일 때에는, 상기 제제는 상기 엑소좀에서 발현되는 단백질에 특이적으로 결합하는 항체일 수 있다.When it is for measuring the expression level of a high-purity exosome by the medium composition of the present invention, the agent may be an antibody that specifically binds to a protein expressed in the exosome.
본 발명에서 사용되는 용어, "항체(antibody)"는 항원성 부위에 특이적으로 결합하는 면역글로불린(immunoglobulin)을 의미한다. 항체는 상기 유전자를 발현 벡터에 클로닝하여 상기 유전자에 의해 암호화되는 단백질을 수득하고, 수득한 단백질로부터 당해 기술분야의 통상적인 방법에 따라 제조할 수 있다. 항원성 부위를 포함하는 상기 유전자 단백질의 단편을 이용하여 단백질 특이적인 항체를 제조할 수도 있다. 본 발명의 항체의 형태는 특별히 제한되지 않으며, 다클론항체(polyclonal antibody) 또는 단일클론항체(monoclonal antibody)를 포함한다. 또한 항원-항체 결합성을 갖는 것이면 전체 항체의 일부도 본 발명의 항체에 포함되며, 상기 유전자에 특이적으로 결합하는 모든 종류의 면역글로불린 항체가 포함된다. 예를 들어 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 갖는 완전한 형태의 항체 뿐 아니라 항체 분자의 기능적인 단편, 즉 항원 결합 기능을 갖는 Fab, F(ab'), F(ab')2 및 Fv 등을 포함한다. 나아가 본 발명의 항체에는 CD9, CD81 단백질에 특이적으로 결합할 수 있는 것이라면 인간화 항체, 키메릭 항체 등의 특수 항체와 재조합 항체도 포함된다. As used herein, the term “antibody” refers to an immunoglobulin that specifically binds to an antigenic site. The antibody can be prepared by cloning the gene into an expression vector to obtain a protein encoded by the gene, and from the obtained protein according to a conventional method in the art. A protein-specific antibody can also be prepared using the fragment of the gene protein containing the antigenic site. The form of the antibody of the present invention is not particularly limited, and includes a polyclonal antibody or a monoclonal antibody. In addition, as long as it has antigen-antibody binding properties, a part of the entire antibody is also included in the antibody of the present invention, and all types of immunoglobulin antibodies that specifically bind to the gene are included. For example, a functional fragment of an antibody molecule as well as a complete antibody having two full-length light chains and two full-length heavy chains, i.e. Fab, F(ab'), F(ab') with antigen-binding function. 2 and Fv, and the like. Furthermore, the antibodies of the present invention include special antibodies such as humanized antibodies and chimeric antibodies, and recombinant antibodies as long as they can specifically bind to CD9 or CD81 proteins.
본 발명에서 상기 배지 첨가제는 동물 혈청을 포함하지 않는 무혈청 배지일 수 있으며, 무혈청 배지를 사용하여 면역원성이 낮고 안전성 및 안정성이 향상될 수 있다.In the present invention, the medium additive may be a serum-free medium that does not contain animal serum, and by using a serum-free medium, immunogenicity may be low and safety and stability may be improved.
본 발명자들은 실시예를 통해 본 발명에 따른 배지 조성물의 고효율 및 고순도 엑소좀 생산능을 확인하였다.The present inventors confirmed the high efficiency and high purity exosome production ability of the medium composition according to the present invention through Examples.
구체적으로, 본 발명의 일실시예에서는 알부민 및 리피드를 포함하는 배지에서 배양된 줄기세포 유래 엑소좀의 시료에서 엑소좀의 순도를 분석한 결과, 본 발명의 조성물이 알부민과 리피드를 포함하고 있지 않은 대조군 비해 분리된 엑소좀의 총단백질 및 RNA가 제거되어 높은 순도를 나타내는 것을 확인하였다(실시예 4 참조). Specifically, in one embodiment of the present invention, as a result of analyzing the purity of exosomes in a sample of stem cell-derived exosomes cultured in a medium containing albumin and lipids, the composition of the present invention does not contain albumin and lipids. It was confirmed that the total protein and RNA of the isolated exosomes were removed compared to the control, indicating high purity (see Example 4).
상기 결과로부터 본 발명의 알부민과 리피드를 포함하는 배지 조성물에서 생산된 엑소좀의 순도가 향상됨을 알 수 있었으며, 이에 본 발명의 배지 조성물을 줄기세포 유래 엑소좀의 고효율 및 고순도 생산에 활용할 수 있다.From the above results, it was found that the purity of the exosomes produced in the medium composition containing the albumin and lipids of the present invention was improved, and thus the medium composition of the present invention could be utilized for high-efficiency and high-purity production of stem cell-derived exosomes.
이에, 본 발명의 다른 양태로써 본 발명은 상기 배지 조성물에 줄기세포를 배양하여 엑소좀을 생산하는 단계를 포함하는, 고효율 및 고순도 엑소좀 생산방법을 제공한다.Accordingly, as another aspect of the present invention, the present invention provides a method for producing exosomes with high efficiency and high purity, comprising the step of culturing stem cells in the medium composition to produce exosomes.
본 발명에서 상기 줄기세포는 인간의 지방조직, 골수, 말초혈액, 또는 제대혈 등에서 분리된 것일 수 있으며, 바람직하게는 인간 유래 중간엽 줄기세포일 수 있고, 더욱 바람직하게는 인간 지방유래 중간엽 줄기세포일 수 있다. In the present invention, the stem cells may be isolated from human adipose tissue, bone marrow, peripheral blood, or umbilical cord blood, preferably human-derived mesenchymal stem cells, and more preferably human adipose-derived mesenchymal stem cells. can be
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
[실시예][Example]
실시예 1. 배양배지 준비Example 1. Preparation of culture medium
알부민은 0.01 - 1 mg/ml 농도로, 리피드를 0.5 - 50 ug/ml 농도로 포함하는 배양배지를 제조하였다.Albumin was prepared at a concentration of 0.01 - 1 mg/ml, and a culture medium containing lipids at a concentration of 0.5 - 50 ug/ml.
실시예 2. 줄기세포 준비 및 엑소좀 분리Example 2. Stem cell preparation and exosome isolation
본 발명자들은 지방 내 존재하는 중간엽줄기세포 (mesenchymal stem cell, MSC)의 빠르고 효율적인 연구를 위해 정상인의 지방 (lipoaspirate) 조직에서 채취하여 일차 배양된 중간엽줄기세포주 (ThermoFisher scientific, 미국)를 구입하여 이용하였다. 중간엽 줄기세포의 일정기간 계대배양을 위하여 실시예 1에 의해 제조된 무혈청 줄기세포 배양배지를 이용하여 배양하였다. 배양조건은 5% 이산화탄소 배양기에 온도 37℃, 습도 95% 이상을 유지하였으며, 3일 배양 후 세포들이 80% 교회하면 (confluent), Accutase (1x accutase enzyme) 처리하여 계대배양 (subcultivation)을 시행하였다. 상기와 같은 방법으로 배양된 줄기세포로 부터 줄기세포로부터 엑소좀 시료를 얻고 시료로부터 엑소좀을 분리하고 발현수준을 측정하였다.The present inventors purchased a primary cultured mesenchymal stem cell line (ThermoFisher scientific, USA) obtained from adipose tissue (lipoaspirate) of normal people for a fast and efficient study of mesenchymal stem cells (MSC) existing in adipose. was used. For subculture of mesenchymal stem cells for a certain period of time, they were cultured using the serum-free stem cell culture medium prepared in Example 1. The culture conditions were maintained at 37°C and humidity of 95% or higher in a 5% carbon dioxide incubator. After 3 days of incubation, when the cells were 80% confluent, subcultivation was performed by treatment with Accutase (1x accutase enzyme). . An exosome sample was obtained from the stem cells from the cultured stem cells in the same manner as above, the exosomes were separated from the sample, and the expression level was measured.
실시예 3. 알부민 및 리피드를 포함하는 배지 조성물에서 배양된 줄기세포 유래 엑소좀 표지 마커의 발현 수준 확인Example 3. Confirmation of expression level of stem cell-derived exosome marker marker cultured in a medium composition comprising albumin and lipid
상기 실시예 1에 제조된 배지에서 배양한 배지를 배지 A로 표기하고, 실시예1의 배지에서 알부민 및 리피드를 제외한 나머지 성분을 포함하는 배지를 배지 B로 표기하였다. 각각의 배지에서 배양된 줄기세포의 배양액을 엑소좀 표지마커인 CD9, CD81항체로 웨스턴블랏팅하여 마커의 발현수준을 확인하였다. The medium cultured in the medium prepared in Example 1 was referred to as medium A, and the medium containing the remaining components except for albumin and lipid in the medium of Example 1 was referred to as medium B. The expression level of the marker was confirmed by Western blotting of the culture medium of stem cells cultured in each medium with CD9 and CD81 antibodies, which are exosome marker markers.
그 결과, 도 1에 나타낸 바와 같이, CD9의 경우 배지 A 및 B 모두에서 발현이 나타났지만 배지 A의 경우 더 발현 수준이 높게 나타나는 것으로 확인되었으며, CD81의 경우 배지 B에서는 발현수준이 미미하나, 배지 A에서 발현 수준이 증가된 것을 확인하였다.As a result, as shown in FIG. 1 , in the case of CD9, expression was shown in both media A and B, but in the case of media A, the expression level was confirmed to be higher, and in the case of CD81, the expression level was insignificant in media B, but the It was confirmed that the expression level was increased.
실시예 4. 알부민 및 리피드를 포함하는 배지 조성물에서 배양된 줄기세포 유래 엑소좀의 순도 확인Example 4. Confirmation of purity of stem cell-derived exosomes cultured in a medium composition comprising albumin and lipid
알부민 및 리피드를 포함하고 있는 배지 A, 및 알부민 및 리피드가 포함되지 않은 배지 B의에서 배양된 줄기세포 배양액으로부터 엑소좀을 분리하는 마지막 단계인 이온 크로마토그래피 분석 결과를 통해, 엑소좀의 순도를 확인한 결과, 도 2a에 나타낸 바와 같이 배지 A의 배양액으로부터 분리된 엑소좀의 경우 총 단백질 및 RNA가 제거되어 높은 순도를 나타내는 것을 확인하였으며 배지 A와 배지 B의 분리양상이 상이하게 나타나는 것을 확인하였다.Through the ion chromatography analysis result, which is the last step of separating the exosomes from the stem cell culture medium cultured in the medium A containing albumin and lipids, and the medium B not containing albumin and lipids, the purity of the exosomes was confirmed. As a result, as shown in FIG. 2a , in the case of exosomes isolated from the culture medium of medium A, it was confirmed that total protein and RNA were removed, indicating high purity, and it was confirmed that the separation patterns of medium A and medium B were different.
또한, 도 2a의 결과를 SDS-PAGE로 확인한 결과 도 2b에 나타낸 바와 같이 PAGE상의 밴드 패턴이 상이하게 나타나는 것을 확인하였으며, 도 2a 의 결과를 엑소좀 표지 마커인 CD9 및 CD81 항체로 웨스턴블랏팅하여 확인한 결과 도 2c에 나타낸 바와 같이 타겟단백질의 발현이 배지 B보다 우수하게 나타남으로써 배지 A에서 불순물이 적고 순도가 높은 것을 확인하였다.In addition, as a result of confirming the result of Fig. 2a by SDS-PAGE, it was confirmed that the band pattern on the PAGE was different as shown in Fig. 2b, and the result of Fig. 2a was western blotted with CD9 and CD81 antibodies, which are exosome marker markers. As a result, as shown in FIG. 2c , the expression of the target protein was superior to that of the medium B, confirming that the medium A had fewer impurities and high purity.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.
Claims (10)
- 알부민 및 리피드를 포함하는, 고효율 및 고순도 엑소좀 생산용 배지 첨가제.A medium additive for high-efficiency and high-purity exosome production, including albumin and lipid.
- 제1항에 있어서,According to claim 1,상기 알부민은 배지 첨가제에 0.01 - 1 mg/ml 농도로 포함되는 것을 특징으로 하는, 배지 첨가제.The albumin is a medium additive, characterized in that it is contained in a concentration of 0.01 - 1 mg / ml in the medium additive.
- 제1항에 있어서,According to claim 1,상기 리피드는 배지 첨가제에 0.5 - 50 ug/ml 농도로 포함되는 것을 특징으로 하는, 배지 첨가제.The lipid is characterized in that contained in the medium additive at a concentration of 0.5 - 50 ug / ml, the medium additive.
- 제1항 내지 제3항 중 어느 한 항에 있어서,4. The method according to any one of claims 1 to 3,상기 알부민은 재조합 알부민인 것을 특징으로 하는, 배지 첨가제.The albumin is characterized in that the recombinant albumin, the medium additive.
- 제1항 내지 제3항 중 어느 한 항에 있어서,4. The method according to any one of claims 1 to 3,상기 리피드는 포스파티딘산(phosphatidic acid) 또는 포스파티딜콜린(phosphatidylcholine)을 포함하는 것을 특징으로 하는, 배지 첨가제.The lipid is phosphatidic acid (phosphatidic acid) or phosphatidylcholine (phosphatidylcholine), characterized in that it comprises a medium additive.
- 제1항 내지 제5항 중 어느 한 항의 배지 첨가제 및 기본 배지를 포함하는, 배지 조성물.A medium composition comprising the medium additive of any one of claims 1 to 5 and a basal medium.
- 제6항에 있어서,7. The method of claim 6,상기 기본 배지는 DMEM (Dulbecco's Modified Eagle's Medium); MEM (Minimal Essential Medium); BME (Basal Medium Eagle); RPMI 1640; DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12); a-MEM(a-Minimal essential Medium); G-MEM(Glasgow's Minimal Essential Medium); IMDM (Isocove's Modified Dulbecco's Medium); 및 KnockOut DMEM (GIBCO, USA)으로 이루어진 군에서 선택되는 어느 하나인 것을 특징으로 하는, 배지 조성물.The basal medium is DMEM (Dulbecco's Modified Eagle's Medium); Minimal Essential Medium (MEM); BME (Basal Medium Eagle); RPMI 1640; DMEM/F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10); DMEM/F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12); a-MEM (a-Minimal essential Medium); Glasgow's Minimal Essential Medium (G-MEM); IMDM (Isocove's Modified Dulbecco's Medium); And KnockOut DMEM (GIBCO, USA) characterized in that any one selected from the group consisting of, the medium composition.
- 제6항에 있어서,7. The method of claim 6,상기 배지 조성물은 무혈청 배지인 것을 특징으로 하는, 배지 조성물.The medium composition is characterized in that the serum-free medium, the medium composition.
- 제6항의 배지 조성물에 줄기세포를 배양하는 단계를 포함하는, 고효율 및 고순도 엑소좀 생산방법.A method for producing exosomes with high efficiency and purity, comprising the step of culturing stem cells in the medium composition of claim 6 .
- 제9항에 있어서,10. The method of claim 9,상기 줄기세포는 인간 지방유래 중간엽 줄기세포인 것을 특징으로 하는, 고효율 및 고순도 엑소좀 생산방법.The stem cells are human adipose-derived mesenchymal stem cells, characterized in that the high-efficiency and high-purity exosome production method.
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| KR20100122087A (en) * | 2008-02-22 | 2010-11-19 | 에이전시 포 사이언스, 테크놀로지 앤드 리서치 | Mesenchymal stem cell particles |
| US20120093885A1 (en) * | 2010-10-18 | 2012-04-19 | Northwestern University | Therapeutic vesicles |
| KR20150004822A (en) * | 2012-04-03 | 2015-01-13 | 레뉴런 리미티드 | Stem cell microparticles |
| US20160108368A1 (en) * | 2014-07-03 | 2016-04-21 | ReCyte Therapeutics, Inc. | Exosomes from clonal progenitor cells |
| US20200017825A1 (en) * | 2017-01-23 | 2020-01-16 | Stemcell Technologies Canada Inc. | Media and methods for enhancing the survival and proliferation of stem cells |
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| KR20100122087A (en) * | 2008-02-22 | 2010-11-19 | 에이전시 포 사이언스, 테크놀로지 앤드 리서치 | Mesenchymal stem cell particles |
| US20120093885A1 (en) * | 2010-10-18 | 2012-04-19 | Northwestern University | Therapeutic vesicles |
| KR20150004822A (en) * | 2012-04-03 | 2015-01-13 | 레뉴런 리미티드 | Stem cell microparticles |
| US20160108368A1 (en) * | 2014-07-03 | 2016-04-21 | ReCyte Therapeutics, Inc. | Exosomes from clonal progenitor cells |
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