WO2022098018A1 - Method of obtaining progenitor cells by using cold shock, and method of producing exosomes by using same - Google Patents
Method of obtaining progenitor cells by using cold shock, and method of producing exosomes by using same Download PDFInfo
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- WO2022098018A1 WO2022098018A1 PCT/KR2021/015574 KR2021015574W WO2022098018A1 WO 2022098018 A1 WO2022098018 A1 WO 2022098018A1 KR 2021015574 W KR2021015574 W KR 2021015574W WO 2022098018 A1 WO2022098018 A1 WO 2022098018A1
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- exosomes
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- exosome
- progenitor cells
- glutamine
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- C12N2523/00—Culture process characterised by temperature
Definitions
- the present invention relates to a method for obtaining progenitor cells by treating mononuclear cells isolated from cells or tissues with cold shock, and a method for culturing the obtained progenitor cells to produce exosomes, and more particularly, to a method of producing exosomes by culturing the obtained progenitor cells. It relates to a method for obtaining human-derived progenitor cells with stable activity.
- exosomes are mostly focused on mesenchymal stem cells, induced pluripotent stem cells, and the 293T cell line.
- Mesenchymal stem cells and induced pluripotent stem cell-derived exosomes are focused on research on their efficacy, and 293T cell-derived exosomes are focused on cargo research that transports anticancer substances.
- 293T cell-derived exosomes are focused on cargo research that transports anticancer substances. In other words, research to use exosomes as biopharmaceutical materials rather than cargo is limited to two cells.
- exosomes secreted by the mesenchymal stem cells show the characteristics of mesenchymal stem cells
- the exosomes secreted by the induced pluripotent stem cells show the characteristics of the induced pluripotent stem cells.
- exosomes are vesicles surrounded by a membrane with a size of 30-150 nm generated from endosomes.
- cells of all living organisms secrete exosomes.
- the size of other extracellular vesicles, such as microvesicals and apoptotic bodies, is similar to that of exosomes, but the biogenesis pathway is different.
- the biggest role of the exosome is as a cargo, that is, a material carrier, which transports nucleic acid information such as proteins, lipids, mRNA, microRNA, and long non-coding RNA.
- exosomes play an important role in cell-to-cell communication by continuously passing these information back and forth between cells.
- microRNAs are the most important among substances transported by exosomes, and are the core that exosomes can exhibit physiological or therapeutic efficacy (Nature Reviews Neurology 15, 193-203 (2019)).
- exosomes differ depending on the derived cells, the size, concentration, and loaded nucleic acid information of the exosomes.
- Existing studies of producing exosomes derived from human beings all use mesenchymal stem cells or 293T cells.
- the average size of the exosomes produced using these cells is about 200 nm, which is often outside the size range defined by the exosomes, and the concentration of exosomes secreted by the cells itself is low, so complex processes such as separation and separation are required.
- the production volume is limited and the production cost is high.
- the average size of the exosomes is about 200 nm, the size is quite large, and when filtration sterilization using a 0.2 ⁇ m filter filter, the loss of exosomes is large and the pores of the filter are clogged, so it is difficult to proceed with filtration sterilization. .
- the problems described above made it difficult to apply the existing exosomes to clinical research.
- the present inventors invented a method for obtaining a new specific progenitor cell, and prepared exosomes with uniform size distribution and high concentration from the cells in the present invention was completed.
- the present invention was created in view of the above circumstances, and its technical purpose is to provide a method for isolating new progenitor cells and to provide a method for culturing the progenitor cells to obtain exosomes of a specific size at a high concentration. .
- the present invention provides a method capable of separating the exosomes of the present invention only by diluting the exosomes so that the volume of the exosomes becomes the same as the starting volume again by adding a stabilization buffer after concentration and diafiltration without additional additives and processing steps. It is another purpose to provide.
- a method for obtaining progenitor cells using cold shock characterized in that the progenitor cells are obtained by a cold shock method in which mononuclear cells are frozen under certain conditions and then thawed.
- the obtained progenitor cells preferably satisfy the following conditions.
- the composition used when freezing cells is glutamine (L-glutamine) or L-glutamine dipeptide form (L-alanyl-L-glutamine dipeptide), DMSO (dimethyl sulfoxide) and knockout serum replacement.
- DMEM Dulbecco's Modified Eagle Medium
- MEM Minimal Essential Medium
- IMDM Iscove's Modified Dulbecco's Medium
- DMEM/F-12 Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
- a medium containing DMEM/F-12 Glutamax supplement medium a medium containing DMEM/F-12 Glutamax supplement medium
- c) is immunologically negative for CD24, CD117 or SSEA-1, or
- the progenitor cells may be derived from umbilical cord blood, bone marrow, fat, umbilical cord, amniotic fluid, dental or peripheral blood.
- a method for producing exosomes characterized in that it comprises the steps of collecting some, and (e) separating the collected exosomes, and exosomes produced by the method.
- the first medium used for attaching the progenitor cells is glutamine (L-glutamine) or a dipeptide form of L-glutamine (L-alanyl-L-glutamine dipeptide), fetal bovine serum (FBS), sodium pyruvate (Sodium pyruvate) ), non-essential amino acids, transferrin, sodium selenite and insulin, Dulbecco's Modified Eagle Medium (DMEM: Dulbecco') 2 Modified Eagle Medium), Advanced DMEM, Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), or DMEM/F12 medium.
- DMEM Dulbecco's Modified Eagle Medium
- MEM Minimal Essential Medium
- IMDM Iscove's Modified Dulbecco's Medium
- DMEM/F12 medium Dulbecco's Modified Eagle Medium
- the second medium used for culturing the progenitor cells is glutathione monosodium, vitamin B12, monosodium hypoxanthine, thymidine, glutamine or L-glutamine.
- the dipeptide form L-alanyl-L-glutamine dipeptide
- at least one selected from the group consisting of knockout serum replacement (DMEM: Dulbecco'2 Modified Eagle Medium), at least Essential medium (MEM: Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) or DMEM/F-12 Glutamax supplement medium may be included.
- the step (e) is, (e-1) passing the progenitor cell culture solution through a 0.45 or 0.8 ⁇ m filter to filter cell debris, wastes and large particles, (e-2) filtering the filtered culture solution through a sterile filter sterilizing with, (e-3) separating the exosomes by concentration and diafiltration of the sterilized culture solution using tangential flow filtration, (e-4) adding a stabilization buffer to the separated exosomes Diluting to the same volume as the initial volume, (e-5) sterilizing the diluted exosomes through a sterile filter using a liquid exchange system, and (e-6) sterilizing the sterilized exosomes It is preferred to include the step of storing in a sealed storage container.
- the concentration and diafiltration in step (e-3) are tangential flow filtration is performed intermittently or continuously, the concentration is concentrated 10 to 25 times with respect to the starting volume, and the diafiltration process preferably includes filtration with a buffer having at least 10 times the volume of the concentrated volume.
- the exosome dilution preferably includes a process of diluting the total volume to the same as the starting volume by adding a stabilization buffer 10 to 25 times the concentration and diafiltration volume.
- the exosome has a concentration of 3 x 10 9 particles/ml to 5 x 10 9 particles/ml, and preferably contains an exosome having a size of 30 to 40 nm.
- Exosomes derived from progenitor cells may have a size of 30 to 200 nm, specifically, may have an average size of 80 to 130 nm.
- the exosomes further contain CD18, CD53, CD87, CD98, and CD155 as CD markers, in addition to CD9, CD63, and CD81, single exosomes, double exosomes, and multiple exosomes. It is preferable to contain (multi exosome), multilayer exosome and compressed exosome (zip exosome).
- exosomes derived from progenitor cells with high concentration, high purity and uniform particle size distribution can be obtained in large quantities at a relatively low cost.
- the present invention can provide a large amount of safe and stable progenitor cell-derived exosomes, can be scaled up, and is also suitable for GMP (Good Manufacturing Practice).
- Figure 1a is a photograph showing the buffy coat layer after centrifugation of umbilical cord blood
- Figure 1b is a photograph showing the mononuclear cell layer after centrifuging the buffy coat layer by the Ficoll density gradient method
- Figure 1c is a photograph of mononuclear cells analyzed by LUNA STEM This is a graph showing the size distribution.
- Figure 2a is a graph showing the distribution of mononuclear cell size analyzed by an automatic fluorescent cell counter after freezing-thawing by cold shock
- Figure 2b is a microscopic photograph of progenitor cells attached to a cell culture flask
- Figure 2c is This is a photograph observed under a microscope after culturing the attached progenitor cells in a stabilizing medium for 2 to 4 weeks.
- FIG. 3 is a flowchart showing the process of separating the exosomes from the culture medium produced using the progenitor cells of the present invention.
- Figure 4a shows the results of the NTA analysis showing the particle size distribution of the exosomes before separation of the exosomes, Figure 4b after separation of the exosomes.
- Figure 5a shows the Western blot results of the exosomes obtained according to an embodiment of the present invention
- Figure 5b is a peptide distribution diagram according to the protein mass spectrometry of the exosomes obtained according to an embodiment of the present invention.
- Figure 6 shows the results of analyzing the exosomes obtained according to an embodiment of the present invention with an electron microscope.
- FIG. 7 is a photograph of the exosome obtained according to an embodiment of the present invention analyzed by cryogenic electron microscopy, FIG. 7a is a single exosome, FIG. 7b is a double exosome, FIG. 7c is a multiple exosome, and FIG. 7d is a multi-membrane. Exosomes, Figure 7e shows the compressed exosomes.
- FIG. 8 is a graph showing the density of exosomes by size as a result of TFF treatment of the exosomes obtained according to an embodiment of the present invention using a 500,000 Da hollow fiber filter
- FIG. 8a is a large group of exosomes
- FIG. 8b is a small exo It represents a small group.
- FIGS. 9 is a test result confirming that there is no toxicity by repeatedly administering the exosome obtained according to an embodiment of the present invention to the skin
- FIGS. 9a and b are body weight
- FIGS. 9c and d are feed intake
- FIGS. 9g and h are blood biochemical tests
- FIGS. 9i and j are test results for organ weight.
- the progenitor cells used in the method of the present invention may be progenitor cells derived from various tissues of origin.
- the progenitor cells include, but are not limited to, progenitor cells in umbilical cord blood, bone marrow, fat, umbilical cord, amniotic fluid, dental or peripheral blood.
- the method for culturing progenitor cells of the present invention can be applied to cord blood-derived progenitor cells.
- the progenitor cells include, but are not limited to, progenitor cells obtained from mammals including humans.
- the method for culturing progenitor cells of the present invention is applied to progenitor cells obtained from humans.
- the method for isolating the progenitor cells may be preferably performed using cold shock.
- the progenitor cell attachment method of the present invention uses isolated progenitor cells with fetal bovine serum (FBS), sodium pyruvate, non-essential amino acids, transferrin, and sodium selenite. And at least one selected from the group consisting of insulin (Insulin) is added Dulbecco's Modified Eagle Medium (DMEM: Dulbecco'2 Modified Eagle Medium), Minimal Essential Medium (MEM: Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium) and In a medium selected from the group consisting of DMEM/F12 medium, 5% CO 2 , and culturing at 37° C. conditions.
- the medium used in the above process is not limited to the commercial medium described above.
- the attachment medium may or may not contain serum as needed, and may contain knockout serum replacement instead of serum.
- the adherent progenitor cells are treated with vitamin B12, monosodium hypoxanthine, thymidine, glutamine dipeptide (L-alanyl-L-glutamine dipeptide), knockout serum replacement Dulbecco'2 Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), DMEM/F12 with one or more selected from (Knockout serum replacement) added (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) and DMEM/F-12 in a medium selected from the group consisting of Glutamax supplement medium, 5% CO 2 , and culturing under 37°C conditions.
- the medium used in the process is not limited to the commercial medium described above.
- Adhered progenitor cells can be stably cultured for up to 4-5 weeks according to the cell state by the culture method of the present invention, and the supernatant (hereinafter, culture solution) is recovered and used for exosome separation.
- the culture medium produced by the above culture method has a uniform particle size distribution and concentration of the exosomes, so there is no need to add additives such as other chemicals or small molecule substances in the separation process of the exosomes.
- the culture medium produced by the culture method of the present invention is filtered through 0.45 or 0.8 ⁇ m and 0.2 ⁇ m filters to remove impurities such as cell debris, and sterilization is performed before separating the exosomes by tangential flow filtration.
- a hollow fiber fiber can be used, and a material selected from among mPES (Modified Polyethersulfone), PS (Polysulfone), PES (Polyethersulfone) or ME (Mixed Cellulose Ester) may be used for the membrane material.
- mPES Modified Polyethersulfone
- PS Polysulfone
- PES Polyethersulfone
- ME Mated Cellulose Ester
- the molecular weight cutoff (MWCO) of the hollow fiber filter may be 10,000 Da, 50,000 Da, or 75,000 Da.
- the culture medium separates the exosomes using a mPES material, a hollow fiber filter with a molecular weight limit of 50,000Da.
- the membrane material of the hollow fiber filter used in the above process is not limited to the commercial hollow fiber filter described above.
- the exosomes separated by the method of the present invention are finally filtered and sterilized with a 0.2 ⁇ m sterile filter.
- the isolated exosomes ensure complete sterility by connecting 0.2 ⁇ m Sartopore 2 XLG Caps (purchased from Sartorius) to the port of a disposable assembly airtight container (purchased from Thermo Fisher Scientific) to ensure complete sterility and pump
- the separated exosomes are transported in an airtight container through a 0.2 ⁇ m sterile filter through a tube.
- the sterilization filter and the disposable assembly airtight container used in the above process are not limited to the above-mentioned commercial products.
- the present invention provides a new progenitor cell and a technology capable of obtaining a large amount of exosomes having a high concentration, high purity, and uniform size from a culture medium obtained by culturing the same.
- the present invention provides an exosome therapeutic agent comprising the exosome obtained by the above method.
- the exosome therapeutic agent of the present invention can be used to suppress and regulate immune cells infiltrating around blood vessels, and to repair and regenerate cells or tissues and blood vessels damaged by immune cells.
- the exosome therapeutic agent of the present invention can be used for inhibiting or treating dermatitis, arthritis, Alzheimer's, cerebral infarction and cerebral hemorrhage.
- the exosome therapeutic agent of the present invention can be used for the treatment of cardiovascular diseases or the central and peripheral nervous system.
- Cord blood from the donor is processed within 12 hours.
- the specific processing process is as follows. After the umbilical cord blood was transferred to a 50 ml conical tube, the conical tube was centrifuged for 30 minutes at a speed of 2000 rpm at room temperature using a centrifuge.
- the supernatant (plasma) shown in FIG. 1A is removed, and a white buffer layer (buffy coat layer) is collected.
- the buffer layer is slowly injected so that the ratio of Ficoll to the buffer layer is 2 : 1 in the 50ml conical tube containing the collected buffer layer.
- the conical tube in which Ficoll and the buffer layer were mixed was centrifuged for 30 minutes at a rotation speed of 2000 rpm at room temperature using a centrifuge.
- the supernatant (plasma) shown in FIG. 1B was removed and a white cell layer was collected.
- Fig. 1c is like the graph of
- the remaining mixture was centrifuged for 5 minutes at a rotation speed of 1500 rpm at room temperature using a centrifuge, then the supernatant was removed and DMEM containing 10% knockout serum replacement and 2 mM Glutamine After resuspending the cells in /F-12 medium, adding 10% dimethyl sulfoxide (DMSO), mixing thoroughly, dispensing into cryo vials, and placing them in a freezing container at -80°C in a deep refrigerator (deep freezer) for 24 hours.
- DMSO dimethyl sulfoxide
- Figure 2a is a graph showing the distribution of mononuclear cell size analyzed by an automatic fluorescent cell counter after freezing-thawing by cold shock
- Figure 2b is a microscopic photograph of progenitor cells attached to a cell culture flask
- Figure 2c is This is a photograph observed under a microscope after culturing the attached progenitor cells in a stabilizing medium for 2 to 4 weeks.
- the cell surface markers CD24, CD31, CD45RA, CD105, CD117, CD146, SSEA-1, and TRA-1-60 of the progenitor cells adhered by the method of the present invention were analyzed with a flow cytometer from Thermo Fisher Scientific, and the results are shown in Table 1 shown.
- the exosomes contained in the culture medium have a high concentration and uniform particle size distribution, so it is easy to separate the exosomes by a tangential flow filtration (TFF) system, and no additives such as chemicals or small molecule substances are added.
- the culture medium was pumped at a rate of 20ml/min and filtered through a filter (using Sartopore 2 XLG SartoScale 25 (0.8 ⁇ m, 0.2 ⁇ m)) to remove impurities such as cell debris, wastes and large particles, and sterilization was carried out.
- the filter-sterilized culture solution was immediately stored frozen or stored in a -20°C freezer and then thawed, and then exosomes were separated using a tangential flow filtration device (TFF).
- concentration and diafiltration were performed by a tangential flow filtration method.
- a hollow fiber filter purchased from Repligen was used as a filter for tangential flow filtration.
- various materials and molecular weight cutoff (MWCO) can be selected according to the properties of the exosomes and the purpose of filtration. Exosomes were selectively separated by the selected molecular weight limit, and all particles, proteins, lipids, nucleic acids, and low molecular weight compounds smaller than the selected molecular weight limit were removed.
- mPES Modified Polyethersulfone
- PS Polysulfone
- PES Polyethersulfone
- ME Mated Cellulose Ester
- 1xPBS without Ca 2+ and Mg 2+ ) (purchased from Corning) was used, and no chemicals or low molecular weight substances were added.
- the exosomes subjected to concentration and diafiltration by tangential flow filtration were diluted by adding a stabilization buffer 10 to 25 times so that the volume of the exosomes became the same as the starting volume.
- exosomes isolated by the method of the present invention are finally filter-sterilized with a 0.2 ⁇ m filter (Sartopore 2 XLG Caps, purchased from Sartorius), and complete sterility is achieved through the port of the disposable assembly sealed container immediately following (purchased from Thermo Fisher Scientific). Transported in a guaranteed sealed container.
- a 0.2 ⁇ m filter Stemopore 2 XLG Caps, purchased from Sartorius
- Exosomes having a uniform particle size of 30 to 200 nm were isolated from the culture medium by the method described above.
- Figure 5a is a result of performing a Western blot on the exosome isolated according to the method of an embodiment of the present invention, it was confirmed the presence of the known exosome markers CD9 and CD63. Antibodies for each marker were purchased from BD Biosciences and used.
- #1 is a mesenchymal stem cell-derived exosome
- #2 is a Western blot result of the exosome produced in the present invention.
- Figure 5b is a peptide analysis by mass spectrometry (using Orbitrap LC-MS analyzer) after extracting proteins from exosomes isolated according to an embodiment of the present invention and labeling them with TMTsixplex Isobaric Label Reagent (purchased from Thermo Fisher Scientific). It is a distribution diagram and Table 2 below is a list of CD-related proteins among the analysis results.
- Table 3 shows the concentrations of cytokines EGF, bFGF, IL-8, MCP-1 (purchased from Thermo Fisher Scientific, R&D systems) present in exosomes purified by tangential flow filtration according to the method of an embodiment of the present invention by ELISA The results confirmed by the kit (Enzyme-Linked Immunosorbent Assay Kit) are shown.
- the exosome production technology of the present invention can economically and efficiently mass-produce exosomes with high concentration, high purity and uniform particle size distribution only by tangential flow filtration without the need for special additives or processes.
- all processes such as cell culture, culture solution filtration, and exosome separation and sterilization through tangential flow filtration of an embodiment of the present disease can be scaled up and are set appropriately for good manufacturing practice (GMP) knew it could be done.
- TEM electron microscope
- Cryo-EM cryogenic electron microscope
- Fig. 6 shows the results of electron micrographs analyzed by staining with negative staining
- Fig. 7 shows the results of rapid freezing and analysis with cryogenic electron microscopy.
- Figure 7a is a single exosome
- Figure 7b is a double exosome
- Figure 7c is a multiple exosome
- Figure 7d is a multi-membrane exosome
- Figure 7e is a compressed exosome.
- the exosomes of the present invention are intensively distributed in the size of 30-150 nm, and the exosomes of the corresponding size are not only single exosomes, but also double exosomes and multiple exosomes. exosome), multilayer exosomes, and compressed exosomes were found to exist in various forms. This proves that the properties of the exosomes obtained by the method of the present invention are different from other exosomes.
- the concentration and size distribution were measured by thawing the exosomes stored at -20 ⁇ 5° C. for 12 months (once/month).
- the concentration and size distribution of exosomes were measured with a nanoparticle tracking analyzer, and the results are shown in Table 4 below.
- the exosome technology of the present invention is a technology capable of producing exosomes with secured stability.
- rats were treated with 500, 1000 and 2000 mg/kg/day of test substances and 4 groups of control (water for injection), each of 10 sexes per group.
- the animals were transdermally administered for 13 weeks. Body weight and feed intake were measured for 13 weeks. After the observation period, hematological examination, blood biochemical examination, organ weight measurement, and macroscopic examination and histopathological examination were performed at autopsy.
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Abstract
The present invention relates to a method of obtaining progenitor cells by treating mononuclear cells isolated from tissues or cells with cold shock, and to production of exosomes by culturing the progenitor cells, and the cold-shock method of the present invention can produce human-derived progenitor cells which are uniform in size and have a stable exosome secretion activity. The exosomes produced by the present invention are distributed in a high concentration, preponderantly on a particular size, and can be used directly with a tangential-flow filtration technique without additives, and may be advantageously utilized to increase purity and yields to produce exosomes of high concentration, high purity, and high yield.
Description
본 발명은 세포 또는 조직에서 분리한 단핵세포를 한랭쇼크로 처리하여 전구세포를 획득하는 방법과 획득된 전구세포를 배양하여 엑소좀을 생산하는 방법에 관한 것으로서, 보다 상세하게는 크기가 균일하고 분비활성이 안정된 인체 유래 전구세포를 획득하는 방법에 관한 것이다.The present invention relates to a method for obtaining progenitor cells by treating mononuclear cells isolated from cells or tissues with cold shock, and a method for culturing the obtained progenitor cells to produce exosomes, and more particularly, to a method of producing exosomes by culturing the obtained progenitor cells. It relates to a method for obtaining human-derived progenitor cells with stable activity.
엑소좀에 대한 연구는 중간엽줄기세포, 유도만능줄기세포와 293T 세포주에 대부분 집중되어 있다. 중간엽줄기세포와 유도만능줄기세포 유래 엑소좀은 그 효능에 대한 연구에 초점이 맞춰져 있고, 293T 세포 유래 엑소좀은 항암물질 등을 운반하는 카고(cargo) 연구에 초점이 맞춰져 있다. 즉, 엑소좀을 카고(cargo)가 아닌 바이오제약 물질로 사용하기 위한 연구는 두 세포에 국한되어 있다. Research on exosomes is mostly focused on mesenchymal stem cells, induced pluripotent stem cells, and the 293T cell line. Mesenchymal stem cells and induced pluripotent stem cell-derived exosomes are focused on research on their efficacy, and 293T cell-derived exosomes are focused on cargo research that transports anticancer substances. In other words, research to use exosomes as biopharmaceutical materials rather than cargo is limited to two cells.
하지만 엑소좀의 성질을 결정하는 하나의 요소가 엑소좀이 유래한 세포이다. 중간엽줄기세포가 분비하는 엑소좀은 중간엽줄기세포의 특성을 나타내고 유도만능줄기세포가 분비하는 엑소좀은 유도만능줄기세포의 특성을 나타낸다. 중간엽줄기세포의 임상적인 효능에 대한 연구가 많이 진행되고 있지만 뛰어난 in vitro 결과에 비해 in vivo 효과가 미미하다. 유도만능줄기세포는 직접적으로 in vivo에 적용하기보다 특정 전구세포 또는 조직으로 분화하여야 그 효과를 나태내기에 엑소좀을 생산하기 위해서는 분화라는 과정이 필요하다는 점에서 비효율적이다. 따라서, 엑소좀을 생산할 수 있는 새로운 세포를 확보하는 것이 아주 중요하다. 그러나 중간엽줄기세포와 유도만능줄기세포 외의 다른 세포로 엑소좀을 연구한다는 보고는 거의 없다. However, one factor that determines the properties of exosomes is the cell from which the exosomes are derived. The exosomes secreted by the mesenchymal stem cells show the characteristics of mesenchymal stem cells, and the exosomes secreted by the induced pluripotent stem cells show the characteristics of the induced pluripotent stem cells. Although many studies have been conducted on the clinical efficacy of mesenchymal stem cells, the in vivo effects are insignificant compared to the excellent in vitro results. Induced pluripotent stem cells must be differentiated into specific progenitor cells or tissues rather than directly applied in vivo to show their effects, so it is inefficient in that a process called differentiation is required to produce exosomes. Therefore, it is very important to secure new cells capable of producing exosomes. However, there are few reports of studying exosomes with cells other than mesenchymal stem cells and induced pluripotent stem cells.
한편, 엑소좀은 엔도솜(endosome)에서 생성되는 30-150nm 크기의 막으로 둘러싸인 소낭체이다. 생리학적 조건에서 모든 생물체의 세포는 엑소좀를 분비한다. 미세소포(microvesicals), 세포자멸사 소체(apoptotic bodies) 등 기타 세포외 소낭체들의 크기도 엑소좀과 비슷하지만 생물발생 경로가 다르다. 엑소좀의 가장 큰 역할은 카고(cargo) 즉 물질운반체로서 단백질, 지질, mRNA, microRNA, long non-coding RNA 등 핵산정보를 운반한다. 생리학적 조건에서 엑소좀은 지속적으로 세포와 세포사이를 오가면서 이런 정보를 전달하여 세포들의 교류에 중요한 역할을 한다. 특히 엑소좀이 운반하는 물질 중에서 microRNA들이 가장 중요하다는 것이 밝혀졌고 엑소좀이 생리학적 또는 치료 효능을 나타낼 수 있는 핵심이다(Nature Reviews Neurology 15, 193-203(2019)). On the other hand, exosomes are vesicles surrounded by a membrane with a size of 30-150 nm generated from endosomes. Under physiological conditions, cells of all living organisms secrete exosomes. The size of other extracellular vesicles, such as microvesicals and apoptotic bodies, is similar to that of exosomes, but the biogenesis pathway is different. The biggest role of the exosome is as a cargo, that is, a material carrier, which transports nucleic acid information such as proteins, lipids, mRNA, microRNA, and long non-coding RNA. Under physiological conditions, exosomes play an important role in cell-to-cell communication by continuously passing these information back and forth between cells. In particular, it has been found that microRNAs are the most important among substances transported by exosomes, and are the core that exosomes can exhibit physiological or therapeutic efficacy (Nature Reviews Neurology 15, 193-203 (2019)).
엑소좀의 특성은 유래 세포, 엑소좀의 크기, 농도, 탑재한 핵산정보 등에 따라 다르다. 현존하는 인체 유래 엑소좀을 제조하는 연구들은 모두 중간엽줄기세포 또는 293T 세포를 사용한다. 이런 세포를 사용하여 제조한 엑소좀의 평균 크기가 대략 200nm 정도로서 엑소좀이 정의한 크기범위에서 벗어나는 경우가 많고, 세포 자체가 분비하는 엑소좀의 농도가 낮아 분리 및 분리과정에서 등 복잡한 과정을 거쳐야 하기에 생산양이 제한적이고 생산단가가 높다. 또한, 엑소좀의 평균크기가 대략 200nm 정도로 그 크기가 상당히 크기에 0.2μm 여과필터를 사용하여 여과멸균할 때 엑소좀 손실이 크고 필터의 구멍(pore)이 막히기에 여과멸균을 진행하는데 어려움이 있다. 위에 기술한 문제들은 현존하는 엑소좀을 임상연구에 적용하기 어렵게 만들었다. The characteristics of exosomes differ depending on the derived cells, the size, concentration, and loaded nucleic acid information of the exosomes. Existing studies of producing exosomes derived from human beings all use mesenchymal stem cells or 293T cells. The average size of the exosomes produced using these cells is about 200 nm, which is often outside the size range defined by the exosomes, and the concentration of exosomes secreted by the cells itself is low, so complex processes such as separation and separation are required. The production volume is limited and the production cost is high. In addition, the average size of the exosomes is about 200 nm, the size is quite large, and when filtration sterilization using a 0.2 μm filter filter, the loss of exosomes is large and the pores of the filter are clogged, so it is difficult to proceed with filtration sterilization. . The problems described above made it difficult to apply the existing exosomes to clinical research.
본 발명자들은 상기 세포들과 엑소좀 생산의 단점을 개선하기 위하여 연구한 결과, 새로운 특이성 전구세포를 획득하는 방법을 발명하였고, 해당 세포로 크기분포가 균일하고 농도가 높은 엑소좀을 제조하여 본 발명을 완성하였다. As a result of the study to improve the disadvantages of the above cells and exosome production, the present inventors invented a method for obtaining a new specific progenitor cell, and prepared exosomes with uniform size distribution and high concentration from the cells in the present invention was completed.
이에, 본 발명은 상기한 사정을 감안하여 창출된 것으로, 새로운 전구세포를 분리하는 방법을 제공하고 해당 전구세포를 배양하여 특정 크기의 엑소좀을 고농도로 획득하는 방법을 제공함에 그 기술적 목적이 있다.Accordingly, the present invention was created in view of the above circumstances, and its technical purpose is to provide a method for isolating new progenitor cells and to provide a method for culturing the progenitor cells to obtain exosomes of a specific size at a high concentration. .
또한, 본 발명은 본 발명의 엑소좀은 추가적인 첨가물 및 처리과정이 필요 없고 농축 및 정용여과후 안정화 버퍼를 추가하여 엑소좀의 부피가 다시 시작 부피와 똑같게 되게끔 희석하는 과정만으로 분리가 가능한 방법을 제공하는 것이 또 다른 목적이다.In addition, the present invention provides a method capable of separating the exosomes of the present invention only by diluting the exosomes so that the volume of the exosomes becomes the same as the starting volume again by adding a stabilization buffer after concentration and diafiltration without additional additives and processing steps. It is another purpose to provide.
상기 목적을 달성하기 위한 본 발명의 일측면에 따르면, 단핵세포(mononuclear cell)를 일정 조건에서 냉동한 후 해동하는 한랭쇼크 방법으로 전구세포를 획득하는 것을 특징으로 하는 한랭쇼크를 이용한 전구세포 획득방법이 제공된다.According to one aspect of the present invention for achieving the above object, a method for obtaining progenitor cells using cold shock, characterized in that the progenitor cells are obtained by a cold shock method in which mononuclear cells are frozen under certain conditions and then thawed. this is provided
여기서, 상기 획득된 전구세포는 다음의 조건을 만족하는 것이 바람직하다. Here, the obtained progenitor cells preferably satisfy the following conditions.
a) 세포를 냉동할 때 사용하는 조성물은 글루타민(L-glutamine) 또는 L-글루타민의 dipeptide형태(L-alanyl-L-glutamine dipeptide), DMSO(Dimethyl sulfoxide) 및 녹아웃 혈청 대체물(Knockout serum replacement)로 이루어진 군에서 선택된 하나 이상이 첨가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove's Modified Dulbecco's Medium), DMEM/F-12(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 또는 DMEM/F-12 Glutamax supplement 배지를 포함하는 배지,a) The composition used when freezing cells is glutamine (L-glutamine) or L-glutamine dipeptide form (L-alanyl-L-glutamine dipeptide), DMSO (dimethyl sulfoxide) and knockout serum replacement. At least one selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM: Dulbecco'2 Modified Eagle Medium), Minimal Essential Medium (MEM: Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), DMEM/F-12 ( Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) or a medium containing DMEM/F-12 Glutamax supplement medium;
b) 세포배양 플라스크에 부착되는 선형 또는 구형의 세포, b) linear or spherical cells attached to the cell culture flask;
c) CD24, CD117 또는 SSEA-1에 대하여 음성의 면역학적 특성을 나타냄, 또는c) is immunologically negative for CD24, CD117 or SSEA-1, or
d) CD31, CD45RA, CD105, CD146 또는 TRA-1-60에 대하여 양성의 면역학적 특성을 나타냄.d) positive immunological properties for CD31, CD45RA, CD105, CD146 or TRA-1-60.
그리고, 상기 전구세포가 제대혈, 골수, 지방, 탯줄, 양수, 치아 또는 말초혈액에서 유래된 것일 수 있다.In addition, the progenitor cells may be derived from umbilical cord blood, bone marrow, fat, umbilical cord, amniotic fluid, dental or peripheral blood.
상기 목적을 달성하기 위한 본 발명의 다른 일측면에 따르면, (a) 단핵세포를 일정 조건에서 냉동한 후 해동하는 한랭쇼크 방법으로 전구세포를 획득하는 단계, (b) 제1배지에서 상기 전구세포를 배양하여 세포배양 플라스크에 부착시키는 단계, (c) (b)의 결과물을 제2배지에서 상기 전구세포를 1주 내지 5주 동안 배양하는 단계, (d) (c)의 결과물이 분비하는 엑소좀을 수집하는 단계, 및 (e) 상기 수집된 엑소좀을 분리하는 단계를 포함하는 것을 특징으로 하는 엑소좀 생산방법 및 상기 방법에 의해 생산된 엑소좀이 제공된다.According to another aspect of the present invention for achieving the above object, (a) obtaining progenitor cells by a cold shock method in which mononuclear cells are frozen under certain conditions and then thawed, (b) the progenitor cells in a first medium culturing and attaching to a cell culture flask, (c) culturing the progenitor cells in a second medium for the result of (b) for 1 to 5 weeks, (d) exo secreted by the result of (c) There is provided a method for producing exosomes, characterized in that it comprises the steps of collecting some, and (e) separating the collected exosomes, and exosomes produced by the method.
여기서, 전구세포를 부착할 때 사용되는 제1배지는 글루타민(L-glutamine) 또는 L-글루타민의 dipeptide형태(L-alanyl-L-glutamine dipeptide), 우태아혈청(FBS), 피루브산 나트륨(Sodium pyruvate), 불필수아미노산(Non-essential amino acids), 트랜스페린(Transferrin), 소듐 셀레나이트(Sodium selenite) 및 인슐린(Insulin)으로 이루어진 군에서 선택된 하나 이상이 추가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), Advanced DMEM, 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove’s Modified Dulbecco’s Medium) 또는 DMEM/F12 배지를 포함할 수 있다.Here, the first medium used for attaching the progenitor cells is glutamine (L-glutamine) or a dipeptide form of L-glutamine (L-alanyl-L-glutamine dipeptide), fetal bovine serum (FBS), sodium pyruvate (Sodium pyruvate) ), non-essential amino acids, transferrin, sodium selenite and insulin, Dulbecco's Modified Eagle Medium (DMEM: Dulbecco') 2 Modified Eagle Medium), Advanced DMEM, Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), or DMEM/F12 medium.
그리고, 전구세포를 배양할 때 사용하는 제2배지는 글루타티온 모노소듐(Glutathione monosodium), 비타민 B12, 모노소듐 하이포크산틴(Monosodium hypoxanthine), 티미딘(Thymidine), 글루타민(L-glutamine) 또는 L-글루타민의 디펩타이드 형태(L-alanyl-L-glutamine dipeptide), 녹아웃 혈청 대체물(Knockout serum replacement)으로 이루어진 군에서 선택된 하나 이상이 추가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove's Modified Dulbecco's Medium), DMEM/F12(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 또는 DMEM/F-12 Glutamax supplement 배지를 포함할 수 있다.And, the second medium used for culturing the progenitor cells is glutathione monosodium, vitamin B12, monosodium hypoxanthine, thymidine, glutamine or L-glutamine. of the dipeptide form (L-alanyl-L-glutamine dipeptide), and at least one selected from the group consisting of knockout serum replacement (DMEM: Dulbecco'2 Modified Eagle Medium), at least Essential medium (MEM: Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), DMEM/F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) or DMEM/F-12 Glutamax supplement medium may be included.
그리고, 상기 (e) 단계는, (e-1) 전구세포 배양액을 0.45 또는 0.8μm 필터를 통과하여 세포 잔해물, 노폐물 및 거대 입자를 여과하는 단계, (e-2) 상기 여과된 배양액을 멸균 필터로 멸균하는 단계, (e-3) 상기 멸균된 배양액을 접선유동여과를 이용하여 농축과 정용여과 과정으로 엑소좀을 분리하는 단계, (e-4) 상기 분리된 엑소좀에 안정화 버퍼를 추가하여 초기 부피와 동일한 부피가 되도록 희석하는 단계, (e-5) 상기 희석된 엑소좀을 액체 교환 시스템을 이용하여 멸균 필터를 통과시켜 멸균처리하는 단계 및 (e-6) 상기 멸균처리된 엑소좀을 밀봉된 저장용기에 저장하는 단계를 포함하는 것이 바람직하다.And, the step (e) is, (e-1) passing the progenitor cell culture solution through a 0.45 or 0.8 μm filter to filter cell debris, wastes and large particles, (e-2) filtering the filtered culture solution through a sterile filter sterilizing with, (e-3) separating the exosomes by concentration and diafiltration of the sterilized culture solution using tangential flow filtration, (e-4) adding a stabilization buffer to the separated exosomes Diluting to the same volume as the initial volume, (e-5) sterilizing the diluted exosomes through a sterile filter using a liquid exchange system, and (e-6) sterilizing the sterilized exosomes It is preferred to include the step of storing in a sealed storage container.
여기서, 상기 접선유동여과를 위하여, 분자량 한계치가 10,000Da, 50,000Da 또는 75,000Da 중 어느 하나인 중공사 필터를 사용하는 것이 바람직하고, 상기 (e-3) 단계의 농축과 정용여과는 접선유동여과를 단속 또는 연속적으로 진행하고, 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과 과정은 농축된 부피에 대하여 적어도 10배의 부피를 갖는 버퍼로 여과하는 과정을 포함하는 것이 바람직하다.Here, for the tangential flow filtration, it is preferable to use a hollow fiber filter having a molecular weight limit of any one of 10,000Da, 50,000Da, or 75,000Da, and the concentration and diafiltration in step (e-3) are tangential flow filtration is performed intermittently or continuously, the concentration is concentrated 10 to 25 times with respect to the starting volume, and the diafiltration process preferably includes filtration with a buffer having at least 10 times the volume of the concentrated volume.
또한, 엑소좀 희석은 농축 및 정용여과된 부피에 대하여 10배 내지 25배의 안정화 버퍼를 첨가하여 전체 부피가 시작 부피와 동일하도록 희석하는 과정을 포함하는 것이 바람직하다.In addition, the exosome dilution preferably includes a process of diluting the total volume to the same as the starting volume by adding a stabilization buffer 10 to 25 times the concentration and diafiltration volume.
상기 엑소좀은 농도가 3 x 109 particles/ml 내지 5 x 109 particles/ml의 농도를 가지고, 30 내지 40nm 크기의 엑소좀을 함유하고 있는 것이 바람직하다. 전구세포로부터 유래하는 엑소좀은 크기가 30 내지 200nm일 수 있으며, 구체적으로 평균 80 내지 130nm 크기를 가질 수 있다.The exosome has a concentration of 3 x 10 9 particles/ml to 5 x 10 9 particles/ml, and preferably contains an exosome having a size of 30 to 40 nm. Exosomes derived from progenitor cells may have a size of 30 to 200 nm, specifically, may have an average size of 80 to 130 nm.
또한, 상기 엑소좀은 CD마커로서, CD9, CD63, CD81 외에 CD18, CD53, CD87, CD98, CD155를 더 함유하고 있고, 단일 엑소좀(single exosome), 이중 엑소좀(double exosome), 다중 엑소좀(multi exosome), 다중막 엑소좀(multilayer exosome) 및 압축 엑소좀(zip exosome)을 함유하고 있는 것이 바람직하다.In addition, the exosomes further contain CD18, CD53, CD87, CD98, and CD155 as CD markers, in addition to CD9, CD63, and CD81, single exosomes, double exosomes, and multiple exosomes. It is preferable to contain (multi exosome), multilayer exosome and compressed exosome (zip exosome).
본 발명에 의하면, 크기가 균일한 전구세포를 분리하고 이를 특정한 조건하에서 부착 및 배양하여 고농도, 고순도이면서 입자크기 분포가 균일한 전구세포 유래의 엑소좀을 상대적으로 낮은 원가에 대량으로 수득할 수 있다. 또한, 최종 단계에서 멸균필터를 통과하여 여과멸균을 할 수 있어 임상적용에 가장 중요한 엑소좀의 멸균문제를 해결하였다. 본 발명은 안전하고 안정적인 전구세포 유래의 엑소좀을 대량으로 제공할 수 있고 스케일업(scale up)이 가능하고 GMP(Good Manufacturing Practice)에도 적합하다. According to the present invention, by separating progenitor cells having a uniform size and attaching and culturing them under specific conditions, exosomes derived from progenitor cells with high concentration, high purity and uniform particle size distribution can be obtained in large quantities at a relatively low cost. . In addition, it was possible to filter sterilize by passing through a sterilization filter in the final step, thereby solving the sterilization problem of exosomes, which is the most important for clinical application. The present invention can provide a large amount of safe and stable progenitor cell-derived exosomes, can be scaled up, and is also suitable for GMP (Good Manufacturing Practice).
도 1a는 제대혈을 원심분리한 후 buffy coat층을 나타낸 사진이고, 도 1b는 buffy coat층을 Ficoll density gradient 방법으로 원심분리한 후 단핵세포층을 나타낸 사진이고, 도 1c는 LUNA STEM으로 분석한 단핵세포 크기의 분포를 나타낸 그래프이다. Figure 1a is a photograph showing the buffy coat layer after centrifugation of umbilical cord blood, Figure 1b is a photograph showing the mononuclear cell layer after centrifuging the buffy coat layer by the Ficoll density gradient method, Figure 1c is a photograph of mononuclear cells analyzed by LUNA STEM This is a graph showing the size distribution.
도 2a는 한랭쇼크로 냉동-해동한 후 자동 형광세포 카운터로 분석한 단핵세포 크기의 분포를 나타낸 그래프이고, 도 2b는 세포 배양 플라스크에 부착된 전구세포를 현미경으로 관찰한 사진이고, 도 2c는 부착된 전구세포를 안정화 배지에서 2~4주 배양한 후 현미경으로 관찰한 사진이다.Figure 2a is a graph showing the distribution of mononuclear cell size analyzed by an automatic fluorescent cell counter after freezing-thawing by cold shock, Figure 2b is a microscopic photograph of progenitor cells attached to a cell culture flask, Figure 2c is This is a photograph observed under a microscope after culturing the attached progenitor cells in a stabilizing medium for 2 to 4 weeks.
도 3은 본 발명의 전구세포를 이용하여 생산한 배양액에서 엑소좀을 분리하는 과정을 나타낸 흐름도이다.3 is a flowchart showing the process of separating the exosomes from the culture medium produced using the progenitor cells of the present invention.
도 4a는 엑소좀을 분리하기 전, 도 4b는 엑소좀을 분리한 후 엑소좀의 입자 크기 분포를 나타내는 NTA분석 결과를 나타낸 것이다.Figure 4a shows the results of the NTA analysis showing the particle size distribution of the exosomes before separation of the exosomes, Figure 4b after separation of the exosomes.
도 5a는 본 발명의 일 실시예에 따라 얻어진 엑소좀의 웨스턴 블랏 결과를 나타낸 것이고, 도 5b는 본 발명의 일 실시예에 따라 얻어진 엑소좀의 단백질 질량분석에 따른 펩타이드 분포도이다.Figure 5a shows the Western blot results of the exosomes obtained according to an embodiment of the present invention, Figure 5b is a peptide distribution diagram according to the protein mass spectrometry of the exosomes obtained according to an embodiment of the present invention.
도 6은 본 발명의 일 실시예에 따라 얻어진 엑소좀을 전자현미경으로 분석한 결과를 나타낸 것이다.Figure 6 shows the results of analyzing the exosomes obtained according to an embodiment of the present invention with an electron microscope.
도 7은 본 발명의 일 실시예에 따라 얻어진 엑소좀을 초저온전자현미경으로 분석한 사진으로, 도 7a는 단일 엑소좀, 도 7b는 이중 엑소좀, 도 7c는 다중 엑소좀, 도 7d는 다중막 엑소좀, 도 7e는 압축 엑소좀을 나타낸 것이다.7 is a photograph of the exosome obtained according to an embodiment of the present invention analyzed by cryogenic electron microscopy, FIG. 7a is a single exosome, FIG. 7b is a double exosome, FIG. 7c is a multiple exosome, and FIG. 7d is a multi-membrane. Exosomes, Figure 7e shows the compressed exosomes.
도 8은 본 발명의 일 실시예에 따라 얻어진 엑소좀을 500,000Da hollow fiber filter를 이용하여 TFF 처리한 결과 엑소좀의 사이즈별 밀도를 나타낸 그래프로서, 도 8a는 큰 엑소좀군을 도 8b는 작은 엑소좀군을 나타낸 것이다. 8 is a graph showing the density of exosomes by size as a result of TFF treatment of the exosomes obtained according to an embodiment of the present invention using a 500,000 Da hollow fiber filter, FIG. 8a is a large group of exosomes, FIG. 8b is a small exo It represents a small group.
도 9는 본 발명의 일 실시예에 따라 얻어진 엑소좀을 경피에 반복투여하여 독성이 없음을 확인한 시험결과로서, 도 9a, b는 체중, 도 9c, d는 사료섭취량, 도 9e, f는 혈액학적 검사, 도 9g, h는 혈액생화학적 검사, 도 9i, j는 장기중량에 대한 시험결과이다.9 is a test result confirming that there is no toxicity by repeatedly administering the exosome obtained according to an embodiment of the present invention to the skin, FIGS. 9a and b are body weight, FIGS. 9c and d are feed intake, and FIGS. Physical examination, FIGS. 9g and h are blood biochemical tests, and FIGS. 9i and j are test results for organ weight.
본 발명에 기재된 실시예 및 도면에 도시된 구성은 본 발명의 바람직한 실시예에 불과할 뿐이고, 본 발명의 기술적 사상을 모두 표현하는 것은 아니므로, 본 발명의 권리범위는 본문에 설명된 실시예 및 도면에 의하여 제한되는 것으로 해석되어서는 아니 된다. 즉, 실시예는 다양한 변경이 가능하고 여러 가지 형태를 가질 수 있으므로 본 발명의 권리범위는 기술적 사상을 실현할 수 있는 균등물들을 포함하는 것으로 이해되어야 한다. 또한, 본 발명에서 제시된 목적 또는 효과는 특정 실시예가 이를 전부 포함하여야 한다거나 그러한 효과만을 포함하여야 한다는 의미는 아니므로, 본 발명의 권리범위는 이에 의하여 제한되는 것으로 이해되어서는 아니 될 것이다.The configuration shown in the embodiments and drawings described in the present invention is only a preferred embodiment of the present invention, and does not express all the technical ideas of the present invention, so the scope of the present invention is the embodiment and drawings described in the text should not be construed as being limited by That is, since the embodiment may have various changes and may have various forms, it should be understood that the scope of the present invention includes equivalents capable of realizing the technical idea. In addition, since the object or effect presented in the present invention does not mean that a specific embodiment should include all of them or only such effects, it should not be understood that the scope of the present invention is limited thereby.
여기서 사용되는 모든 용어들은 다르게 정의되지 않는 한, 본 발명이 속하는 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가진다. 일반적으로 사용되는 사전에 정의되어 있는 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 것으로 해석되어야 하며, 본 발명에서 명백하게 정의하지 않는 이상적이거나 과도하게 형식적인 의미를 지니는 것으로 해석될 수 없다.All terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs, unless otherwise defined. Terms defined in general used in the dictionary should be interpreted as having the meaning consistent with the context of the related art, and cannot be interpreted as having an ideal or excessively formal meaning not explicitly defined in the present invention.
본 발명의 방법에 사용되는 전구세포는 다양한 기원 조직으로부터 유래된 전구세포일 수 있다. 상기 전구세포의 예로는 제대혈, 골수, 지방, 탯줄, 양수, 치아 또는 말초혈액 등에서 전구세포를 들 수 있으나, 이에 제한되지 않는다. 본 발명의 바람직한 실시예에서, 본 발명의 전구세포의 배양방법은 제대혈 유래 전구세포에 적용될 수 있다. 상기 전구세포의 예로는 인간을 포함한 포유동물로부터 수득한 전구세포를 들 수 있으나, 이에 제한되지는 않는다. 본 발명의 바람직한 실시예에서, 본 발명의 전구세포의 배양방법은 인간으로부터 수득한 전구세포에 적용된다.The progenitor cells used in the method of the present invention may be progenitor cells derived from various tissues of origin. Examples of the progenitor cells include, but are not limited to, progenitor cells in umbilical cord blood, bone marrow, fat, umbilical cord, amniotic fluid, dental or peripheral blood. In a preferred embodiment of the present invention, the method for culturing progenitor cells of the present invention can be applied to cord blood-derived progenitor cells. Examples of the progenitor cells include, but are not limited to, progenitor cells obtained from mammals including humans. In a preferred embodiment of the present invention, the method for culturing progenitor cells of the present invention is applied to progenitor cells obtained from humans.
본 발명의 방법에서 상기 전구세포의 분리방법은 바람직하게 한랭쇼크를 이용하여 수행될 수 있다. In the method of the present invention, the method for isolating the progenitor cells may be preferably performed using cold shock.
본 발명의 전구세포 부착방법은 분리된 전구세포를 우태아혈청(FBS), 피루브산 나트륨(Sodium pyruvate), 불필수아미노산(Non-essential amino acids), 트랜스페린(Transferrin), 소듐 셀레나이트(Sodium selenite) 및 인슐린(Insulin)으로 이루어진 군에서 선택된 하나 이상이 추가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove’s Modified Dulbecco’s Medium) 및 DMEM/F12배지로 이루어진 군에서 선택된 배지에서 5% CO2, 37℃ 조건 하에서 배양하는 단계이다. 상기 과정에서 사용되는 배지는 위에 기술한 상업용 배지에 제한되는 것은 아니다. 또한 상기 부착용 배지는 필요에 따라 혈청을 포함하거나 포함하지 않을 수 있고 혈청 대신에 대체물(Knockout serum replacement)을 포함할 수 있다. The progenitor cell attachment method of the present invention uses isolated progenitor cells with fetal bovine serum (FBS), sodium pyruvate, non-essential amino acids, transferrin, and sodium selenite. And at least one selected from the group consisting of insulin (Insulin) is added Dulbecco's Modified Eagle Medium (DMEM: Dulbecco'2 Modified Eagle Medium), Minimal Essential Medium (MEM: Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium) and In a medium selected from the group consisting of DMEM/F12 medium, 5% CO 2 , and culturing at 37° C. conditions. The medium used in the above process is not limited to the commercial medium described above. In addition, the attachment medium may or may not contain serum as needed, and may contain knockout serum replacement instead of serum.
본 발명의 전구세포 배양방법은 부착된 전구세포를 비타민 B12, 모노소듐 하이포크산틴(Monosodium hypoxanthine), 티미딘(Thymidine), 글루타민의 디펩타이드 형태(L-alanyl-L-glutamine dipeptide), 녹아웃 혈청 대체물(Knockout serum replacement)에서 선택된 하나 이상이 추가된 둘베코변형 이글 배지(DMEM: Dulbecco'2 Modified Eagle Medium), 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove's Modified Dulbecco's Medium), DMEM/F12(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 및 DMEM/F-12 Glutamax supplement 배지로 이루어진 군에서 선택된 배지에서 5% CO2, 37℃ 조건하에서 배양하는 단계이다. 해당 과정에서 사용되는 배지는 위에 기술한 상업용 배지에 제한되는 것은 아니다. In the progenitor cell culture method of the present invention, the adherent progenitor cells are treated with vitamin B12, monosodium hypoxanthine, thymidine, glutamine dipeptide (L-alanyl-L-glutamine dipeptide), knockout serum replacement Dulbecco'2 Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), DMEM/F12 with one or more selected from (Knockout serum replacement) added (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) and DMEM/F-12 in a medium selected from the group consisting of Glutamax supplement medium, 5% CO 2 , and culturing under 37°C conditions. The medium used in the process is not limited to the commercial medium described above.
본 발명의 배양방법으로 부착된 전구세포를 세포의 상태에 따라 최대 4-5주까지 안정적으로 배양할 수 있으며, 그 동안 상층액(이하, 배양액)을 회수하여 엑소좀 분리에 사용한다. 상기 배양방법으로 생산한 배양액은 엑소좀의 입자크기 분포와 농도가 균일하여 엑소좀의 분리과정에 기타 화학물질 또는 소분자 물질 등 첨가물을 추가할 필요가 없다. Adhered progenitor cells can be stably cultured for up to 4-5 weeks according to the cell state by the culture method of the present invention, and the supernatant (hereinafter, culture solution) is recovered and used for exosome separation. The culture medium produced by the above culture method has a uniform particle size distribution and concentration of the exosomes, so there is no need to add additives such as other chemicals or small molecule substances in the separation process of the exosomes.
본 발명의 배양방법으로 생산한 배양액은 0.45 또는 0.8μm 및 0.2μm 필터로 여과하여 세포 잔해물 등 불순물을 제거하고 접선유동여과 방식으로 엑소좀을 분리하기 전에 멸균을 진행한다. 본 발명의 배양양액에서 엑소좀을 분리하기 위하여 중공사 섬유를 사용할 수 있으며, 맴브레인 재질은 mPES(Modified Polyethersulfone), PS(Polysulfone), PES(Polyethersulfone) 또는 ME(Mixed Cellulose Ester) 중 선택된 재질이 사용될 수 있다. 또한 중공사 필터의 분자량 한계치(MWCO : molecular weight cutoff)는 10,000 Da, 50,000Da 또는 75,000Da을 사용할 수 있다. 본 발명의 바람직한 실시예에서, 배양액은 mPES재질, 50,000Da 분자량 한계치의 중공사 필터를 사용하여 엑소좀을 분리한다. 상기 과정에서 사용되는 중공사 필터의 맴브레인 재질은 위에 기술한 상업용 중공사 필터에 제한되는 것은 아니다.The culture medium produced by the culture method of the present invention is filtered through 0.45 or 0.8 μm and 0.2 μm filters to remove impurities such as cell debris, and sterilization is performed before separating the exosomes by tangential flow filtration. In order to separate exosomes from the nutrient solution of the present invention, a hollow fiber fiber can be used, and a material selected from among mPES (Modified Polyethersulfone), PS (Polysulfone), PES (Polyethersulfone) or ME (Mixed Cellulose Ester) may be used for the membrane material. can In addition, the molecular weight cutoff (MWCO) of the hollow fiber filter may be 10,000 Da, 50,000 Da, or 75,000 Da. In a preferred embodiment of the present invention, the culture medium separates the exosomes using a mPES material, a hollow fiber filter with a molecular weight limit of 50,000Da. The membrane material of the hollow fiber filter used in the above process is not limited to the commercial hollow fiber filter described above.
본 발명의 방법으로 분리한 엑소좀을 최종적으로 0.2μm 멸균필터로 여과멸균한다. 본 발명의 가장 바람직한 실시예에서, 상기 분리된 엑소좀은 0.2μm Sartopore 2 XLG Caps(Sartorius에서 구매)를 일회용 조립 밀폐용기의 포트(Thermo Fisher Scientific에서 구매)에 연결하여 완전한 무균상태를 보장하며 펌프로 분리된 엑소좀을 튜브를 통해 0.2μm 멸균필터를 거쳐 밀폐용기에 수송한다. 상기 과정에서 사용되는 멸균필터와 일회용 조립 밀폐용기는 위에 기술한 사상업용 제품에 제한되는 것은 아니다.The exosomes separated by the method of the present invention are finally filtered and sterilized with a 0.2 μm sterile filter. In the most preferred embodiment of the present invention, the isolated exosomes ensure complete sterility by connecting 0.2 μm Sartopore 2 XLG Caps (purchased from Sartorius) to the port of a disposable assembly airtight container (purchased from Thermo Fisher Scientific) to ensure complete sterility and pump The separated exosomes are transported in an airtight container through a 0.2 μm sterile filter through a tube. The sterilization filter and the disposable assembly airtight container used in the above process are not limited to the above-mentioned commercial products.
이에 따라, 본 발명은 새로운 전구세포 및 이를 배양하여 획득한 배양액에서 고농도, 고순도, 크기가 균일한 엑소좀을 대량으로 획득할 수 있는 기술을 제공한다. Accordingly, the present invention provides a new progenitor cell and a technology capable of obtaining a large amount of exosomes having a high concentration, high purity, and uniform size from a culture medium obtained by culturing the same.
또한, 본 발명은 상기 방법에 의해 수득된 엑소좀을 포함하는 엑소좀치료제를 제공한다. 본 발명의 엑소좀치료제는 혈관주위로 침투한 면연세포를 억제 및 조절하고 면역세포에 의해 손상된 세포 또는 조직 및 혈관을 복구 및 재생하는데 이용될 수 있다. 또한, 본 발명의 엑소좀치료제는 피부염, 관절염, 알츠하이머, 뇌경색 및 뇌출혈의 억제 또는 치료에 사용될 수 있다. 나아가, 본 발명의 엑소좀치료제는 심혈관 질환 또는 중추 및 말초신경계의 치료에 사용될 수 있다. In addition, the present invention provides an exosome therapeutic agent comprising the exosome obtained by the above method. The exosome therapeutic agent of the present invention can be used to suppress and regulate immune cells infiltrating around blood vessels, and to repair and regenerate cells or tissues and blood vessels damaged by immune cells. In addition, the exosome therapeutic agent of the present invention can be used for inhibiting or treating dermatitis, arthritis, Alzheimer's, cerebral infarction and cerebral hemorrhage. Furthermore, the exosome therapeutic agent of the present invention can be used for the treatment of cardiovascular diseases or the central and peripheral nervous system.
이하 본 발명의 실시예를 단계적으로 상세히 설명하지만, 본 발명의 범위가 본 실시예에 의해 한정되는 것은 아니다. Hereinafter, the embodiment of the present invention will be described in detail step by step, but the scope of the present invention is not limited by the present embodiment.
(1) 전구세포 분리(1) Isolation of progenitor cells
기증자로부터 얻은 제대혈을 12시간 내에 처리한다. 구체적인 처리과정은 아래와 같다. 제대혈을 50ml 용량의 원추형 튜브에 옮긴 후, 원추형 튜브를 원심분리기를 이용하여 실온조건에서 2000rpm의 속도로 30분 동안 원심분리하였다. Cord blood from the donor is processed within 12 hours. The specific processing process is as follows. After the umbilical cord blood was transferred to a 50 ml conical tube, the conical tube was centrifuged for 30 minutes at a speed of 2000 rpm at room temperature using a centrifuge.
원심분리 후 도 1a에서 보이는 상층액(혈장)을 제거하고 흰색으로 보이는 완충층(buffy coat layer)을 수거한다. 수거한 완충층을 피콜(Ficoll)이 담겨있는 50ml 용량의 원추형 튜브에 피콜과 완충층의 비율이 2 : 1이 되도록 완충층을 천천히 주입한다. 피콜과 완충층이 혼합된 원추형 튜브를 원심분리기를 이용하여 실온조건에서 2000rpm의 회전속도로 30분 동안 원심분리하였다. 원심분리 후 도 1b에서 보이는 상층액(혈장)을 제거하고 흰색으로 보이는 세포층을 수거하였다. After centrifugation, the supernatant (plasma) shown in FIG. 1A is removed, and a white buffer layer (buffy coat layer) is collected. The buffer layer is slowly injected so that the ratio of Ficoll to the buffer layer is 2 : 1 in the 50ml conical tube containing the collected buffer layer. The conical tube in which Ficoll and the buffer layer were mixed was centrifuged for 30 minutes at a rotation speed of 2000 rpm at room temperature using a centrifuge. After centrifugation, the supernatant (plasma) shown in FIG. 1B was removed and a white cell layer was collected.
수거한 세포를 1배 인산완충생리식염수(1xPBS, 칼슘과 마그네슘이 없음)에 충분히 혼합하고 10μl의 샘플을 채취하여 자동 형광세포 카운터(LUNA STEM장비)로 셀 카운팅을 진행하였고, 그 결과는 도 1c의 그래프와 같다.The collected cells were sufficiently mixed with 1x phosphate buffered saline (1xPBS, without calcium and magnesium), 10 μl of a sample was collected, and cell counting was performed with an automatic fluorescent cell counter (LUNA STEM equipment), and the results are shown in Fig. 1c is like the graph of
나머지 혼합액은 원심분리기를 이용하여 실온조건에서 1500rpm의 회전속도에서 5분 동안 원심분리한 후, 상층액을 제거하고 10% 녹아웃 혈청 대체물(Knockout serum replacement)과 2mM의 글루타민(Glutamine)이 포함된 DMEM/F-12배지로 세포를 재부유시켜 10% 디메틸술폭시드(DMSO : Dimethyl sulfoxide)를 추가한 후 충분히 혼합하여 저온 유리병(cryo vial)에 분주하여 냉동 컨테이너에 넣고 -80℃ 초저온 냉장고(deep freezer)에서 24시간 동안 보관하였다. The remaining mixture was centrifuged for 5 minutes at a rotation speed of 1500 rpm at room temperature using a centrifuge, then the supernatant was removed and DMEM containing 10% knockout serum replacement and 2 mM Glutamine After resuspending the cells in /F-12 medium, adding 10% dimethyl sulfoxide (DMSO), mixing thoroughly, dispensing into cryo vials, and placing them in a freezing container at -80℃ in a deep refrigerator (deep freezer) for 24 hours.
(2) 전구세포 부착 및 배양(2) Progenitor cell attachment and culture
-80℃ 초저온 냉장고에 보관한 세포를 37℃ 항온수조에서 5분 동안 급속 해동하였다. 해동한 세포를 차가운 1배 인산완충생리식염수에 충분히 혼합하고 40μm 셀 스트레이너(cell strainer)를 통과하여 뭉쳐져 있을 수 있는 세포를 제거한 후 10μl의 샘플을 채취하여 자동 형광세포 카운터로 셀 카운팅을 진행하였고, 그 결과가 도 2a의 그래프에 나타나 있다, 나머지 샘플은 원심분리기로 상온에서 1200rpm의 회전속도에서 10분 동안 원심분리한 후, 상층액을 제거하고 20% 우태아혈청(FBS : fetal bovine serum)을 함유한 배지(Advanced DMEM 사용)로 재부유시켜 세포배양접시에 접종한 후 5% CO2, 37℃ 조건 하에서 배양하였다. 75% 이상의 세포가 부착된 후, 1배 인산완충생리식염수로 충분히 세척 후, 10% 녹아웃 혈청 대체물(Knockout serum replacement), 2mM L-글루타민의 디펩타이드 형태(L-alanyl-L-glutamine dipeptide)를 함유한 DMEM/F-12 배지로 교체하여 4주 내지 5주간 배양하고 그 배양액을 회수하였다. 도 2a는 한랭쇼크로 냉동-해동한 후 자동 형광세포 카운터로 분석한 단핵세포 크기의 분포를 나타낸 그래프이고, 도 2b는 세포 배양 플라스크에 부착된 전구세포를 현미경으로 관찰한 사진이고, 도 2c는 부착된 전구세포를 안정화 배지에서 2~4주 배양한 후 현미경으로 관찰한 사진이다.Cells stored in a -80°C cryogenic refrigerator were rapidly thawed in a 37°C constant temperature water bath for 5 minutes. The thawed cells were sufficiently mixed with cold 1x phosphate buffered saline, passed through a 40 μm cell strainer to remove cells that may have clumped, and then 10 μl of a sample was collected and cell counting was performed with an automatic fluorescent cell counter. The results are shown in the graph of FIG. 2a. The remaining samples were centrifuged for 10 minutes at a rotation speed of 1200 rpm at room temperature with a centrifuge, then the supernatant was removed and 20% fetal bovine serum (FBS) was added. After resuspension with the containing medium (Advanced DMEM) and inoculated in a cell culture dish, 5% CO 2 , and cultured under 37°C conditions. After more than 75% of cells have adhered, wash thoroughly with 1x phosphate buffered saline, 10% knockout serum replacement, and 2 mM L-glutamine dipeptide (L-alanyl-L-glutamine dipeptide) It was replaced with the containing DMEM/F-12 medium and cultured for 4 to 5 weeks, and the culture medium was recovered. Figure 2a is a graph showing the distribution of mononuclear cell size analyzed by an automatic fluorescent cell counter after freezing-thawing by cold shock, Figure 2b is a microscopic photograph of progenitor cells attached to a cell culture flask, Figure 2c is This is a photograph observed under a microscope after culturing the attached progenitor cells in a stabilizing medium for 2 to 4 weeks.
본 발명의 방법으로 부착한 전구세포의 세포표면 표지자 CD24, CD31, CD45RA, CD105, CD117, CD146, SSEA-1, TRA-1-60를 Thermo Fisher Scientific사의 유세포 분석기로 분석하였고 그 결과를 표 1에 도시하였다.The cell surface markers CD24, CD31, CD45RA, CD105, CD117, CD146, SSEA-1, and TRA-1-60 of the progenitor cells adhered by the method of the present invention were analyzed with a flow cytometer from Thermo Fisher Scientific, and the results are shown in Table 1 shown.
CD 마커CD marker | CD24CD24 | CD31CD31 | CD45RACD45RA | CD105CD105 | CD117CD117 | CD146CD146 | SSEA-1SSEA-1 | TRA-1-60TRA-1-60 |
TCP세포TCP cell | -- | ++ | ++ | ++ | -- | ++ | -- | ++ |
(3) 접선유동여과(TFF : Tangential Flow Filtration)를 이용한 엑소좀 분리(3) Separation of exosomes using Tangential Flow Filtration (TFF)
위의 방법에 의해 수득된 엑소좀의 분리과정은 도 3의 흐름도에 따라 진행하였다. The separation process of the exosomes obtained by the above method was carried out according to the flowchart of FIG.
배양액에 함유된 엑소좀은 농도가 높고, 입자크기 분포가 균일하기에 접선유동여과(TFF) 시스템으로 엑소좀을 분리하기 용이하고 화학물질 또는 소분자 물질 등 첨가물을 추가하지 않았다. 우선 배양액을 20ml/min의 속도로 펌핑하여 필터(Sartopore 2 XLG SartoScale 25(0.8μm, 0.2μm) 사용)로 여과하여 세포 잔해물, 노폐물 및 거대입자 등 불순물을 제거하고 멸균까지 진행하였다. 여과멸균된 배양액은 즉시 또는 -20℃ 냉동고에 동결보관하였다가 해동시킨 후 접선유동여과장치(TFF : Tangential Flow Filtration)를 이용하여 엑소좀을 분리하였다. The exosomes contained in the culture medium have a high concentration and uniform particle size distribution, so it is easy to separate the exosomes by a tangential flow filtration (TFF) system, and no additives such as chemicals or small molecule substances are added. First, the culture medium was pumped at a rate of 20ml/min and filtered through a filter (using Sartopore 2 XLG SartoScale 25 (0.8μm, 0.2μm)) to remove impurities such as cell debris, wastes and large particles, and sterilization was carried out. The filter-sterilized culture solution was immediately stored frozen or stored in a -20°C freezer and then thawed, and then exosomes were separated using a tangential flow filtration device (TFF).
상기 여과멸균된 배양액에서 엑소좀을 분리하기 위하여 접선유동여과 방법으로 농축 및 정용여과(diafiltration)를 진행하였다. 접선유동여과를 위한 필터로는 중공사 필터(Repligen에서 구매)를 사용하였다. 중공사 필터는 엑소좀의 성질과 여과목적에 따라 다양한 재질과 분자량 한계치(MWCO : Molecular weight cutoff)을 선택할 수 있다. 선택된 분자량 한계치에 의해 선별적으로 엑소좀을 분리하였고 선택된 분자량 한계치보다 작은 입자나 단백질, 지질, 핵산, 저분자 화합물 등은 모두 제거하였다. In order to separate the exosomes from the filter-sterilized culture medium, concentration and diafiltration were performed by a tangential flow filtration method. A hollow fiber filter (purchased from Repligen) was used as a filter for tangential flow filtration. For the hollow fiber filter, various materials and molecular weight cutoff (MWCO) can be selected according to the properties of the exosomes and the purpose of filtration. Exosomes were selectively separated by the selected molecular weight limit, and all particles, proteins, lipids, nucleic acids, and low molecular weight compounds smaller than the selected molecular weight limit were removed.
엑소좀을 분리하기 위한 중공사로서는 mPES(Modified Polyethersulfone), PS(Polysulfone), PES(Polyethersulfone) 또는 ME(Mixed Cellulose Ester)재질이 사용되고, 분자량 한계치 10,000Da, 50,000Da 또는 75,000Da를 사용하였다. 접선유동여과 방식을 이용한 농축 및 정용여과(diafiltration)과정은 단속 또는 연속적으로 수행하였고 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과는 농축한 볼륨에 대하여 적어도 10배, 바람직하게는 12배 내지 15배 이상의 부피를 갖는 완충용액을 이용하여 수행하였다. 완충용액은 1xPBS(without Ca2+ and Mg2+)(Corning에서 구매)를 사용하였고 어떠한 화학물질 또는 저분자 물질도 첨가하지 않았다. 접선유동여과 방식으로 농축과 정용여과를 거친 엑소좀은 안정화 버퍼를 10배 내지 25배 추가하여 엑소좀의 부피가 시작 부피와 똑같게 되도록 희석하였다.As a hollow fiber for separating the exosomes, mPES (Modified Polyethersulfone), PS (Polysulfone), PES (Polyethersulfone) or ME (Mixed Cellulose Ester) materials were used, and molecular weight limit values of 10,000 Da, 50,000 Da or 75,000 Da were used. Concentration and diafiltration using the tangential flow filtration method were performed intermittently or continuously, and the concentration was concentrated 10 to 25 times relative to the starting volume, and the diafiltration was at least 10 times the concentrated volume, preferably This was performed using a buffer solution having a volume of 12 to 15 times or more. As the buffer solution, 1xPBS (without Ca 2+ and Mg 2+ ) (purchased from Corning) was used, and no chemicals or low molecular weight substances were added. The exosomes subjected to concentration and diafiltration by tangential flow filtration were diluted by adding a stabilization buffer 10 to 25 times so that the volume of the exosomes became the same as the starting volume.
본 발명의 방법으로 분리한 엑소좀을 최종적으로 0.2μm 필터(Sartopore 2 XLG Caps, Sartorius에서 구매)로 여과멸균하고 바로 이어지는 일회용 조립 밀폐용기의 포트(Thermo Fisher Scientific에서 구매)를 통해 완전한 무균상태가 보장된 밀폐용기에 수송하였다. The exosomes isolated by the method of the present invention are finally filter-sterilized with a 0.2 μm filter (Sartopore 2 XLG Caps, purchased from Sartorius), and complete sterility is achieved through the port of the disposable assembly sealed container immediately following (purchased from Thermo Fisher Scientific). Transported in a guaranteed sealed container.
상기 기술한 방법으로 배양액에서 30 내지 200nm 균일한 입자크기의 엑소좀을 분리하였다. Exosomes having a uniform particle size of 30 to 200 nm were isolated from the culture medium by the method described above.
(4) 전구세포 유래 엑소좀의 특성 분석(4) Characterization of progenitor cell-derived exosomes
본 발명의 일 실시예의 접선유동여과 방법에 의해 수득된 엑소좀의 입자의 크기 및 농도를 분석한 결과 세 배치(three batches)에서 생산성과 재연성을 확인하였다. 본 발명에서 분리한 엑소좀은 나노입자 추적 분석기(NanoSight NS300 제품 사용)로 측정하였고 그 결과는 도 4a, 4b에 도시하였다. 도 4a는 엑소좀을 정제하기 전, 도 4b는 엑소좀을 정제한 후 엑소좀의 입자의 크기 및 농도를 도시하였다. As a result of analyzing the particle size and concentration of exosomes obtained by the tangential flow filtration method of an embodiment of the present invention, productivity and reproducibility were confirmed in three batches. The exosomes isolated in the present invention were measured with a nanoparticle tracking analyzer (using a NanoSight NS300 product), and the results are shown in FIGS. 4a and 4b. Figure 4a shows the size and concentration of the particles of the exosomes before purification of the exosomes, Figure 4b shows the exosomes after purification of the exosomes.
도 5a는 본 발명의 일 실시예의 방법에 따라 분리된 엑소좀에 대해 웨스턴 블랏을 수행한 결과로서, 기존에 알려져 있은 엑소좀 마커인 CD9, CD63의 존재를 확인하였다. 각 마커에 대한 항체는 BD Biosciences의 항체를 구매하여 사용하였다. 도 5a에서 #1은 중간엽줄기세포 유래 엑소좀, #2는 본 발명에서 생산된 엑소좀의 웨스턴 블랏 결과이다. 도 5b는 본 발명의 일 실시예의 방법에 따라 분리된 엑소좀으로부터 단백질을 추출하여 TMTsixplex Isobaric Label Reagent (Thermo Fisher Scientific에서 구매)로 라벨링한 후 질량 분석기(Orbitrap LC-MS analyzer 사용)로 분석한 펩타이드 분포도이고 아래 표 2는 분석결과 중 CD관련 단백질 리스트이다. Figure 5a is a result of performing a Western blot on the exosome isolated according to the method of an embodiment of the present invention, it was confirmed the presence of the known exosome markers CD9 and CD63. Antibodies for each marker were purchased from BD Biosciences and used. In Figure 5a, #1 is a mesenchymal stem cell-derived exosome, and #2 is a Western blot result of the exosome produced in the present invention. Figure 5b is a peptide analysis by mass spectrometry (using Orbitrap LC-MS analyzer) after extracting proteins from exosomes isolated according to an embodiment of the present invention and labeling them with TMTsixplex Isobaric Label Reagent (purchased from Thermo Fisher Scientific). It is a distribution diagram and Table 2 below is a list of CD-related proteins among the analysis results.
Protein ID Protein ID |
CD9CD9 | CD63CD63 | CD81CD81 | CD18CD18 | CD53CD53 | CD87CD87 | CD98CD98 | CD155CD155 |
TCP세포TCP cell | ++ | ++ | ++ | ++ | ++ | ++ | ++ | ++ |
표 3은 본 발명의 일 실시예의 방법에 따라 접선유동여과로 정제한 엑소좀 속에 존재하는 사이토카인 EGF, bFGF, IL-8, MCP-1 (Thermo Fisher Scientific, R&D systems에서 구매)의 농도를 ELISA kit(Enzyme-Linked Immunosorbent Assay Kit)로 확인한 결과를 나타낸 것이다.Table 3 shows the concentrations of cytokines EGF, bFGF, IL-8, MCP-1 (purchased from Thermo Fisher Scientific, R&D systems) present in exosomes purified by tangential flow filtration according to the method of an embodiment of the present invention by ELISA The results confirmed by the kit (Enzyme-Linked Immunosorbent Assay Kit) are shown.
내용 detail | EGFEGF | bFGF bFGF | IL-8IL-8 | MCP-1MCP-1 | PDGFbbPDGFbb |
농도(pg/ml)Concentration (pg/ml) | 00 | 00 | 00 | 00 | 00 |
상기 결과들을 통해, 본 발명의 엑소좀 생산 기술은 특별한 첨가물이나 과정이 필요없이 접선유동여과만으로 고농도, 고순도이면서 입자크기 분포가 균일한 엑소좀을 경제적이면서 효율적으로 대량으로 생산할 수 있는 기술임을 확인하였다. 또한, 본 발병의 일 실시예의 세포배양, 배양액 여과, 접선유동여과를 통한 엑소좀 분리 및 멸균 등 모든 공정이 스케일업이 가능하고 우수 의약품 제조·관리 기준(GMP : Good Manufacturing Practice)에도 적합하게 셋팅할 수 있음을 알 수 있었다.Through the above results, it was confirmed that the exosome production technology of the present invention can economically and efficiently mass-produce exosomes with high concentration, high purity and uniform particle size distribution only by tangential flow filtration without the need for special additives or processes. . In addition, all processes such as cell culture, culture solution filtration, and exosome separation and sterilization through tangential flow filtration of an embodiment of the present disease can be scaled up and are set appropriately for good manufacturing practice (GMP) knew it could be done.
본 발명에 따른 엑소좀의 모양을 확인하기 위하여 전자현미경(TEM)과 초저온전자현미경(Cryo-EM)으로 분석하였다. 음성염색(negative staining)법으로 염색하여 분석한 전자현미경 사진결과는 도 6, 급속냉동하여 초저온전자현미경으로 분석한 결과는 도 7과 같다. 도 7에서 도 7a는 단일 엑소좀, 도 7b는 이중 엑소좀, 도 7c는 다중 엑소좀, 도 7d는 다중막 엑소좀, 도 7e는 압축 엑소좀을 나타낸 것이다.In order to confirm the shape of the exosome according to the present invention, it was analyzed with an electron microscope (TEM) and a cryogenic electron microscope (Cryo-EM). Fig. 6 shows the results of electron micrographs analyzed by staining with negative staining, and Fig. 7 shows the results of rapid freezing and analysis with cryogenic electron microscopy. In Figure 7, Figure 7a is a single exosome, Figure 7b is a double exosome, Figure 7c is a multiple exosome, Figure 7d is a multi-membrane exosome, Figure 7e is a compressed exosome.
상기 결과를 통해, 본 발명의 엑소좀은 30-150nm 크기에 집중적으로 분포되어 있고, 해당 크기의 엑소좀이 단일 엑소좀(single exosome)뿐만 아니라 이중 엑소좀(double exosome), 다중 엑소좀(multi exosome), 다중막 엑소좀(multilayer exosome) 및 압축 엑소좀(zip exosome) 등 다양한 형태로 존재한다는 것을 밝혔다. 이는 본 발명의 방법으로 획득한 엑소좀의 특성 및 기타 엑소좀과 다르다는 것을 입증한다.Through the above results, the exosomes of the present invention are intensively distributed in the size of 30-150 nm, and the exosomes of the corresponding size are not only single exosomes, but also double exosomes and multiple exosomes. exosome), multilayer exosomes, and compressed exosomes were found to exist in various forms. This proves that the properties of the exosomes obtained by the method of the present invention are different from other exosomes.
(5) 전구세포 유래 엑소좀의 서브그룹(5) Subgroup of progenitor cell-derived exosomes
본 발명의 엑소좀을 나노입자 추적분석기로 분석한 결과, 입자크기 분포가 30 내지 200nm임을 확인하였다. 따라서, 접선유동여과 방법으로 상기 엑소좀을 크기에 따라 서브그룹(subgroup)으로 나눌 수 있을 가능성에 대하여 실험을 진행하였다. As a result of analyzing the exosomes of the present invention with a nanoparticle tracking analyzer, it was confirmed that the particle size distribution was 30 to 200 nm. Therefore, an experiment was conducted on the possibility of dividing the exosomes into subgroups according to the size by the tangential flow filtration method.
위에서 여과멸균한 엑소좀을 접선유동여과 방법으로 500,000Da 중공사(Repligen에서 구매)를 적용하여 각각 유지 성분(여과 후 남은 부분)과 침투성분(여과된 부분)의 용액을 수거하여 엑소좀의 특성을 분석하였다. 그 결과, 유지성분에서 입자 크기가 약 105nm인 엑소좀, 침투 성분에서 입자 크기가 33nm인 엑소좀이 발견되었다. 따라서, 본 발명의 방법으로 분리한 엑소좀은 큰 입자크기(약 105nm)의 엑소좀(도 8a)과 작은 입자크기(약 33nm)의 엑소좀(도 8b)을 함유하고 있는 것을 특징으로 한다는 것을 밝혔다. Characteristics of exosomes by applying a 500,000 Da hollow fiber (purchased from Repligen) to the exosomes filtered and sterilized above by tangential flow filtration method was analyzed. As a result, an exosome having a particle size of about 105 nm in the oil component and an exosome having a particle size of 33 nm in the penetrating component were found. Therefore, the exosomes isolated by the method of the present invention are characterized in that they contain exosomes having a large particle size (about 105 nm) (Fig. 8a) and exosomes having a small particle size (approximately 33 nm) (Fig. 8b). said.
(6) 전구세포 유래 엑소좀의 안정성 시험(6) Stability test of progenitor cell-derived exosomes
본 발명의 방법으로 분리한 전구세포 유래 엑소좀의 안정성을 테스트하기 위하여 12개월간(1회/월) -20±5℃에 보관한 엑소좀을 해동하여 농도와 크기 분포를 측정하였다. 엑소좀의 농도와 크기 분포는 나노입자 추적분석기로 측정하였고 그 결과는 아래 표 4와 같다. In order to test the stability of the progenitor cell-derived exosomes isolated by the method of the present invention, the concentration and size distribution were measured by thawing the exosomes stored at -20±5° C. for 12 months (once/month). The concentration and size distribution of exosomes were measured with a nanoparticle tracking analyzer, and the results are shown in Table 4 below.
상기 결과를 통해, 본 발명의 엑소좀 기술은 안정성이 확보된 엑소좀을 생산할 수 있는 기술임을 확인하였다.Through the above results, it was confirmed that the exosome technology of the present invention is a technology capable of producing exosomes with secured stability.
항목 |
0개월 0 |
3개월3 |
6개월6 |
9개월9 |
12개월12 months |
농도(RATIO, %)Concentration (RATIO, %) | 100%100% | 100%100% | 100%100% | 100%100% | 100%100% |
평균크기(nm)Average size (nm) | 97.997.9 | 97.197.1 | 97.797.7 | 98.398.3 | 97.997.9 |
(7) 전구세포 유래 엑소좀의 독성 시험(7) Toxicity test of progenitor cell-derived exosomes
본 발명의 일 실시예의 방법에 따라 수득된 엑소좀의 독성을 평가하기 위해 랫드(Rat)를 시험물질 500, 1000 및 2000mg/kg/day 투여군과 대조군(주사용수)의 4군으로 군당 암수 각 10마리에 13주간 경피 투여하였다. 13주 동안 체중 및 사료섭취량 측정을 실시하였고, 관찰기간 종료 후 혈액학적 검사, 혈액생화학적 검사, 장기의 중량측정, 부검시 육안적 검사 및 조직병리학적 검사를 수행하였다. In order to evaluate the toxicity of exosomes obtained according to the method of an embodiment of the present invention, rats were treated with 500, 1000 and 2000 mg/kg/day of test substances and 4 groups of control (water for injection), each of 10 sexes per group. The animals were transdermally administered for 13 weeks. Body weight and feed intake were measured for 13 weeks. After the observation period, hematological examination, blood biochemical examination, organ weight measurement, and macroscopic examination and histopathological examination were performed at autopsy.
투여기간 동안, 암수 500, 1,000 및 2,000mg/kg/day 투여군에서 시험물질 투여에 기인한 사망 사례는 관찰되지 않았다. 체중(도 9a, b), 사료섭취량(도 9c, d), 혈액학적 검사(도 9e, f), 혈액생화학적 검사(도 9g, h), 장기중량(도 9i, j)에 있어서 본 발명의 엑소좀에 기인한 변화는 관찰되지 않았다. 또한, 부검 및 조직병리학적 검사 결과, 암수 투여군에서 본 발명의 엑소좀에 의한 독성학적 영향은 관찰되지 않았다. During the administration period, no deaths due to test substance administration were observed in the 500, 1,000, and 2,000 mg/kg/day administration groups. The present invention in body weight (FIG. 9a, b), feed intake (FIG. 9c, d), hematological test (FIG. 9e, f), blood biochemical test (FIG. 9g, h), organ weight (FIG. 9i, j) No change due to exosomes was observed. In addition, as a result of autopsy and histopathological examination, toxicological effects by the exosomes of the present invention were not observed in the male and female administration groups.
본 발명의 기술로 획득한 엑소좀을 랫드에 13주 반복 경피투여한 결과 시험된 용량 범위 내에서 본 발명의 엑소좀에 의한 독성이 나타나지 않음을 확인하였다. As a result of repeated transdermal administration of the exosomes obtained by the technique of the present invention to rats for 13 weeks, it was confirmed that toxicity by the exosomes of the present invention did not appear within the tested dose range.
Claims (14)
- 단핵세포(mononuclear cell)를 일정 조건에서 냉동한 후 해동하는 한랭쇼크 방법으로 전구세포를 획득하는 것을 특징으로 하는 한랭쇼크를 이용한 전구세포 획득방법.A method for obtaining progenitor cells using cold shock, characterized in that the progenitor cells are obtained by a cold shock method in which mononuclear cells are frozen under certain conditions and then thawed.
- 제1항에 있어서,According to claim 1,상기 획득된 전구세포는 다음의 조건을 만족하는 것을 특징으로 하는 한랭쇼크를 이용한 전구세포 획득방법.The method for obtaining progenitor cells using cold shock, characterized in that the obtained progenitor cells satisfy the following conditions.a) 세포를 냉동할 때 사용하는 조성물은 글루타민(L-glutamine) 또는 L-글루타민의 dipeptide형태(L-alanyl-L-glutamine dipeptide), DMSO(Dimethyl sulfoxide) 및 녹아웃 혈청 대체물(Knockout serum replacement)로 이루어진 군에서 선택된 하나 이상이 첨가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove's Modified Dulbecco's Medium), DMEM/F-12(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 또는 DMEM/F-12 Glutamax supplement배지를 포함하는 배지,a) The composition used for freezing the cells is glutamine (L-glutamine) or L-glutamine dipeptide form (L-alanyl-L-glutamine dipeptide), DMSO (dimethyl sulfoxide) and knockout serum replacement. At least one selected from the group consisting of Dulbecco's Modified Eagle Medium (DMEM: Dulbecco'2 Modified Eagle Medium), Minimal Essential Medium (MEM: Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), DMEM/F-12 ( Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) or a medium containing DMEM/F-12 Glutamax supplement medium;b) 세포배양 플라스크에 부착되는 선형 또는 구형의 세포,b) linear or spherical cells attached to the cell culture flask;c) CD24, CD117 또는 SSEA-1에 대하여 음성의 면역학적 특성을 나타냄, 또는c) is immunologically negative for CD24, CD117 or SSEA-1, ord) CD31, CD45RA, CD105, CD146 또는 TRA-1-60에 대하여 양성의 면역학적 특성을 나타냄d) exhibits positive immunological properties against CD31, CD45RA, CD105, CD146 or TRA-1-60
- 제1항 또는 제2항에 있어서,3. The method of claim 1 or 2,상기 전구세포가 제대혈, 골수, 지방, 탯줄, 양수, 치아 또는 말초혈액에서 유래된 것을 특징으로 하는 전구세포 획득방법.The method for obtaining progenitor cells, characterized in that the progenitor cells are derived from umbilical cord blood, bone marrow, fat, umbilical cord, amniotic fluid, dental or peripheral blood.
- (a) 단핵세포를 일정 조건에서 냉동한 후 해동하는 한랭쇼크 방법으로 전구세포를 획득하는 단계;(a) obtaining progenitor cells by a cold shock method in which mononuclear cells are frozen under certain conditions and then thawed;(b) 제1배지에서 상기 전구세포를 배양하여 세포배양 플라스크에 부착시키는 단계;(b) culturing the progenitor cells in a first medium and attaching them to a cell culture flask;(c) (b)의 결과물을 제2배지에서 상기 전구세포를 1주 내지 5주 동안 배양하는 단계; (c) culturing the resultant of (b) in a second medium for 1 to 5 weeks;(d) (c)의 결과물이 분비하는 엑소좀을 수집하는 단계; 및(d) collecting the exosomes secreted by the result of (c); and(e) 상기 수집된 엑소좀을 분리하는 단계를 포함하는 것을 특징으로 하는 엑소좀 생산방법.(E) exosome production method comprising the step of separating the collected exosomes.
- 제4항에 있어서,5. The method of claim 4,상기 제1배지는 글루타민(L-glutamine) 또는 L-글루타민의 dipeptide형태(L-alanyl-L-glutamine dipeptide), 우태아혈청(FBS), 피루브산 나트륨(Sodium pyruvate), 불필수아미노산(Non-essential amino acids), 트랜스페린(Transferrin), 소듐 셀레나이트(Sodium selenite) 및 인슐린(Insulin)으로 이루어진 군에서 선택된 하나 이상이 추가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), Advanced DMEM, 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove’s Modified Dulbecco’s Medium) 또는 DMEM/F12 배지를 포함하는 것을 특징으로 하는 엑소좀 생산방법.The first medium is glutamine (L-glutamine) or L-glutamine dipeptide form (L-alanyl-L-glutamine dipeptide), fetal bovine serum (FBS), sodium pyruvate (Sodium pyruvate), non-essential amino acids (Non-essential) Dulbecco'2 Modified Eagle Medium (DMEM) with at least one selected from the group consisting of amino acids), transferrin, sodium selenite and insulin, Advanced DMEM, Minimum essential medium (MEM: Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium) or DMEM / F12 exosome production method comprising a medium.
- 제4항에 있어서,5. The method of claim 4,상기 제2배지는 Glutathione monosodium, 비타민 B12, 모노소듐 하이포크산틴(Monosodium hypoxanthine), 티미딘(Thymidine), 글루타민(L-glutamine) 또는 L-글루타민의 디펩타이드 형태(L-alanyl-L-glutamine dipeptide) 및 녹아웃 혈청 대체물(Knockout serum replacement)으로 이루어진 군에서 선택된 하나 이상이 추가된 둘베코변형 이글 배지(DMEM: Dulbecco’2 Modified Eagle Medium), 최소 필수 배지(MEM: Minimal Essential Medium), IMDM(Iscove's Modified Dulbecco's Medium), DMEM/F12(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 또는 DMEM/F-12 Glutamax supplement 배지를 포함하는 것을 특징으로 하는 엑소좀 생산방법.The second medium is Glutathione monosodium, vitamin B12, monosodium hypoxanthine, thymidine, glutamine (L-glutamine) or dipeptide form of L-glutamine (L-alanyl-L-glutamine dipeptide) And at least one selected from the group consisting of knockout serum replacement, Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Iscove's Modified (IMDM) added Dulbecco's Medium), DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) or DMEM / F-12 Exosome production method comprising a Glutamax supplement medium.
- 제4항에 있어서,5. The method of claim 4,상기 (e) 단계는,Step (e) is,(e-1) 전구세포 배양액을 0.45 또는 0.8μm 필터를 통과하여 세포 잔해물, 노폐물 및 거대 입자를 여과하는 단계;(e-1) filtering the progenitor cell culture medium through a 0.45 or 0.8 μm filter to filter out cell debris, waste products and large particles;(e-2) 상기 여과된 배양액을 멸균 필터로 멸균하는 단계;(e-2) sterilizing the filtered culture solution with a sterile filter;(e-3) 상기 멸균된 배양액을 접선유동여과를 이용하여 농축과 정용여과 과정으로 엑소좀을 분리하는 단계;(e-3) separating the exosomes from the sterilized culture solution by concentration and diafiltration using tangential flow filtration;(e-4) 상기 분리된 엑소좀에 안정화 버퍼를 추가하여 초기 부피와 동일한 부피가 되도록 희석하는 단계;(e-4) adding a stabilization buffer to the isolated exosome and diluting it to the same volume as the initial volume;(e-5) 상기 희석된 엑소좀을 액체 교환 시스템을 이용하여 멸균 필터를 통과시켜 멸균처리하는 단계; 및 (e-5) sterilizing the diluted exosomes through a sterile filter using a liquid exchange system; and(e-6) 상기 멸균처리된 엑소좀을 밀봉된 저장용기에 저장하는 단계를 포함하는 것을 특징으로 하는 엑소좀 생산방법.(e-6) Exosome production method comprising the step of storing the sterilized exosomes in a sealed storage container.
- 제7항에 있어서,8. The method of claim 7,상기 접선유동여과를 위하여, 분자량 한계치가 10,000 Da, 50,000 Da 또는 75,000 Da 중 어느 하나인 중공사 필터를 사용하는 것을 특징으로 하는 엑소좀 생산방법.For the tangential flow filtration, exosome production method, characterized in that using a hollow fiber filter having a molecular weight limit of any one of 10,000 Da, 50,000 Da, or 75,000 Da.
- 제7항에 있어서,8. The method of claim 7,상기 (e-3) 단계의 농축과 정용여과는 접선유동여과를 단속 또는 연속적으로 진행하고, 농축은 시작 부피에 대하여 10배 내지 25배 농축하고, 정용여과 과정은 농축된 부피에 대하여 적어도 10배의 부피를 갖는 버퍼로 여과하는 과정을 포함하는 것을 특징으로 하는 엑소좀 생산방법.The concentration and diafiltration in step (e-3) are performed intermittently or continuously through tangential flow filtration, the concentration is concentrated 10 to 25 times relative to the starting volume, and the diafiltration process is at least 10 times relative to the concentrated volume. Exosome production method comprising the step of filtration with a buffer having a volume of.
- 제7항에 있어서,8. The method of claim 7,엑소좀 희석은 농축 및 정용여과된 부피에 대하여 10배 내지 25배의 안정화 버퍼를 첨가하여 전체 부피가 시작 부피와 동일하도록 희석하는 과정을 포함하는 것을 특징으로 하는 엑소좀 생산방법.Exosome dilution is a method for producing exosomes, characterized in that it comprises a process of diluting the total volume to the same as the starting volume by adding a stabilization buffer of 10 to 25 times to the concentrated and diafiltered volume.
- 제7항에 있어서,8. The method of claim 7,상기 엑소좀은 농도가 3 x 109 particles/ml 내지 5 x 109 particles/ml의 농도를 가지는 것을 특징으로 하는 엑소좀 생산방법.The exosomes have a concentration of 3 x 10 9 particles/ml to 5 x 10 9 particles/ml Exosome production method, characterized in that it has a concentration.
- 제7항에 있어서,8. The method of claim 7,상기 엑소좀은 30 내지 40nm 크기의 엑소좀을 함유하고 있는 것을 특징으로 하는 엑소좀 생산방법.The exosomes are exosomes production method, characterized in that it contains the exosomes having a size of 30 to 40 nm.
- 제7항에 있어서,8. The method of claim 7,상기 엑소좀은 CD마커로서, CD9, CD63, CD81 외에 CD18, CD53, CD87, CD98 또는 CD155를 더 함유하고 있는 것을 특징으로 하는 엑소좀 생산방법.The exosome is a CD marker, CD9, CD63, CD81 in addition to CD18, CD53, CD87, CD98 or CD155 exosome production method, characterized in that it further contains.
- 제7항에 있어서,8. The method of claim 7,상기 엑소좀은 단일 엑소좀(single exosome), 이중 엑소좀(double exosome), 다중 엑소좀(multi exosome), 다중막 엑소좀(multilayer exosome) 및 압축 엑소좀(zip exosome)을 함유하고 있는 것을 특징으로 하는 엑소좀 생산방법.The exosome is characterized in that it contains a single exosome, a double exosome, a multi exosome, a multilayer exosome, and a compressed exosome. A method for producing exosomes.
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Title |
---|
EMELYANOV ANTON, SHTAM TATIANA, KAMYSHINSKY ROMAN, GARAEVA LUIZA, VERLOV NIKOLAI, MILIUKHINA IRINA, KUDREVATYKH ANASTASIA, GAVRILO: "Cryo-electron microscopy of extracellular vesicles from cerebrospinal fluid", PLOS ONE, vol. 15, no. 1, 30 January 2020 (2020-01-30), pages e0227949, XP055928523, DOI: 10.1371/journal.pone.0227949 * |
MEYER T.P.H., HOFMANN B., ZAISSERER J., JACOBS V.R., FUCHS B., RAPP S., WEINAUER F., BURKHART J.: "Analysis and cryopreservation of hematopoietic stem and progenitor cells from umbilical cord blood", CYTOTHERAPY, ISIS MEDICAL MEDIA, OXFORD,, GB, vol. 8, no. 3, 1 January 2006 (2006-01-01), GB , pages 265 - 276, XP055928527, ISSN: 1465-3249, DOI: 10.1080/14653240600735685 * |
QIU GUANGUAN, ZHENG GUOPING, GE MENGHUA, WANG JIANGMEI, HUANG RUOQIONG, SHU QIANG, XU JIANGUO: "Functional proteins of mesenchymal stem cell-derived extracellular vesicles", STEM CELL RESEARCH & THERAPY, vol. 10, no. 1, 1 December 2019 (2019-12-01), XP055928524, DOI: 10.1186/s13287-019-1484-6 * |
REKA AGNES HARASZTI, RACHAEL MILLER, MATTEO STOPPATO, YVES Y. SERE, ANDREW COLES, MARIE-CECILE DIDIOT, RACHEL WOLLACOTT, ELLEN SAP: "Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity", MOLECULAR THERAPY, ELSEVIER INC., US, vol. 26, no. 12, 1 December 2018 (2018-12-01), US , pages 2838 - 2847, XP055751842, ISSN: 1525-0016, DOI: 10.1016/j.ymthe.2018.09.015 * |
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