KR102284304B1 - Isolation method of progenitor cells using cold shock and method for producing exosome using these progenitor cells - Google Patents

Abstract

The present invention relates to a method for obtaining progenitor cells by treating monocytes isolated from cells or tissues with a cold shock, and to exosomes produced by culturing the progenitor cells. A cold shock method of the present invention can obtain human-derived progenitor cells with uniform size and stable activity to secrete exosomes. The exosomes produced by the present invention are densely distributed in a specific size at high concentration, can be directly applied to tangential flow filtration without additives, and be usefully used for mass production of high-concentration, high-purity, and high-yield exosomes by increasing purity and yield.

Description

Method for obtaining progenitor cells using cold shock and method for producing exosomes using same {Isolation method of progenitor cells using cold shock and method for producing exosome using these progenitor cells}

The present invention relates to a method for obtaining progenitor cells by treating monocytes 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 with uniform size and secretory activity. It relates to a method for obtaining this stable human-derived progenitor cell.

Research on exosomes is mostly focused on mesenchymal stem cells, induced pluripotent stem cells, and 293T cell lines. Mesenchymal stem cell 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.

However, one factor that determines the properties of exosomes is exosome-derived cells. 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 effect, 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.

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. Other extracellular vesicles, such as microvesicals and apoptotic bodies, are similar in size to exosomes, but differ in their biogenesis pathways. The biggest role of exosomes 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 exosomes are the key to showing physiological or therapeutic efficacy (Nature Reviews Neurology 15, 193-203 (2019)).

The characteristics of exosomes differ depending on the derived cells, the size, concentration, and loaded nucleic acid information of the exosomes. Existing studies to manufacture human-derived exosomes 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 exosomes is about 200 nm, and the size of the exosomes is quite large. 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.

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 of the present invention was completed.

Korean Patent No. 2063571 (Method for producing surface-modified exosomes from various cells) Japanese Patent Laid-Open No. 2020-072718 (Stem Cell Microparticles)

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. .

In addition, the present invention provides a method capable of separating the exosomes of the present invention only by the process of dilution 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. Another purpose is to provide.

According to one aspect of the present invention for achieving the above object, there is provided a progenitor cell procurement method using cold shock, wherein progenitor cells are obtained by a cold shock method in which monocytes are frozen under certain conditions and then thawed.

Here, the obtained progenitor cells preferably satisfy the following conditions.

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), knockout serum replacement Added Dulbecco'2 Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle Medium: Nutrient Mixture (DMEM/F-12) F-12) and a medium containing a medium selected from DMEM/F-12 Glutamax supplement medium

b) linear or spherical cells attached to the cell culture flask

c) show negative immunological properties for CD24, CD117, SSEA-1

d) show positive immunological properties for CD31, CD45RA, CD105, CD146, TRA-1-60

In addition, the progenitor cells may be derived from umbilical cord blood, bone marrow, fat, umbilical cord, amniotic fluid, dental or peripheral blood.

According to another aspect of the present invention for achieving the above object, (a) obtaining progenitor cells by a cold shock method in which monocytes are frozen under a certain condition and then thawed, (b) the progenitor cells are prepared in a first medium Culturing and attaching the progenitor cells to a cell culture flask, (c) culturing the progenitor cells in a second medium for 1 to 5 weeks, (d) collecting exosomes secreted by the progenitor cells And (e) there is provided an exo-some production method comprising the step of isolating the collected exosomes.

Here, (b) the first medium used in the step of culturing progenitor cells and attaching them to the flask is glutamine (L-glutamine) or a dipeptide form of L-glutamine (L-alanyl-L-glutamine dipeptide), fetal bovine serum ( FBS), sodium pyruvate, non-essential amino acids, transferrin, sodium selenite, and insulin (Insulin) added at least one selected from Dulbecco's Modified Eagle medium (Dulbecco'2 Modified Eagle Medium (DMEM)), Advanced DMEM, Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), and DMEM/F12 medium.

And, the second medium used in the step of culturing the resultant of (b) for 1 to 5 weeks in the second medium is Glutathione monosodium, vitamin B12, monosodium hypoxanthine, thymidine, glutamine. Dulbecco's medium (DMEM: Dulbecco'2) to which one or more selected from (L-glutamine) or L-glutamine dipeptide (L-alanyl-L-glutamine dipeptide), knockout serum replacement is added Modified Eagle Medium), Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12), and DMEM/F-12 Glutamax supplement medium selected from media may be included.

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) the filtered culture solution is subjected to a sterile filter sterilizing with, (e-3) separating the exosomes from the sterilized culture solution by concentration and diafiltration 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.

Here, for the tangential flow filtration, it is preferable to use a hollow fiber filter having a molecular weight limit of any one of 10,000 Da, 50,000 Da, or 75,000 Da, and the concentration and diafiltration in step (e-3) are tangential flow filtration. intermittently or continuously, the concentration is concentrated 10 to 25 times the starting volume, and the diafiltration process preferably includes filtration with a buffer having at least 10 times the volume of the concentrated volume.

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 of 10 to 25 times to the concentrated and diafiltered volume.

The exosome has a concentration of 3 x 10 ⁹ particles/ml to 5x10 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.

In addition, as a CD marker, the exosome further contains CD18, CD53, CD87, CD98, and CD155 in addition to CD9, CD63, and CD81, single exosome, double exosome, multiple exosome It is preferable to contain (multi exosome), a multilayer exosome and a compressed exosome (zip exosome).

According to the present invention, by separating progenitor cells having a uniform size, attaching them 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 sterilizing filter in the final step, thus solving the most important problem of sterilization of exosomes 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).

Figure 1a is a photograph showing the buffy coat layer after centrifugation of cord blood, Figure 1b is a photograph showing the monocyte layer after centrifuging the buffy coat layer by the Ficoll density gradient method, Figure 1c is the size of monocytes analyzed by LUNA STEM is a graph showing the distribution of
Figure 2a is a graph showing the distribution of the size of monocytes 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 adhesion This is a photograph of the progenitor cells observed under a microscope after culturing for 2 to 4 weeks in a stabilized medium.
Figure 3 is a flow chart showing the process of separating the exosomes from the culture medium produced using the progenitor cells of the present invention.
Figure 4a shows the NTA analysis results showing the particle size distribution of the exosomes before separating the exosomes, Figure 4b after separating 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.
7 is a photo 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.
8 is a graph showing the density of exosomes by size as a result of TFF treatment of 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.
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, FIG. 9a is body weight, FIG. 9b is feed intake, FIG. 9c is a hematological test, FIG. Blood biochemical test, FIG. 9E is a test result for organ weight.

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.

All terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, unless otherwise defined. Terms defined in a commonly used dictionary should be interpreted as being consistent with the meaning of 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.

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.

In the method of the present invention, 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. ), Dulbecco'2 Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Iscove's Modified Dulbecco's Medium (IMDM) and DMEM/ This is a step of culturing in a selected medium among F12 medium under conditions of 5% CO2 and 37°C. 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.

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 Glutamax supplement medium is a step of culturing under the conditions of 5% CO2 and 37°C. 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 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 the exosomes from the nutrient solution of the present invention, a hollow fiber fiber may 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.

The exosomes isolated 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 are completely sterile by connecting 0.2 μm Sartopore 2 XLG Caps (purchased from Sartorius) to the port (purchased from Thermo Fisher Scientific) of a disposable assembled airtight container 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 disposable assembly sealed container used in the above process are not limited to the above-mentioned commercial products.

Accordingly, the present invention provides a new progenitor cell and a technology for obtaining a large amount of exosomes having a high concentration, high purity, and uniform size from a culture medium obtained by culturing the same.

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 control 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.

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) Isolation of progenitor cells

Cord blood from the donor is processed within 12 hours. The specific processing process is as follows. After transferring the umbilical cord blood to a conical tube with a capacity of 50 ml, the conical tube was centrifuged for 30 minutes at a speed of 2000 rpm at room temperature using a centrifuge.

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 to 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.

The collected cells were sufficiently mixed with 1x phosphate buffered saline (1xPBS, no calcium and magnesium), and 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

The remaining mixture was centrifuged for 5 minutes at a rotation speed of 1500 rpm at room temperature using a centrifuge, and 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 putting them in a freezing container at -80℃ in a deep refrigerator (deep freezer) for 24 hours.

(2) Progenitor cell attachment and culture

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 and passed through a 40 μm cell strainer to remove any cells that may have clumped. The results are shown in the graph of Figure 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, it was cultured under the conditions of 5% CO2 and 37°C. After more than 75% of cells have adhered, wash thoroughly with 1x phosphate buffered saline, and then 10% knockout serum replacement and 2 mM L-glutamine dipeptide form (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 the size of monocytes 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 adhesion This is a photograph of the progenitor cells observed under a microscope after culturing for 2 to 4 weeks in a stabilized medium.

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 by a flow cytometer from Thermo Fisher Scientific, and the results are shown in Table 1 shown.

(3) Exosome separation using Tangential Flow Filtration (TFF)

The separation process of the exosomes obtained by the above method was carried out according to the flowchart of FIG.

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 20 ml/min and filtered through a filter (using Sartopore 2 XLG SartoScale 25 (0.8 μm, 0.2 μm)) to remove impurities such as cell debris, wastes and large particles, and sterilization was performed. The filter-sterilized culture solution was immediately or frozen in a -20 °C freezer and thawed, and then exosomes were separated using a tangential flow filtration device (TFF).

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.

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. The concentration and diafiltration process using the tangential flow filtration method were performed intermittently or continuously, and the concentration was concentrated 10 to 25 times 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 Ca2+andMg2+) (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.

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.

Exosomes having a uniform particle size of 30 to 200 nm were isolated from the culture medium by the method described above.

(4) Characterization of progenitor cell-derived exosomes

As a result of analyzing the particle size and concentration of the 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 particles of the exosomes before purification of the exosomes, Figure 4b shows the exosomes after purification of the exosomes.

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, CD63. Antibodies for each marker were purchased and used from BD Biosciences. In FIG. 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 analyzed by a mass spectrometer (using Orbitrap LC-MS analyzer) after extracting the protein from the exosome isolated according to the method of an embodiment of the present invention and labeling it 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 It shows the results confirmed by the kit (Enzyme-Linked Immunosorbent Assay Kit).

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 medium 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.

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 a negative staining method, and Fig. 7 shows the results of rapid freezing and analysis by cryogenic electron microscopy. In FIG. 7, FIG. 7a shows a single exosome, FIG. 7b shows a double exosome, FIG. 7c shows a multiple exosome, FIG. 7d shows a multi-membrane exosome, and FIG. 7e shows a compressed exosome.

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) Subgroup of progenitor cell-derived exosomes

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.

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, exosomes having a particle size of about 105 nm in the oil component and exosomes 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 by containing 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) Stability test of progenitor cell-derived exosomes

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.

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.

(7) Toxicity test of progenitor cell-derived exosomes

In order to evaluate the toxicity of the 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 male and female 10 per group The animals were transdermally administered for 13 weeks. Body weight and feed intake were measured for 13 weeks, and after the observation period, hematological examination, blood biochemical examination, organ weight measurement, and gross examination and histopathological examination were performed at autopsy.

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. Changes due to the exosomes of the present invention in body weight (FIG. 9a), feed intake (FIG. 9b), hematology (FIG. 9c), blood biochemical testing (FIG. 9d), and organ weight (FIG. 9e) were not observed. didn't 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.

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)

delete delete delete (a) A cold shock method in which mononuclear cells are frozen and then thawed, which shows negative immunological properties for CD24, CD117 and SSEA-1, and CD31, CD45RA, CD105, CD146 and TRA-1-60 obtaining progenitor cells exhibiting positive immunological properties with respect to;
(b) culturing the progenitor cells in a first medium and attaching them to a cell culture flask;
(c) culturing the resultant of (b) in a second medium for 1 to 5 weeks;
(d) collecting the exosomes secreted by the result of (c); and
(E) exosome production method comprising the step of separating the collected exosomes.
5. The method of claim 4,
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- Dulbecco'2 Modified Eagle Medium (DMEM) with at least one selected from the group consisting of essential 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.
5. The method of claim 4,
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) , Knockout serum replacement (Knockout serum replacement) Dulbecco's Modified Eagle Medium (DMEM: Dulbecco'2 Modified Eagle Medium), minimal essential medium (MEM: Minimal Essential Medium), IMDM (Iscove's Modified) added at least one selected from the group consisting of 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.
5. The method of claim 4,
Step (e) is,
(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) sterilizing the filtered culture solution with a sterile filter;
(e-3) separating the exosomes from the sterilized culture solution by concentration and diafiltration using tangential flow filtration;
(e-4) adding a stabilization buffer to the isolated exosome and diluting it 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) Exosome production method comprising the step of storing the sterilized exosomes in a sealed storage container.
8. The method of claim 7,
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.
8. The method of claim 7,
The concentration and diafiltration in step (e-3) are performed intermittently or continuously with 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 process of filtration with a buffer having a volume of.
8. The method of claim 7,
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.
8. The method of claim 7,
The exosomes have a concentration of 3 x 10 ⁹ particles/ml to 5x10 ⁹ particles/ml Exosome production method, characterized in that it has a concentration.
8. The method of claim 7,
The exosomes are exosomes production method, characterized in that it contains the exosomes having a size of 30 to 40 nm.
8. The method of claim 7,
The exosomes are CD markers, CD9, CD63, CD81, CD18, CD53, CD87, CD98 and CD155 exosome production method, characterized in that it further contains.
8. The method of claim 7,
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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