WO2022098018A1 - Procédé d'obtention de cellules progénitrices par choc à froid et procédé de production d'exosomes l'utilisant - Google Patents

Procédé d'obtention de cellules progénitrices par choc à froid et procédé de production d'exosomes l'utilisant Download PDF

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
exosomes
medium
exosome
progenitor cells
glutamine
Prior art date
Application number
PCT/KR2021/015574
Other languages
English (en)
Korean (ko)
Inventor
쵄소왠
김창영
Original Assignee
주식회사 타임바이오
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 주식회사 타임바이오 filed Critical 주식회사 타임바이오
Publication of WO2022098018A1 publication Critical patent/WO2022098018A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/10—Separation or concentration of fermentation products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/12—Purification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2511/00—Cells for large scale production
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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.

Landscapes

  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Sustainable Development (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une méthode d'obtention de cellules progénitrices par traitement de cellules mononucléaires isolées de tissus ou de cellules par un choc à froid, et la production d'exosomes par culture des cellules progénitrices, et le procédé de choc à froid de la présente invention peut produire des cellules progénitrices d'origine humaine de taille uniforme et ayant une activité stable de sécrétion d'exosomes. Les exosomes produits par la présente invention sont distribués à une concentration élevée, de manière prépondérante sur une taille particulière, et peuvent être utilisés directement avec une technique de filtration à flux tangentiel sans additifs, et être avantageusement utilisés pour augmenter la pureté et les rendements afin de produire des exosomes de haute concentration, haute pureté, et à haut rendement.
PCT/KR2021/015574 2020-11-05 2021-11-01 Procédé d'obtention de cellules progénitrices par choc à froid et procédé de production d'exosomes l'utilisant WO2022098018A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2020-0146938 2020-11-05
KR1020200146938A KR102284304B1 (ko) 2020-11-05 2020-11-05 한랭쇼크를 이용한 전구세포 획득 방법 및 이를 이용한 엑소좀 생산 방법

Publications (1)

Publication Number Publication Date
WO2022098018A1 true WO2022098018A1 (fr) 2022-05-12

Family

ID=77315613

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2021/015574 WO2022098018A1 (fr) 2020-11-05 2021-11-01 Procédé d'obtention de cellules progénitrices par choc à froid et procédé de production d'exosomes l'utilisant

Country Status (2)

Country Link
KR (1) KR102284304B1 (fr)
WO (1) WO2022098018A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102284304B1 (ko) * 2020-11-05 2021-08-02 주식회사 타임바이오 한랭쇼크를 이용한 전구세포 획득 방법 및 이를 이용한 엑소좀 생산 방법

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101895916B1 (ko) * 2017-08-11 2018-09-07 주식회사 엑소코바이오 엑소좀 및/또는 세포외 소포체 및 이를 포함하는 조성물의 제조 방법
KR102284304B1 (ko) * 2020-11-05 2021-08-02 주식회사 타임바이오 한랭쇼크를 이용한 전구세포 획득 방법 및 이를 이용한 엑소좀 생산 방법

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2691296T3 (es) 2012-04-03 2018-11-26 Reneuron Limited Micropartículas de células madre
KR102063571B1 (ko) 2017-06-12 2020-01-09 성균관대학교산학협력단 다양한 세포로부터의 표면개질된 엑소좀의 제조방법

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101895916B1 (ko) * 2017-08-11 2018-09-07 주식회사 엑소코바이오 엑소좀 및/또는 세포외 소포체 및 이를 포함하는 조성물의 제조 방법
KR102284304B1 (ko) * 2020-11-05 2021-08-02 주식회사 타임바이오 한랭쇼크를 이용한 전구세포 획득 방법 및 이를 이용한 엑소좀 생산 방법

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
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 *

Also Published As

Publication number Publication date
KR102284304B1 (ko) 2021-08-02

Similar Documents

Publication Publication Date Title
US9982233B2 (en) Method for promoting generation of stem cell-derived exosome by using thrombin
WO2018182356A1 (fr) Culture contenant une concentration élevée d'exosome de haute pureté dérivé de cellules souches mésenchymateuses, et son procédé de préparation
US7897388B2 (en) Growth of neural precursor cells using umbilical cord blood serum and a process for the preparation for therapeutic purposes
Parker et al. Low serum and serum-free culture of multipotential human adipose stem cells
WO2018226051A2 (fr) Composition de milieu sans sérum pour la culture de cellules comprenant un exosome dérivé d'une cellule souche humaine
JP2022520420A (ja) 幹細胞の凍結保存
AU2015200117A1 (en) Methods of upscaling mesenchymal stromal cell production, compositions and kit thereof
CN112608894A (zh) 一种间充质干细胞培养基
EP3943597B1 (fr) Méthode de préparation d'un exosome dérivé d'une cellule souche mésenchymateuse et culture cellulaire préparée à partir dudit exosome
WO2022098018A1 (fr) Procédé d'obtention de cellules progénitrices par choc à froid et procédé de production d'exosomes l'utilisant
CN113736738B (zh) 一种胃癌微肿瘤细胞模型的培养方法
CN112961825A (zh) 一种无血清培养基及其制备方法
CN110974944A (zh) 间充质干细胞复合活性因子冻干粉及其制备方法和应用
CN115011553A (zh) 一种躯干神经嵴来源骨髓间充质干细胞的制备方法及用途
CN113755441B (zh) 一种肺癌微肿瘤细胞模型的培养方法
CN114177203A (zh) 脐带血浓缩细胞治疗卵巢早衰的细胞治疗剂
Costa et al. Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell‐derived extracellular vesicles in stirred‐tank bioreactors
CN112080469B (zh) T1肽在体外促进脐带血造血干细胞增殖中的应用
KR101626217B1 (ko) 타우로우루소데옥시콜릭산을 유효성분으로 함유하는 조혈모세포 증식용 조성물
WO2024045404A1 (fr) Surnageant de moelle osseuse et son utilisation dans la culture cellulaire
CN114507642B (zh) 一种动物神经系统周细胞单细胞的分离方法
WO2020161748A1 (fr) Méthode d'isolement de cellules souches mésenchymateuses et de différenciation d'ostéoblastes
CN113073077A (zh) 一种使用封闭式系统培养临床级脐带血间充质干细胞的方法
CN109722418B (zh) 一种获取和纯化新生小鼠少突胶质前体细胞的方法
CN116396930B (zh) 间充质干细胞无血清培养基及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21889483

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21889483

Country of ref document: EP

Kind code of ref document: A1