WO2018226051A2 - Composition de milieu sans sérum pour la culture de cellules comprenant un exosome dérivé d'une cellule souche humaine - Google Patents

Composition de milieu sans sérum pour la culture de cellules comprenant un exosome dérivé d'une cellule souche humaine Download PDF

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WO2018226051A2
WO2018226051A2 PCT/KR2018/006488 KR2018006488W WO2018226051A2 WO 2018226051 A2 WO2018226051 A2 WO 2018226051A2 KR 2018006488 W KR2018006488 W KR 2018006488W WO 2018226051 A2 WO2018226051 A2 WO 2018226051A2
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serum
stem cells
cell culture
cell
medium
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Korean (ko)
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WO2018226051A3 (fr
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조용우
최지숙
이찬미
김재동
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주식회사 엑소스템텍
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • 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
    • 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
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

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  • the present invention relates to a serum-free medium composition for culturing cells containing exosomes derived from human stem cells as an active ingredient, more specifically, containing exosomes derived from human stem cells, it is possible to minimize cell damage And a new serum-free medium composition for cell culture that can aid in cell adhesion and growth and induce and promote cell proliferation.
  • the present invention also relates to an animal serum replacement agent comprising an exosome derived from human stem cells, a serum-free medium composition for cell culture containing the same, and a method of culturing cells using the same.
  • the present invention can overcome the shortcomings of the serum by using a medium in which exosomes are added instead of animal serum, and can be applied to a serum-free medium composition for stem cell culture, thereby culturing stem cells without animal serum. And finally to a method for culturing stem cells for cell therapy for clinical applications.
  • Serum contains a variety of hormones in the insulin or polypeptide family that promote cell growth and function, growth factors that regulate cell division and function, and adhesion and diffusion factors such as fibronectin, transferrin as a binding protein, lipids and minerals. Many trace elements are included. Serum is also a buffer that regulates the osmotic pressure and pH of the culture, and has a viscous action that protects cells from proteolytic inhibition and physical shock. Cells can be cultured using serum containing these various functions.
  • Stem cells have the property of continuously producing the same cells as themselves for a certain period of time in an undifferentiated state, and differentiate into specific cells under suitable conditions.
  • Adult stem cells can be obtained from bone marrow, blood, brain, skin, and fat, and are known to be able to differentiate into various tissue cells.
  • adult stem cells such as adipose derived stem cells, bone marrow derived stem cells, umbilical cord or umbilical-cord blood derived stem cells Research into the development of autologous or other cell therapy is also actively underway.
  • animal serum may have a change in composition depending on the time and place of collection, it is difficult to administer to the patient stem cells cultured in a serum-containing medium.
  • the method of culturing stem cells by adding hormones, growth factors or purified proteins instead of serum is most commonly used. Although it is added to the basic medium by combining components necessary for adhesion and growth of stem cells such as amino acids, vitamins, insulin-like factors, epidermal growth factor, fibroblast growth factor, protease inhibitors, proteins and selenium, And growth factors are sold at a very high price, it is difficult to use a combination of various components.
  • exosomes are vesicles with the same membrane structure as cell membranes and are known to deliver membrane components, proteins, RNA, and the like to other cells and tissues.
  • exosomes secreted from stem cells contain various growth factors and cytokines secreted by stem cells, and are known to regulate cell adhesion, growth, and differentiation.
  • exosomes are removed impurities such as cell waste, antibiotics, serum in the cell culture in the separation process, it can be used safely and equivalent to the effect of the cell culture.
  • Stem cell exosomes contain complex components such as protein, insulin-like growth factor, epithelial cell growth factor, fibroblast growth factor, vascular epithelial cell growth factor, various fatty acids and antioxidants to help stem cell adhesion and growth.
  • the present invention has developed a serum-free culture medium containing stem cell-derived exosomes that can minimize cell damage of stem cells and help adhesion and growth.
  • An object of the present invention is to isolate the exosomes that induce cell attachment and proliferation from human stem cells, and to provide a new serum-free medium composition for culturing cells containing exosomes in the basal medium. It is an object of the present invention to provide a serum-free medium composition for stem cell culture comprising extracting an exosome having the efficacy of stem cell proliferation and culture from human adipose stem cells and a mixture thereof.
  • an object of the present invention contains exosomes derived from human stem cells, which can minimize cell damage, aid in cell adhesion and growth, and induce and promote cell proliferation.
  • Serum medium composition is provided.
  • an object of the present invention can overcome the shortcomings of serum by using a medium in which exosomes are added instead of animal serum, and can be applied to serum-free medium composition for stem cell culture to culture stem cells without animal serum. And finally, to provide a method for culturing stem cells for cell therapy for clinical applications.
  • the present invention is to isolate the exosomes to induce cell adhesion and proliferation from human stem cells, animal serum replacement agent comprising the isolated exosomes, cell culture containing exosomes in the basal medium Serum-free medium composition, and a method for culturing cells using the same.
  • exosome refers to a membrane vesicle that is secreted from various cell types, and serves various roles such as delivering membrane components, proteins, and RNA to other cells and tissues. It is known.
  • the stem cells derived from exosomes may be adult stem cells, more specifically, may be selected from the group consisting of adipose derived stem cells, bone marrow stem cells and umbilical cord blood stem cells, Preferably, it may be selected from the group consisting of human adipose derived stem cells, human bone marrow stem cells, and human umbilical cord blood stem cells, and more preferably human adipose derived stem cells, but is not limited thereto.
  • the animal serum replacement agent and / or serum-free medium composition for cell culture of one embodiment of the present invention is preferably used for culturing animal cells, more preferably for culturing stem cells.
  • the serum-free medium composition for cell culture of one embodiment of the present invention is characterized in that it contains exosomes derived from human stem cells, preferably human fat-derived stem cells, as an active ingredient, and does not include animal serum.
  • Exosomes derived from human stem cells, preferably human adipose-derived stem cells are used by mixing in a basic medium at a constant rate to maximize the proliferation effect of animal cells, preferably stem cells.
  • human stem cell-derived exosomes isolated from serum-free, antibiotic-free medium contain growth factors effective for promoting growth and maintenance of various proliferation-related cells, such as cell activators or growth factors. It can be effectively applied as a serum-free medium composition for culturing animal cells, preferably stem cells, without additives. Therefore, the animal serum replacement agent and / or serum-free medium composition for cell culture of one embodiment of the present invention can culture animal cells, preferably stem cells, without animal serum.
  • basal medium' refers to a culture medium containing essential ingredients necessary for growth and proliferation of cells in vitro.
  • the basal medium may be artificially synthesized or commercially prepared medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • RPMI 1640 F-10, F-12
  • G-MEM Glasgow's Mineral Essential Medium
  • IMDM Iscove's Modified Dulbecco's Medium
  • the exosomes may be included in the medium at a concentration of 0.01 to 1,000 ⁇ g / mL.
  • the exosomes can be prepared using a conventional exosome separation method, for example
  • exosomes are not only loaded with genetic information, proteins, and growth factors of stem cells, but the exosomes themselves may serve as carriers.
  • Exosomes consisting of lipids of about 50-150 nm in size are biocompatible because they are cell-derived materials, and the uptake of cells is also very good.
  • a serum-free medium composition for cell culture containing such exosomes as an active ingredient promotes the attachment and proliferation of animal cells, preferably stem cells.
  • the serum-free medium composition for cell culture of one embodiment of the present invention can be used as a composition for stem cell culture for clinical application.
  • Another aspect of the invention is the step of separating the exosomes from human stem cells; Preparing a serum-free medium composition for cell culture comprising the isolated exosomes; And it provides a cell culture method comprising culturing the cells in the serum-free medium composition for cell culture.
  • the human stem cells may be selected from the group consisting of human adipose derived stem cells, human bone marrow stem cells and human umbilical cord blood stem cells, more preferably human adipose derived stem cells It may be, but is not limited thereto.
  • the serum-free medium composition for cell culture is preferably used for the culturing of animal cells, more preferably for the culturing of stem cells derived from exosomes.
  • the cell culture-free serum medium composition is characterized in that it does not contain animal serum.
  • the cell culture-free serum medium composition is characterized in that it contains substantially no animal serum.
  • the term “substantially free of” may be completely devoid of the component, or contain little of the component to the extent that the effect may be the same as if the component was completely lacking. In other words, a composition that is "substantially free” of components or elements may still actually contain these items unless there is a measurable effect thereof.
  • the term “substantially free of” refers to up to 5 wt%, up to 4 wt%, up to 3 wt% or up to 2 wt%, preferably up to 1 wt%, more preferably 0.1, based on the total content of the composition unless otherwise indicated. It may mean up to weight percent, even more preferably up to 0.01 weight percent.
  • Animal serum substitutes comprising exosomes derived from human stem cells of the present invention, and serum-free medium composition for cell culture containing the same contains genes, proteins, growth factors, etc., which are related to cell growth, and thus cell activators or growth factors. Without other additives, it can help cells attach and grow and induce cell proliferation.
  • Human stem cell-derived exosomes used in the present invention can overcome the problems of animal serum because it is a purified component that does not contain antibiotics, serum, or harmful eyeballs of the culture medium, cell penetration and effective factors because of cell-derived lipid transporter The transmission efficiency is very good.
  • the present invention can overcome the shortcomings of the serum by using a medium in which exosomes are added instead of animal serum, and can be applied to a serum-free medium composition for stem cell culture, thereby culturing stem cells without animal serum. Finally, it can be used for culturing stem cells for cell therapy for clinical applications.
  • Figure 1 shows the results for the characterization of exosomes produced from human adipose derived stem cells
  • Figure 1a is the concentration of stem cell exosomes confirmed using a nanoparticle tracking analysis (Nanoparticle Tracking Analysis, NTA),
  • Figure 1b is the form of stem cell exosomes confirmed using a transmission electron microscope (Transparent Electron Microscope, TEM), and
  • the scale bar of the image is 100 nm (black) and 20 nm (white), respectively.
  • Figure 2 shows the results of the cell proliferation evaluation of human adipose derived stem cells according to the exosome concentration.
  • Cell proliferation was evaluated by incubating human adipose derived stem cells (ASC, 1 ⁇ 10 4 cell / well) for 3 days in a 48-well plate, and the degree of cell proliferation was determined by CCK-8 reagent (Dojindo, cat no. DJB4000X) was used for absorbance analysis.
  • CCK-8 reagent Dojindo, cat no. DJB4000X
  • Exo-proliferation efficiency than some containing the cultured cells in serum-free medium resulting positive control (growth medium) is natatjiman, it was confirmed that the growth rate is improved as compared to the negative control (serum-free medium, SFM) in all experimental groups (P ⁇ 0.001).
  • stem cells cultured in a medium containing 50 ⁇ g / mL of exosomes were confirmed to improve cell growth efficiency more than two times compared to the negative control group
  • Figure 3 shows the results of evaluating the cell proliferation induction of exosomes in culture medium containing a low concentration of serum (Serum 1%, 3%, 5%).
  • Culture medium was prepared by adding 50 ⁇ g / mL of exosomes to all culture conditions except for positive control (Serum 10%).
  • ASC Human adipose derived stem cells
  • n Human adipose derived stem cells
  • Figure 4 shows the results of the differentiation effect of stem cells cultured in a serum-free medium containing exosomes to adipocytes.
  • (a) is an optical micrograph of human adipose derived stem cells cultured in a normal culture medium and a serum-free medium containing exosomes
  • (b) is a fat cell induced differentiation induced through differentiation induction medium in (a) It is an optical microscope photograph evaluated for differentiation ability by red O staining. Scale bars are 200 ⁇ m (white) and 50 ⁇ m (black) respectively. Evaluation of differentiation ability into adipocytes confirmed that the differentiation function of stem cells cultured in serum-free medium containing exosomes was well maintained.
  • Human adipose stem cell-derived exosomes were extracted during the process of culturing human adipose stem cells.
  • human adipose stem cells (Company: CellBio, Cat #: CB-ADMSC-001) are DMEM (Dulbecco) containing 10% FBS (fetal bovine serum) and 1% penicillin / streptomycin (penicillin / streptomycin). Modified Eagle Medium high glucose; Gibco, Cat #: 11995065). 24 hours before extracting the exosomes, the cells were replaced with serum-free, antibiotic-free, phenol-free medium (Company: Gibco, Cat #: 31053028) and cultured for 24 hours, and then cell culture supernatants were recovered. After the supernatant was recovered, normal culture medium was added to the stem cells and cultured. This process was repeated until passage 7.
  • DMEM Dulbecco
  • FBS fetal bovine serum
  • penicillin / streptomycin penicillin / streptomycin
  • Modified Eagle Medium high glucose Gibco, Cat #: 11995065
  • the recovered cell culture supernatant was centrifuged at 2,000 ⁇ g, 4 ° C. for 5 minutes, primary filtering step using a cell strainer (pore size; 40 ⁇ m), and a bottle top filter (pore size). ; 0.22 ⁇ m) to remove cell debris and wastes by a second filtering step.
  • tangential flow filtration THF was used for exosome enrichment and further purification.
  • the filter specification used was MWCO 300 kDa (Molecular Weight Cut-Off).
  • the exosomes extracted in the process of proliferation were used as the stem cell culture additive composition.
  • Exosomes extracted from human adipose derived stem cells of Example 1 were exosomes using a nanoparticle tracking analysis (NTA), a transmission electron microscope (TEM), and a flow cytometry. The concentration and purity of, size, morphology and CD surface markers were confirmed ( Figure 1).
  • exosomes are measured by performing sample dilution (dilution solution; 1X phosphate buffer saline (PBS) solution) so that the number of nanoparticles identified on the screen per frame can be within the range of 30 to 40 (FIG. 1A).
  • the exosome sample was treated with 1% phosphotungstic acid solution for 1 minute and subjected to negative staining. As a result, spherical nanoparticle morphology was observed, and the average particle size was about 100 nm (FIG. 1B).
  • CD9, CD63, and CD81 known as exosome membrane surface markers
  • flow rate analyzer After attaching the reagent (SBI, CD9 / 63 / 81-EXO FLOW Capture kit) and the fluorescent marker to which the surface marker specific antibody is bound to the magnetic bead on the surface of the exosome, the expression of the surface marker of the exosome was measured using a flow rate analyzer. It was confirmed (FIG. 1C).
  • Serum-free culture medium containing exosomes was prepared by mixing as shown in Table 1 below.
  • GM group Stem cell proliferation medium
  • SFM group fetal bovine serum 10%, penicillin / streptomycin 1%) for positive control group
  • SFM group serum-free medium
  • Example 2 Increasing the induction of cell proliferation of human adipose derived stem cells by adding 50 ⁇ g / mL of the exosomes isolated in Example 1 to a culture medium containing low serum, specifically 1%, 3%, and 5% serum Evaluation was performed (Table 2, FIG. 3).
  • GM group human adipose derived stem cells
  • SFM group serum-free medium
  • human adipose derived stem cells (ASC, 1 ⁇ 10 4 cell / well) are radish containing proliferation medium (DMEM + FBS 10% + P / S 1%) and exosomes.
  • DMEM Dulbecco Modified Eagle Medium high glucose; Gibco, Cat #: 11995065
  • FBS fetal bovine serum
  • IBMX Isobutyl-1-methylxanthine; Sigma, Cat # I5879-1G
  • 10 ⁇ g / mL Insulin Sigma, Cat # I16634-100
  • 1 ⁇ M Dexamethasone Abcam, Cat # ab12074
  • 100 ⁇ M Indomethacin Sigma, Cat # I7378-5
  • exosomes produced from human adipose derived stem cells were found to be effective ingredients that help cell proliferation while maintaining the differentiation ability of human adipose derived stem cells.

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Abstract

La présente invention concerne une nouvelle composition de milieu sans sérum pour la culture de cellules, un exosome qui induit l'adhésion et la prolifération cellulaires étant séparé d'une cellule souche humaine, et l'exosome étant contenu dans un milieu basique. Une composition de milieu sans sérum pour la culture de cellules selon la présente invention contient des gènes, des protéines et des facteurs de croissance associés à la croissance cellulaire, et peut ainsi favoriser l'adhésion et à la croissance de cellules même sans autres additifs tels que des activateurs cellulaires ou des facteurs de croissance, et peut induire et activer la prolifération cellulaire. La présente invention peut réduire les inconvénients du sérum à l'aide d'un milieu dans lequel un exosome est ajouté au lieu de sérum animal, ou en réduisant au minimum la quantité de sérum utilisée, et peut enfin être utilisée dans la culture de cellules souches pour une thérapie cellulaire dans une application clinique.
PCT/KR2018/006488 2017-06-07 2018-06-07 Composition de milieu sans sérum pour la culture de cellules comprenant un exosome dérivé d'une cellule souche humaine WO2018226051A2 (fr)

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CN114207116A (zh) * 2019-07-30 2022-03-18 韩国外泌体生技有限公司 新的外排体产生方法及其应用
CN114207116B (zh) * 2019-07-30 2024-03-15 韩国外泌体生技有限公司 新的外排体产生方法及其应用
CN110938593A (zh) * 2020-01-02 2020-03-31 张伟 一种鳄鱼脂肪干细胞来源外泌体提取及冻干粉制备方法
CN114836375A (zh) * 2020-12-31 2022-08-02 国典(北京)医药科技有限公司 一种外泌体分泌诱导剂、诱导培养基及其应用
CN114836375B (zh) * 2020-12-31 2023-07-21 国典(北京)医药科技有限公司 一种外泌体分泌诱导剂、诱导培养基及其应用
CN113234663A (zh) * 2021-05-10 2021-08-10 博品(上海)生物医药科技有限公司 一种培养子宫内膜细胞的方法

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