WO2019103528A2 - Composition de milieu de culture asérique - Google Patents

Composition de milieu de culture asérique Download PDF

Info

Publication number
WO2019103528A2
WO2019103528A2 PCT/KR2018/014531 KR2018014531W WO2019103528A2 WO 2019103528 A2 WO2019103528 A2 WO 2019103528A2 KR 2018014531 W KR2018014531 W KR 2018014531W WO 2019103528 A2 WO2019103528 A2 WO 2019103528A2
Authority
WO
WIPO (PCT)
Prior art keywords
medium
stem cells
serum
cells
cell
Prior art date
Application number
PCT/KR2018/014531
Other languages
English (en)
Korean (ko)
Other versions
WO2019103528A3 (fr
Inventor
유지민
정아름
Original Assignee
주식회사 차바이오랩
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 차바이오랩 filed Critical 주식회사 차바이오랩
Publication of WO2019103528A2 publication Critical patent/WO2019103528A2/fr
Publication of WO2019103528A3 publication Critical patent/WO2019103528A3/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
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0037—Serum-free medium, which may still contain naturally-sourced components
    • 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
    • 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
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • 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
    • C12N2500/36—Lipids
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/21—Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)

Definitions

  • a serum-free medium composition for cell culture and a method for culturing cells using the same.
  • FBS fetal bovine serum
  • Animal-derived serum plays an important role in adherence and growth of cells, but in the cell separation step, a heterogenous population is proliferated together to lower the purity of stem cells, and a lot-to-lot variation , Decrease the ability to proliferate, and decrease the ability to differentiate. It also affects the viability, safety and therapeutic efficacy of cells by increasing the antigenic response.
  • One aspect provides a serum-free medium composition for cell culture.
  • Another aspect provides a method of culturing cells in the medium composition.
  • One aspect provides a serum-free medium composition for cell culture.
  • cell culture refers to the process of cultivating living cells artificially in vitro under controlled conditions.
  • a part of the individual tissue can be aseptically removed, and the suspension in which the intercellular connective material is degraded by an enzyme can be streaked on a flat bottom of a culture dish such as a bottle or a Petri dish to grow and proliferate the cells.
  • culture media refers to materials that enable growth and survival of cells, including stem cells, in vitro.
  • the composition may be selected from the group consisting of insulin, transferrin, selenium, platelet-derived growth factor-AB (PDGF-AB), human serum albumin (HAS) (FGF), Epidermal Growth Factor (EGF), Transforming growth factor beta-1 (TGFbeta-1), heparin, Lysophosphatidic acid, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), chemokine (CXC motif) ligand 12, CXCL12) Or a combination thereof.
  • PDGF-AB platelet-derived growth factor-AB
  • HAS human serum albumin
  • EGF Epidermal Growth Factor
  • TGFbeta-1 Transforming growth factor beta-1
  • Lysophosphatidic acid human granulocyte-macrophage colony-stimulating factor
  • hGM-CSF human granulocyte-macrophage colony-stimulating factor
  • CXC motif chemokine
  • Insulin is a hormone that promotes the uptake of sugar and amino acids into cells and promotes cell proliferation. It can promote cell proliferation in association with somatomedin C (C).
  • Transferrin is a protein that transports iron to cells and can detoxify oxygen radicals and peroxides in the medium.
  • Selenium is a substance that causes selenium-dependent enzymes to reduce fat peroxidation, and plays a role in protecting the cell membrane.
  • the selenium may be in the form of selenium itself or a selenium salt, such as organic and inorganic forms.
  • the organic form of the selenium salt may be an amino acid L (+) - selenomethionine, L (+) - methyl selenocysteine or L (+) - selenocysteine.
  • the inorganic form of the selenium salt may be sodium selenite, calcium selenite, or potassium selenite.
  • the insulin, the transferrin and the selenium may be contained in the medium in an amount of about 0.05% to about 20%, or about 0.1% to about 10%.
  • % means v / v%.
  • the platelet-derived growth factor-AB is a growth factor that regulates cell growth and cleavage.
  • the platelet-derived growth factor-AB (PDGF-AB) is added to the medium in an amount of about 0.5 ng / ml to about 200 ng / To about 100 ng / ml.
  • the human serum albumin (HAS) is a protein which is present in a large amount in human plasma and transports a compound such as a hormone or a fatty acid, and regulates pH and osmotic pressure.
  • the protein is contained in the medium in an amount of about 0.0125% to about 5.0% 0.025% to about 2.5%.
  • % means v / v%.
  • the lipid may regulate the growth and maintenance of cells and may include saturated fatty acids, unsaturated fatty acids, or combinations thereof.
  • the lipid may be included in the medium at about 0.005% to about 2% or about 0.01% to about 1%.
  • % means v / v%.
  • the Fibroblast Growth Factor (FGF) induces signal transduction for proliferation by providing a signal for growth and proliferation of cells.
  • the fibroblast growth factor is one comprising Fibroblast Growth Factor-4 (FGF-4), Fibroblast Growth Factor-2 (FGF-2) .
  • the Fibroblast Growth Factor-4 may be contained in the medium at about 0.25 ng / ml to about 100 ng / ml, or about 0.5 ng / ml to about 50 ng / ml.
  • the Fibroblast Growth Factor-2 may be contained in the medium at about 0.25 ng / ml to about 100 ng / ml, or about 0.5 ng / ml to about 50 ng / ml.
  • the Epidermal Growth Factor binds to the receptor EGFR to stimulate proliferation and differentiation and is contained in the medium at about 0.5 ng / ml to about 200 ng / ml or about 1 ng / ml to about 100 ng / ml .
  • the transforming growth factor beta-1 regulates cell growth, cell proliferation, cell differentiation and apoptosis, and is administered to the medium in an amount of about 0.25 ng / ml to about 100 ng / ml or about 0.5 ng / ml to about 50 ng / ml.
  • the heparin binds to the fibroblast growth factor and promotes cell proliferation.
  • the heparin may be contained in the medium at about 0.05 ⁇ g / ml to 20 ⁇ g / ml or about 0.1 ⁇ g / ml to 10 ⁇ g / ml .
  • the lysophosphatidic acid is a kind of phospholipid derivative acting as a signal molecule and can act as a mitogen and can be used in a concentration of about 0.1 ng / ml to 40 ng / ml or about 0.2 ng / ml To 20 ng / ml.
  • the human granulocyte-macrophage colony-stimulating factor is a cytokine secreted from macrophages, T cells, mast cells, natural killer cells and the like and promotes differentiation maturation of progenitor cells And from about 0.05 ng / ml to about 20 ng / ml or from about 0.1 ng / ml to about 10 ng / ml in the medium.
  • CXCL12 is a chemokine that is mixed with stromal cell-derived factor 1 (SDF1) and promotes the proliferation of B precursor cells, From about 1 ng / ml to about 400 ng / ml or from about 2 ng / ml to about 200 ng / ml in the medium.
  • SDF1 stromal cell-derived factor 1
  • the animal-derived serum can be substituted because the productivity and purity are high while securing safety in cell culture.
  • the animal-derived serum refers to a serum derived from an individual other than a human.
  • the medium composition is not particularly limited as long as it can be used for cell culture.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Base Medium Eagle
  • F-12 F-12
  • DMEM / F12 a-Minimal essential medium
  • G-MEM Iscove's modified Dulbecco's medium
  • MacCoy's 5A medium AmnioMax complete medium
  • AminoMax II complete MECB + Dulbecco ' s Modified Eagle ' s medium low glucose
  • EBM Endothelial Basal Medium
  • Chang's Medium MesenCult-XF medium
  • Dulbecco's modified Eagle medium high glucose medium And the like.
  • the cells may be undifferentiated cells, differentiated cells, or differentiated cells.
  • the cell may be a stem cell.
  • stem cell means a cell having potency and self-renewal ability. Stem cells are divided into pluripotency, multipotency, or unipotency depending on the differentiation ability.
  • the stem cells include embryonic stem cells (ESCs), adult stem cells (undifferentiated cells present in each tissue and organs), and stem cells (induced a pluripotent stem cell (iPSC) (a cell in which a gene and / or a protein is inserted into somatic cells to induce differentiation, or an induced pluripotent stem cell).
  • ESCs embryonic stem cells
  • adult stem cells undifferentiated cells present in each tissue and organs
  • iPSC induced a pluripotent stem cell
  • the stem cells may be mesenchymal stem cells.
  • mesenchymal stem cell MSC
  • MSC mesenchymal stem cell
  • the mesenchymal stem cells are cells that aid in the production of fat, cartilage, bone, bone marrow stroma, muscle, nerve, etc. and can differentiate into various cells such as adipocytes, cartilage cells, skin cells and bone cells .
  • the types and origins of the stem cells are not limited as long as they have the ability to differentiate and self-regenerate.
  • the stem cells may be derived from, for example, mammals, humans, monkeys, pigs, horses, cows, sheep, dogs, cats, mice or rabbits.
  • the stem cells may be derived from isolated umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid.
  • isolated means that it is present in an environment different from that of a naturally occurring cell or tissue. Separation of umbilical cord, placenta, fat, bone marrow, umbilical cord blood or amniotic fluid and obtaining stem cells therefrom may be performed by conventional anatomical methods and known methods.
  • the culture may be growth and proliferation.
  • the term " growth and proliferation" means an increase in the number of cells.
  • the culture may be undifferentiated growth.
  • the undifferentiated proliferation refers to the proliferation of stem cells into cells having the same properties as the original cells without being differentiated into specific cells, that is, cells having potency and self-renewal ability .
  • differentiation means that the structure or function of one another is specialized while the cells are growing by proliferation and proliferation, that is, the cells and tissues of the organism are changed in shape or function . Measuring or determining the degree of differentiation into a particular cell type may be performed by methods well known in the art.
  • the differentiation can be effected by cell surface markers (e.g., staining cells with a tissue-specific or cell-specific antibody) using techniques such as flow cytometry or immunocytochemistry and changes in cell morphology
  • cell surface markers e.g., staining cells with a tissue-specific or cell-specific antibody
  • techniques such as flow cytometry or immunocytochemistry and changes in cell morphology
  • Another aspect provides a method of culturing cells in the medium composition.
  • the culture may be growth and proliferation.
  • the method of culturing may include subculturing.
  • subculture refers to a method of cell proliferation, in which cells are periodically transplanted into new medium every few days to preserve the cells and continue the cell cycle.
  • a " passage” may be a growth and proliferation of stem cells from the initial seed culture in the culture vessel to the confluence of the cells in the same culture vessel.
  • the method of culturing the cells and the method of subculturing may be carried out by a conventional culture method and a known method.
  • the cell may be a stem cell.
  • the stem cells may be embryonic stem cells, mesenchymal stem cells, or degenerated stem cells.
  • the cells that can be cultured are the same as described above.
  • the serum-free medium composition for cell culture of the present invention When used, it is economical to replace the expensive cell culture medium containing serum, and it is possible to reduce side effects such as infection that may occur when existing serum is contained. In addition, it exhibits similar cell proliferation rate and gene expression ratio as the cell culture medium containing the existing serum.
  • Figure 1 shows the proliferation rate of stem cells according to the basal medium. That is, the result of measuring the proliferation rate of stem cells in a cell culture medium containing 6 kinds of basic medium is shown.
  • Fig. 2 shows the morphology and degree of proliferation of stem cells after culturing the stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • Fig. 3 shows the size of stem cells after culturing stem cells in medium containing normal animal-derived serum and the medium of the present invention.
  • Fig. 4 shows the proliferation rate of stem cells after culturing stem cells in a medium containing normal animal-derived serum, normal serum-free medium and medium of the present invention.
  • Fig. 5 shows results of the surface antigen characteristics of stem cells after culturing stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • FIG. 6A is a graph showing the expression characteristics of stem cell gene expression after culturing stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • FIG. 6B is a bar graph showing the expression characteristics of stem cell genes after culture of stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • FIG. 7 shows results of identity-by-state (IBS) analysis of gene expression of stem cells in stem cells after culturing stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • FIG. 8 is a result of comparing secreted proteins secreted from stem cells after culture of stem cells in medium containing normal animal-derived serum and the medium of the present invention.
  • FIG. 9 shows results of staining of calcium salts secreted from stem cells with Alizarin Red S after culture of stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • FIG. 10 shows the result of staining stem cells with Oil Red O after culturing stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • 11 is a graph quantitatively evaluating the degree of lipolysis after discoloration of stem cells stained with Oil Red O with isopropyl alcohol.
  • Fig. 12 is a result of staining of stem cells with Alcian blue after culturing stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • FIG. 13A is a graph showing the expression pattern of the TNFAIP6 gene in stem cells after culturing a medium containing normal animal-derived serum and a stem cell cultured in the medium of the present invention.
  • FIG. 13B is a graph showing the expression patterns of PTGS2 gene in stem cells after culturing a medium containing normal animal-derived serum and a stem cell cultured in the medium of the present invention.
  • 13C is a graph showing the expression patterns of the IDO1 gene in stem cells after culturing a medium containing normal animal-derived serum and a stem cell cultured in the medium of the present invention.
  • FIG. 13D is a graph showing the expression pattern of TNF-.alpha. After culturing a medium containing normal animal-derived serum and cultured stem cells in the medium of the present invention and injecting it into a mouse.
  • Example One Serum-free Identification of the shape, size, proliferation and expression antigen characteristics of the stem cell cultured in the preparation of the cell culture medium composition and the culture medium composition
  • cell culture medium compositions having various compositions and composition ratios were prepared. Accordingly, a variety of cell culture medium compositions were given lot numbers (CHA-SFM-000), and the basic medium, insulin, transferrin, selenium, platelet-derived growth factor-AB , fibroblast growth factor 4 (FGF4), fibroblast growth factor 2 (FGF2), fibroblast growth factor 2 (FGF2), fibroblast growth factor 2 (FGF2), Epidermal Growth Factor (EGF), Transforming growth factor beta-1 (TGFbeta-1), heparin, lysophosphatidic acid, A CHA-SFM-035 lot containing a human granulocyte-macrophage colony-stimulating factor (hGM-CSF) and a chemokine (CXC motif) ligand 12: CXCL12 was selected Respectively.
  • hGM-CSF human granulocyte-macrophage colony-stimulating factor
  • CXC motif chemokine
  • optimized media were selected using a total of six media (MCDB + DMEM / LG, EBM, DMEM / F12, IMDM, DMEM / HG and MEM alpha).
  • the umbilical cord-derived mesenchymal stem cells cultured at 1 in the above-mentioned six cell culture mediums were cultured for about 72 hours, and the cell proliferation rate of the stem cells was measured by Cell Counting Kit-8 (CCK-8) Respectively.
  • Figure 1 shows the proliferation rate of stem cells according to the basal medium. That is, the result of measuring the proliferation rate of stem cells in a cell culture medium containing 6 kinds of basic medium is shown.
  • the proliferation rate is about 25% to about 25% as compared with the case where the stem cells are cultured in the cell culture medium containing other basic medium 400% increase. Therefore, DMEM / F12 was selected as the basic medium of (2.1).
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured in a 4 to 7 passages in a cell culture medium (CHA-SFM-035) containing DMEM / F12 as the basic medium in (2.1).
  • the positive control group was obtained from 1 in ⁇ -MEM medium (hereinafter referred to as CCM) containing 10% fetal bovine serum (FBS) and 25 ng / ml of FGF4 and 2 ug / ml of heparin
  • CCM ⁇ -MEM medium
  • FBS fetal bovine serum
  • one umbilical cord-derived mesenchymal stem cells were cultured to 4 to 7 passages.
  • the culture was performed by dividing stem cells into fibronectin-coated plates, growing and proliferating them, and then adding trypsin to recover the cell suspension. After the subculture, the shape and proliferation of the stem cells were confirmed in the experimental group and the positive control group.
  • Fig. 2 shows the morphology and degree of proliferation of stem cells after culturing the stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • the shape of the stem cells maintained a spindle shape, which was the same or similar to that of the stem cells cultured in the cell culture medium (CCM) containing the animal-derived serum.
  • the degree of proliferation was similar in the experimental group and the positive control group. It can be understood that the cell culture medium of the present invention has cell proliferation power similar to that of the cell culture medium containing animal-derived serum.
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured to 4 to 7 passages.
  • Positive control cells were obtained by culturing umbilical cord-derived mesenchymal stem cells obtained in 1 to 4 to 7 passages in CCM.
  • the size of the obtained cells was confirmed by LUNA analysis.
  • Fig. 3 shows the size of stem cells after culturing stem cells in medium containing normal animal-derived serum and the medium of the present invention. As shown in Fig. 3, the sizes of the cultured stem cells were about 15 to 17 mu m, and the sizes of the stem cells in the experimental group and the positive control group were similar. When the cells were cultured under the culture medium for cell culture of the present invention, the size of the cells was not significantly reduced or maintained.
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured for about 72 hours. Positive control cells were obtained by culturing the umbilical cord-derived mesenchymal stem cells obtained in 1 in CCM for about 72 hours.
  • the negative control group was obtained by culturing the umbilical cord-derived mesenchymal stem cells obtained in 1 in a commercially available serum-free medium (medium containing normal animal-derived serum) for about 72 hours. Cell proliferation rates of stem cells were measured by Cell Counting Kit-8 (CCK-8) analysis after about 24 hours, 48 hours, and about 72 hours.
  • Fig. 4 shows the proliferation rate of stem cells after culturing stem cells in a medium containing normal animal-derived serum, normal serum-free medium and medium of the present invention.
  • the growth rate was 63% as compared with the case where stem cells were cultured in CCM.
  • the proliferation rate was improved by about 27% as compared with the case of culturing the stem cells in the commercial serum-free medium.
  • the growth rate was about 90% as compared with that in the culture medium. It can be seen that the cell culture medium of the present invention has a level of proliferation similar to that of the medium containing serum.
  • Fig. 5 shows results of the surface antigen characteristics of stem cells after culturing stem cells in a medium containing normal animal-derived serum and the medium of the present invention.
  • the stem cells were cultured in the culture medium of CHA-SFM-035, which is the culture medium of the present invention, and in the medium containing normal animal-derived serum, all of the cells were cultured in CD44, CD73, CD105 , CD29, CD49 and CD90 were high (positive), the expression levels of CD45, CD34, CD31 and HLA-DR were low (negative, negative), and the expression levels were also similar. It can be seen that the medium for cell culture of the present invention does not change the surface antigen characteristics of the stem cells.
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured in a 4 to 7 passages in a cell culture medium (CHA-SFM-035) containing DMEM / F12 as the basic medium in (2.1). Positive control cells were cultured in serum-containing medium (CCM). Expression patterns of multiple genes were simultaneously confirmed by Affymetrix Human Gene 2.0 ST Array test method to evaluate the expression of equivalent genes in the experimental group and the positive control group. Total RNA was extracted from the passaged cells of the experimental group and the positive control group and synthesized with cDNA to confirm the difference in expression.
  • CHAM serum-containing medium
  • Fig. 6 shows the results of comparing the expression equivalency of genes in medium containing normal animal-derived serum and in stem cells cultured in the medium of the present invention.
  • FIG. 6A it was confirmed that the plot signal of the gene expression level of the test group and the positive control group was close to 1.
  • the plot signals of the experimental group and the positive control group were similar. That is, the cell culture medium of the present invention shows the level of cell gene expression equivalent to the serum medium.
  • the medium contained the composition of (2.1), it was determined whether the stem cells cultured in CHA-SFM-035 did not show any genetic variation and the DNA remained the same. Infinium® Global Screening Array-24 v1.0 test method.
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured in a 4 to 7 passages in a cell culture medium (CHA-SFM-035) containing DMEM / F12 as the basic medium in (2.1). Positive control cells were cultured in serum-containing medium (CCM).
  • FIG. 7 shows results of identity-by-state (IBS) analysis by analyzing the expression of gene by gene in a medium containing serum from normal animals and stem cells cultured in the medium of the present invention.
  • IBS identity-by-state
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured in a 4 to 7 passages in a cell culture medium (CHA-SFM-035) containing DMEM / F12 as the basic medium in (2.1). Positive control cells were cultured in serum-containing medium (CCM).
  • CCM serum-containing medium
  • Fig. 8 shows the results obtained by comparing the secretory proteins secreted from stem cells cultured in the culture medium of the present invention and the culture medium containing normal animal-derived serum. As shown in Fig. 8, it was confirmed that the scatter plot of the cytokine was close to 1 in the experimental group and the positive control group. In the image analysis, the expression patterns of cytokines in the experimental group and the positive control group were similar. That is, the cell culture medium of the present invention shows the level of cytokine expression equivalent to the serum medium.
  • the medium contained the composition of (2.1), it was confirmed whether the bone marrow differentiation potential of the subcultured stem cells in CHA-SFM-035 was changed.
  • CHAM-0305 containing DMEM / F12 as a basic medium in step (2.1)
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured to passages 4 to 6. Subsequently, the cells in passage 6 were transferred to a commercial bone differentiation induction medium and cultured for another 3 weeks.
  • 6 subcultured cells cultured in serum-containing medium (CCM) were transferred to commercial bone differentiation induction medium and cultured for another 3 weeks.
  • Fig. 9 shows results obtained by staining with calcium-containing Alizarin Red S in a medium containing normal animal-derived serum and in calcium-containing cells secreted from stem cells cultured in the medium of the present invention.
  • cells cultured in the cell culture medium CH-SFM-035) and serum-containing medium (CCM) were not stained, but in the experimental group and the control group, And it was confirmed to be stained with red color. That is, the cell culture medium of the present invention has the same bone differentiation ability as the serum medium when the stem cells are subcultured.
  • the medium contained the composition of (2.1)
  • the fat differentiation potential of the subcultured stem cells in CHA-SFM-035 was changed.
  • CHASH-SFM-035) containing DMEM / F12 as the primary medium in the culture medium (2.1) the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured to passages 4 to 6. Subsequently, the cells in passage 6 were transferred to a commercial fat-differentiation induction medium and cultured for another 3 weeks.
  • the passage 6 cells cultured in CCM were transferred to a commercial fat differentiation induction medium and cultured for another 3 weeks.
  • Fig. 10 shows results obtained by staining a medium containing normal animal-derived serum and stem cells cultured in the medium of the present invention with Oil Red O.
  • Fig. As shown in FIG. 10, the cells cultured in the cell culture medium (CHA-SFM-035) and the serum-containing medium (CCM) were not stained. However, in the experimental group and the control group, And cholesterol ester were stained red. In addition, it was confirmed that the phospholipids and cerebrosides were stained with light pink.
  • 11 is a graph quantitatively evaluating the degree of lipolysis after discoloration of stem cells stained with Oil Red O with isopropyl alcohol. As shown in Fig.
  • the medium contained the composition of (2.1), it was confirmed whether the chondrogenic differentiation potential of the subcultured stem cells in CHA-SFM-035 was changed.
  • CHAM-035 containing DMEM / F12 as a basic medium in step (2.1)
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured to passages 4 to 6. Subsequently, the cells of passage 6 were transferred to commercial cartilage differentiation induction medium and cells cultured for another 3 weeks were used as an experimental group.
  • the control group was prepared by transferring 6 subcultured cells cultured in serum-containing medium (CCM) to a commercial bone differentiation induction medium for an additional 3 weeks.
  • CCM serum-containing medium
  • FIG. 12 shows results obtained by staining a medium containing serum derived from an ordinary animal and stem cells cultured in the medium of the present invention with Alcian blue.
  • the cell size of the experimental group and the control group was larger than that of the cells cultured in the culture medium (CHA-SFM-035) and serum-containing medium (CCM).
  • the shape of nuclei red staining
  • the culture medium for cell culture of the present invention has the same cartilage differentiation ability as the serum medium when the stem cells are subcultured.
  • TNFAIP6, PTGS2, and IDO1 which are inflammatory factors of stem cells cultured subcultured in CHA-SFM-035, was confirmed as the medium contained the composition of (2.1).
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured in a 4 to 7 passages in a cell culture medium (CHA-SFM-035) containing DMEM / F12 as the basic medium in (2.1). Positive control cells were cultured in serum-containing medium (CCM). Immunomodulation of each cell line was carried out using TNF-a, and quantitative polymerase chain reaction (qPCR) was performed.
  • FIGS. 13A to 13C are graphs showing the expression patterns of inflammatory factors in medium containing normal animal-derived serum and in stem cells cultured in the medium of the present invention.
  • Figs. 13A to 13C it was confirmed that expression of TNFAIP6, PTGS2, and IDO1 genes in the experimental group was similar or significantly increased compared with the positive control group. Particularly, it can be confirmed that the temperature rises remarkably in the passage 5. That is, the cell culture medium of the present invention shows that the genes are equivalent in the subculture of the stem cells.
  • the medium contained the composition of (2.1), immune regulation in vivo of stem cells transplanted in CHA-SFM-035 was confirmed.
  • the umbilical cord-derived mesenchymal stem cells obtained in 1 were cultured in a 4 to 7 passages in a cell culture medium (CHA-SFM-035) containing DMEM / F12 as the basic medium in (2.1).
  • Cells cultured in serum-containing medium (CCM) were used for the negative control group and PBS for the positive control group.
  • intraperitoneal inflammation was induced with zymosan using a zymosan induced peritonitis mouse model.
  • experimental group, negative control group and positive control group were injected. After 4 hours, peritoneal exudates were extracted and subjected to ELISA.
  • FIG. 13D is a graph showing the expression pattern of TNF-.alpha. After culturing a medium containing normal animal-derived serum and cultured stem cells in the medium of the present invention and injecting it into a mouse. As shown in Fig. 13 (d), the expression of mTNF-a was decreased in the mice injected with the experimental group. That is, it can be seen that the subcultured cells in the cell culture medium of the present invention have an anti-inflammatory effect of the cell gene showing mTNF-a expression equivalent or improved as compared with the serum-containing medium.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Reproductive Health (AREA)
  • Transplantation (AREA)
  • Rheumatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne une composition de milieu de culture asérique pour culture cellulaire, et un procédé de culture de cellules dans ladite composition. Si la composition de milieu de culture asérique pour culture cellulaire selon la présente invention est utilisée, la composition remplace un milieu de culture cellulaire de coût élevé contenant du sérum et s'avère donc économique, peut réduire un effet secondaire classique tel que l'infection susceptible de survenir quand le sérum est inclus, et présente un taux de prolifération cellulaire similaire à celui d'un milieu de culture cellulaire classique contenant du sérum.
PCT/KR2018/014531 2017-11-24 2018-11-23 Composition de milieu de culture asérique WO2019103528A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR20170157938 2017-11-24
KR10-2017-0157938 2017-11-24

Publications (2)

Publication Number Publication Date
WO2019103528A2 true WO2019103528A2 (fr) 2019-05-31
WO2019103528A3 WO2019103528A3 (fr) 2019-07-18

Family

ID=66631631

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/014531 WO2019103528A2 (fr) 2017-11-24 2018-11-23 Composition de milieu de culture asérique

Country Status (2)

Country Link
KR (2) KR20190060716A (fr)
WO (1) WO2019103528A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021248675A1 (fr) * 2020-06-09 2021-12-16 生物岛实验室 Inducteur pour la différenciation de cellules souches en chondrocytes et application de celui-ci
CN114231485A (zh) * 2021-10-29 2022-03-25 南京国青血液净化科技有限公司 免疫细胞体外诱导扩增以及保存方法

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102428940B1 (ko) * 2019-11-25 2022-08-03 한국해양과학기술원 온도안정성 및 단백질 분해효소 저항성을 향상시킨 fgf2 폴리펩타이드 및 그 용도

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007080919A1 (fr) * 2006-01-13 2007-07-19 Japan Science And Technology Agency Additif pour milieu de culture pour culture sans serum de cellule animale, kit et utilisation de l’additif ou du kit
KR20110097064A (ko) * 2010-02-24 2011-08-31 (주) 아크스템 무혈청 줄기 세포 배양 조성물, 이를 함유한 조직 재생용 조성물 및 이를 이용한 조직 재생 방법
CN103068969A (zh) * 2010-06-17 2013-04-24 思迪公司 化学成分确定的无血清细胞培养基
KR101738508B1 (ko) * 2015-12-16 2017-05-22 강원대학교산학협력단 혈관외피세포를 이용한 무혈청조건배지 및 이를 이용한 전분화능 줄기세포로부터 조혈모세포의 분화 증진 방법
KR101928488B1 (ko) * 2016-01-15 2018-12-12 가톨릭대학교 산학협력단 페록시다신을 포함하는 무혈청 배지 첨가제 조성물 및 이의 이용

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021248675A1 (fr) * 2020-06-09 2021-12-16 生物岛实验室 Inducteur pour la différenciation de cellules souches en chondrocytes et application de celui-ci
CN114231485A (zh) * 2021-10-29 2022-03-25 南京国青血液净化科技有限公司 免疫细胞体外诱导扩增以及保存方法

Also Published As

Publication number Publication date
KR20190060716A (ko) 2019-06-03
KR20210044749A (ko) 2021-04-23
KR102292132B1 (ko) 2021-08-23
WO2019103528A3 (fr) 2019-07-18

Similar Documents

Publication Publication Date Title
US10568911B2 (en) Multipotent stem cells and uses thereof
WO2013081436A1 (fr) Composition de milieu de culture pour rajeunir des cellules souches
Teramura et al. Induction of mesenchymal progenitor cells with chondrogenic property from mouse-induced pluripotent stem cells
WO2018226051A2 (fr) Composition de milieu sans sérum pour la culture de cellules comprenant un exosome dérivé d'une cellule souche humaine
KR102292132B1 (ko) 무혈청 배지 조성물
WO2012008813A2 (fr) Composition de milieu de culture pour mise en culture de cellules souches mésenchymateuses issues de membrane amniotique, et procédé de mise en culture de celles-ci utilisant cette composition
WO2011052818A1 (fr) Procédé de production de cellules souches mésenchymateuses à partir de cellules souches pluripotentes humaines, et cellules souches mésenchymateuses ainsi produites
WO2010107159A1 (fr) Procédé d'isolement et procédé de culture en masse pour des cellules souches de sang de cordon ombilical exprimant znf281
WO2016068596A1 (fr) Composition de milieu pour la culture de cellules souches
WO2010013906A2 (fr) Procédé de séparation de cellules souches fortement actives de cellules souches humaines et cellules ainsi séparées
WO2012008733A2 (fr) Cellules souches issues de tissu de placenta primaire et agent thérapeutique contenant celles-ci
CN111826348A (zh) 一种人诱导性多能干细胞来源的间充质干细胞体外高效制备方法和应用
WO2019198962A1 (fr) Procédé pour favoriser la prolifération et la différenciation de cellules souches pluripotentes induites préparées à partir de cellules souches issues d'urine
WO2013119026A1 (fr) Procédé de différenciation de cellules souches en neurones
WO2010107285A2 (fr) Procédé de production en grandes quantités s'appliquant au facteur de croissance et utilisant des cellules souches mésenchymateuses foetales du le liquide amniotique
WO2022025598A1 (fr) Composition pour induire la différenciation de cellules souches pluripotentes en cellules souches mésenchymateuses comprenant un activateur smad1/5/8 et utilisation associée
WO2010008157A2 (fr) Procédé permettant de différencier des cellules souches de cellules ectodermiques
WO2019221477A1 (fr) Composition pour favoriser la différenciation de cellules souches, comprenant une solution de culture de cellules progénitrices et un film de graphène multicouche, et son utilisation
WO2017039251A1 (fr) Cellule adhérente postnatale améliorée, et utilisation de celle-ci
WO2022103129A1 (fr) Cellules souches mésenchymateuses précoces présentant un vieillissement réduit et une capacité de cellule souche préservée et procédé de culture s'y rapportant
WO2018186649A1 (fr) Procédé de différenciation en cellules souches mésenchymateuses par sous-culture continue de cellules souches dédifférenciées
WO2018101723A1 (fr) Nouvelles cellules progénitrices mésenchymateuses fonctionnellement améliorées, composition d'agent thérapeutique cellulaire anti-inflammatoire les contenant, et procédé de préparation de cellules progénitrices mésenchymateuses
WO2016190704A1 (fr) Cellules adhérentes postnatales améliorées et procédé de préparation associé
WO2021206402A1 (fr) Procédé de culture de cellules souches pour favoriser le rendement initial de cellules souches
WO2019004800A2 (fr) Milieu de culture de cellules précurseurs de peau dérivées de cellules souches et son procédé de préparation

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18880547

Country of ref document: EP

Kind code of ref document: A2