WO2013009100A2 - 탯줄 추출물의 제조방법 및 그의 용도 - Google Patents
탯줄 추출물의 제조방법 및 그의 용도 Download PDFInfo
- Publication number
- WO2013009100A2 WO2013009100A2 PCT/KR2012/005514 KR2012005514W WO2013009100A2 WO 2013009100 A2 WO2013009100 A2 WO 2013009100A2 KR 2012005514 W KR2012005514 W KR 2012005514W WO 2013009100 A2 WO2013009100 A2 WO 2013009100A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- umbilical cord
- stem cells
- cells
- cell
- extract
- Prior art date
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
- C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
- A61K8/985—Skin or skin outgrowth, e.g. hair, nails
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- 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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/10—General cosmetic use
-
- 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
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells
- C12N2502/025—Coculture with; Conditioned medium produced by embryonic cells extra-embryonic cells, e.g. amniotic epithelium, placental cells, Wharton's jelly
-
- 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
Definitions
- the present invention relates to a method of preparing the umbilical cord extract and the use of such a serum substitute of the umbilical cord extract, a composition for stem cell isolation and culture, and a wound repair filler.
- FBS fetal bovine serum
- a medium containing fetal bovine serum is commonly used, and an animal-derived protein source including fetal bovine serum is used in the culture process, thereby making stem cells for clinical application.
- an animal-derived protein source including fetal bovine serum is used in the culture process, thereby making stem cells for clinical application.
- a method using a serum-free medium and a method using a medium containing human serum can be used.
- the method using a serum-free medium is complicated and uneconomical because it requires culturing stem cells by containing a large amount of cytokines (cytokine) including growth hormone.
- the method using a medium containing human serum also has to use a large amount of cytokines made by recombinant methods, and expensive human serum is used as a protein source for culture, which causes a lot of economic burden.
- stem cells are divided into adult stem cells (adult stem cells) found in various tissues and organs of the adult and embryonic stem cells obtained from blastocyst stage cells during the developmental stage.
- Embryonic stem cells have pluripotency to differentiate into various cells such as nerves, blood cells, and pancreas, but there are ethical problems because they must be obtained from embryos that can grow into humans. Therefore, adult stem cells, which can not only eliminate ethical problems but also are easily separated and cultured and can be differentiated into various cells, have been spotlighted as materials for cell therapy.
- BM-MSCs bone marrow-derived mesenchymal stem cells
- bone marrow-derived mesenchymal stem cells decrease in the number and proliferative capacity of stem cells as the donor ages, and pain is involved in harvesting the bone marrow.
- it is attempting to separate mesenchymal stem cells from other tissues, and recently, reports that can be separated from adult and fetal peripheral blood, umbilical cord, placenta, umbilical cord blood, etc. It is attracting attention as a source of new cell therapeutics.
- the umbilical cord is made up of blood vessels and connective tissue called Wharton's jelly around it.
- the length of the umbilical cord during pregnancy is 30-60 cm. It is 40-50 g in weight, rich in nutrients to the fetus, and contains stem and progenitor cells.
- cells derived from umbilical cords have been reported to have characteristics of mesenchymal stem cells derived from bone marrow.
- umbilical cord-derived stem cells express CD73, CD90, CD105, CD10, CD13, CD29, CD44, and HLA-ABC cell surface proteins similar to bone marrow and other tissue-derived mesenchymal stem cells, and CD34, CD45, which are hematopoietic markers And histocompatibility antigens CD14, CD31, CD33, HLA-DR ⁇ cell surface proteins are not expressed.
- stem cells isolated from the umbilical cord have been reported to express embryonic stem cell markers Oct4, Sox2, and Nanog at the same time.
- Umbilical cord-derived stem cells can differentiate into bone cells, chondrocytes, and adipocytes, and are superior to bone marrow or adipose derived stem cells when cultured in vitro. It has been reported that the cells can be used to differentiate into cardiomyocytes and neurons. As such, the umbilical cord is expected to be a tissue capable of supplying stem cells for clinical use, and may be used as a cell therapy.
- umbilical stem-derived stem cells In order to efficiently use umbilical stem-derived stem cells, a large number of umbilical stem-derived stem cells are required. However, there is a limit to the number of stem cells that can be obtained from the umbilical cord, and the stem cells often lose differentiation ability during culture. However, there is no known method for efficiently separating and culturing umbilical cord-derived stem cells in a serum-free medium. In order to solve this problem, the present invention was completed by confirming that umbilical cord-derived stem cells can be effectively isolated and cultured when using umbilical cord-derived extracts.
- Another object is to provide a serum substitute comprising the umbilical cord extract.
- Another object is to provide a cell culture containing the serum replacement.
- Still another object relates to a method for separating umbilical cord derived stem cells comprising the umbilical cord extract from the umbilical cord and a method of culturing the umbilical cord derived stem cells.
- One aspect is to provide a method of making a umbilical cord extract.
- One embodiment includes cutting the umbilical cord; Placing the cut umbilical cord in a buffer; Stirring the umbilical cord impregnated with the buffer; And centrifuging the product obtained by stirring to obtain a supernatant as a umbilical cord extract.
- the present invention solves the problems of the conventional method by separating the glycoproteins, such as growth factors, cytokines, chemokines and GAGs (glycaosminoglycan), which are bound to the extracellular matrix, in a relatively simple process, thereby enabling the active components to be useful in the natural state. It is to provide a method that can be obtained (natural-occurring).
- glycoproteins such as growth factors, cytokines, chemokines and GAGs (glycaosminoglycan)
- the umbilical cord is first cut.
- the cutting of the umbilical cord is performed to facilitate the elution of useful substances present in the umbilical cord tissue by increasing the contact area with the buffer.
- Umbilical cords that can be used in one embodiment include umbilical cords of various mammals. Preferably humans, pigs, horses, cows, mice, rats, hamsters, rabbits, goats and sheep, more preferably humans, pigs, horses and cows, most preferably humans.
- Umbilical cord cutting can be carried out through various methods known in the art.
- the umbilical cord is cut to a length of 0.5-3.0 cm, more preferably 0.7-2.5 cm, even more preferably 1-2 cm.
- the umbilical cord extract is preferably extracted from Wharton's jelly portion of the umbilical cord tissue. Therefore, the step of removing blood and / or blood vessels in the umbilical cord may be additionally included.
- the cut umbilical cord may include the step of buffering.
- Buffers used in the present invention can be used any buffer having a buffering power (buffering power) and include, for example, sodium acetate, sodium phosphate, glycine-HCl, Tris-HCl and Phosphate buffered saline (PBS).
- PBS Phosphate buffered saline
- the pH is 2-11, preferably 4-10, more preferably 5-8 and even more preferably 6.8-7.6.
- the amount of buffer impregnating the cleaved umbilical cord is not particularly limited, and preferably 2-5 times the weight of the umbilical cord, more preferably 2-4 times, even more preferably 2.5-3.2 times the buffer.
- the cleaved umbilical cord may be washed with a suitable solution (eg, buffer) before being placed in the buffer.
- the method may include stirring the umbilical cord impregnated with the buffer.
- the agitation is carried out at 4 ° C-10 ° C, preferably 4 ° C-6 ° C.
- Stirring can also be carried out for 7-24 hours, preferably 12-24 hours, more preferably 18-24 hours.
- Stirring may be carried out through various methods known in the art, and may be stirred using a magnetic bar.
- the product obtained by stirring may be centrifuged to obtain a supernatant as a umbilical cord extract.
- the product obtained by stirring is centrifuged to finally obtain a supernatant as a umbilical cord extract.
- the supernatant contains various useful proteins bound to extracellular matrix such as growth factors and cytokines.
- the centrifugation is carried out at conditions of 3,000-6,000 rpm, preferably 4,000-4,500 rpm. Centrifugation is carried out at 4 ° C-10 ° C, preferably 4 ° C-6 ° C. According to one embodiment, the centrifugation is carried out for 2 minutes-30 minutes, preferably 5 minutes-20 minutes, more preferably 10 minutes-15 minutes.
- Another aspect provides an umbilical cord extract prepared by the method of one aspect described above.
- the umbilical cord extract is IGFBP-7 (insulin-like growth factor binding protein-7), Lipocallin-1, CXCL14, Leptin R, IL-23, MIP-1a, Angiogenin, Thrombospondin-2, IL- 29, IL-4R and the like (FIG. 31) as main components.
- IGFBP-7 insulin-like growth factor binding protein-7
- Lipocallin-1 CXCL14
- Leptin R lipocallin-1
- IL-23 lipocallin-1
- MIP-1a angiogenin
- Thrombospondin-2 IL- 29, IL-4R and the like
- the main cytokines of umbilical cord extracts are well known for their effects on anti-angiogenesis, anti-apoptosis, growth and inflammation.
- Another aspect is to provide a serum replacement comprising umbilical cord extract.
- serum refers to the remainder of the removal of fibrin from plasma. Serum has been commonly used to promote animal cell proliferation since the animal cell culture system was established in vitro.
- serum substitute refers to a substance that can be used to obtain the same or similar effect as serum instead of serum, and the same or superior effect without the use of serum such as fetal bovine serum (FBS). Means the material to obtain.
- FBS fetal bovine serum
- the umbilical cord extract may be an umbilical cord extract obtained by a general method, but is preferably obtained through one embodiment described above. In particular, it is desirable to remove blood from the umbilical cord so that serum components are not included.
- the serum substitute may be applied to any field where serum can be applied.
- it can be used for the culturing of cells, preferably for the culturing of animal cells.
- it can be used for isolation, culture, and the like of stem cells.
- the stem cells may include all kinds of stem cells, such as adult stem cells, mesenchymal stem cells, dedifferentiated stem cells and tissue-derived stem cells.
- Another aspect is to provide a cell culture comprising the serum replacement.
- the cell culture of one embodiment is a cell of various animals, preferably mammalian cells, more preferably human, pig, horse, cow, mouse, rat, hamster, rabbit, goat and sheep cells, even more preferably Human, pig, horse and bovine cells, most preferably human cells.
- Culture medium of one embodiment may be applied to stem cell culture.
- Stem cells to which the present invention is applied are not limited, and cells having characteristics of stem cells, that is, undifferentiated, infinitely proliferating, and differentiating into specific cells are cells to which the present invention can be applied.
- the stem cells may include pluripotent stem cells and multipotent stem cells, including embryonic stem cells (ES) and embryonic germ cells (EG).
- ES embryonic stem cells
- EG embryonic germ cells
- the stem cells may be umbilical cord-derived stem cells.
- the culture solution may include, in addition to the umbilical cord extract as a serum substitute, the basic composition of the medium for animal cell culture.
- the culture of the present invention is Eagle's MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), ⁇ -MEM (Stanner, CP et al., Nat. New Biol. 230: 52 ( 1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147: 923 (1978)), 199 medium (Morgan et al., Proc. Soc.Exp.
- tissue repair filler comprising the umbilical cord extract.
- tissue repair filler refers to a pharmaceutical composition or cosmetic composition used for the purpose of effectively hiding wrinkles and fine lines of the skin.
- the tissue repair filler comprises, in addition to the umbilical cord extract, the basic components of the filler, preferably collagen, hyaluronic acid, polyacrylamide gels, atechol, autogen (auto collagen) or a mixture of polymethylmethacrylate and collagen do.
- Fillers of one embodiment may include waxes, elastomers, higher alcohols, surfactants, oils, powders, humectants, water dispersible polymers, skin protectants, preservatives and / or fragrances.
- the umbilical cord extract may be a umbilical cord extract obtained by a general method, but is preferably obtained through one embodiment described above. In particular, it is desirable to remove blood from the umbilical cord so that serum components are not included.
- Another aspect provides a dressing comprising the umbilical cord extract.
- dressing means a pharmaceutical composition that is applied to a part of a human or animal body for the treatment of skin for therapeutic or cosmetic purposes.
- it is a dressing for the treatment of damaged skin, skin lesions, any interruption of the skin surface (eg skin ulcers, burns, cut wounds, punctures, torn wounds, blunt trauma, acne lesions and boils).
- the dressing may include patches, plasters, bandages and gauze to facilitate delivery of the formulation.
- the dressing is preferably applicable to the internal and external tissues of the body, more preferably to the body surface.
- the umbilical cord extract may be a umbilical cord extract obtained by a general method, but is preferably obtained through one embodiment described above. In particular, it is desirable to remove blood from the umbilical cord so that serum components are not included.
- Another aspect provides a cosmetic composition for improving wrinkles or improving skin aging comprising the umbilical cord extract.
- Cosmetic compositions of one embodiment can be used to improve various skin conditions.
- the composition of the present invention is effective for improving wrinkles and improving skin aging.
- Ingredients included in the cosmetic composition of one embodiment include ingredients commonly used in cosmetic compositions in addition to growth factors as active ingredients, and include, for example, conventional auxiliaries such as stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. do.
- auxiliaries such as stabilizers, solubilizers, vitamins, pigments and flavors, and carriers. do.
- Cosmetic compositions of one embodiment may be prepared in any formulation commonly prepared in the art, including, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.
- the formulation of one embodiment is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components.
- animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components.
- animal oils vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide
- cellulose derivatives polyethylene glycols
- silicones bentonites
- silicas talc or zinc oxide
- lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.
- a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.
- liquid carrier diluents such as water, ethanol or propylene glycol
- suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.
- the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide.
- Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.
- Another aspect provides a method of separating stem cells from the umbilical cord.
- One embodiment includes the steps of culturing the culture medium composition and the fragmented umbilical cord tissue containing the umbilical cord extract of the mammal in a cell culture vessel; Treating a stem cell separation enzyme in the umbilical cord tissue; And it provides a method for separating stem cells from the umbilical cord of mammals comprising the step of separating the stem cells from the umbilical cord tissue.
- the mammal may be human, pig, horse, cow, mouse, rat, hamster, earthenware, goat or sheep.
- the tissue may be selected from the group consisting of fat, umbilical cord, liver and periosteum.
- the cell culture vessel may be a cell culture vessel coated with a cell adhesion protein.
- the cell adhesion protein may be, but is not limited to, mammalian umbilical cord-derived collagen, gelatin, gebronectin, fibronectin, laminin, or poly-D-lysine.
- the mammal's umbilical cord-derived collagen may comprise: (i) pulverizing the umbilical cord tissue of the mammal treated with hydrogen peroxide; (ii) treating the ground cord tissue with acetic acid and pepsin and then centrifuging; (iii) adjusting the pH of the supernatant obtained by centrifugation to 7 and precipitating collagen by adding NaCl; And (iv) can be produced by a method of producing a umbilical cord-derived collagen of mammal comprising the step of separating the precipitated collagen.
- the step (ii) is preferably performed 1 day to 3 days after the start of the step (i).
- the stem cell separation enzyme of step (ii) may be collagenase, and preferably, the collagenase may be type I collagenase. More preferably, in the step (ii), the type I collagenase is included 180 U / ml to 220 U / ml, can be treated for 2 to 6 hours.
- the umbilical cord extract is preferably prepared by the method according to the above embodiments.
- Another aspect provides a method of culturing stem cells using umbilical cord extracts.
- One embodiment comprises the steps of adding the umbilical cord extract to the stem cell culture; It provides a stem cell culture method comprising the step of culturing stem cells using the cell culture in a cell culture vessel.
- the cell culture vessel may be coated with a cell adhesion protein.
- the stem cells may be stem cells that are tissue-derived stem cells.
- the stem cells may be animal stem cells, preferably human-derived stem cells.
- the stem cells may include all kinds of stem cells, such as adult stem cells, mesenchymal stem cells, dedifferentiated stem cells and tissue-derived stem cells.
- cell adhesion protein may be collagen, gelatin, gebronectin, fibronectin, laminin, or poly-D-lysine, but is not limited thereto.
- the tissue may be any one selected from the group consisting of fat, umbilical cord, liver and periosteum, but is not limited thereto.
- the stem cell culture may not contain serum.
- the stem cells may be all animal stem cells, embryonic stem cells, adult stem cells, and dedifferentiated stem cells, the expression of at least one gene in the group consisting of Oxt4, Sox2, KLF4 and Nanog gene. May be a cell. In particular, even when subcultured, it may be a cell in which at least one gene is continuously expressed in a group consisting of Oxt4, Sox2, KLF4 and Nanog, which are genes specific for the embryonic stem cells.
- the stem cells may be a cell selectively showing positive for CD29, CD73, CD90, CD105 and CD166, and may be a cell selectively showing negative for CD34 and CD45.
- a stem cell having excellent survival rate, proliferative capacity, and differentiation capacity using a medium without FBS in primary culture for separating stem cells from umbilical cord tissues for a short period of time (15 days) It is possible to obtain the maximum amount (about 2.0 ⁇ 10 8 cells), and very large amount (1.0 ⁇ 10 10 cells from about 50 g of umbilical cord tissue) can be separated in 2-3 passages. It can be used in ways that are useful for development.
- 1 is a graph showing protein elution with stirring time.
- Figure 2 is a graph comparing the amount of protein elution with agitation time when the medium is replaced and not replaced.
- 3 and 4 are graphs showing protein elution according to umbilical cord size.
- 5 and 6 are diagrams showing the SDS-PAGE results in order to confirm the difference between the protein elution amount and the protein eluted according to the pH of the buffer.
- FIG. 7 and 8 are diagrams showing the protein elution amount according to the elution method.
- 9 and 10 are diagrams showing the difference between the protein eluted from the umbilical cord tissue and fresh umbilical cord tissue.
- 11 is a diagram showing the amount of protein elution according to the amount of buffer.
- UCE umbilical cord extract
- 19 and 20 are graphs showing the results of treatment of markers of mesenchymal stem cells and FACS analysis after culturing bone marrow and cord-derived stem cells in serum-free medium containing umbilical cord extracts, respectively.
- the x-axis is the intensity (intensity)
- the Y-axis is the number of cells (count). Changes in the x- and y-axes can identify the CD markers described above. In the figure is a comparison of cells grown in the addition of umbilical cord and cells grown in the addition of FBS.
- 21 and 22 are the umbilical cord extracts as a result of performing hematoxylin & Eosin staining after subcutaneous injection of a hyaluronic acid derivative (HAD) filler containing a umbilical cord extract (UCE) into the mouse
- HAD hyaluronic acid derivative
- UCE umbilical cord extract
- Figure 23 shows the umbilical cord-derived stem cell proliferation effect according to the concentration of the umbilical cord extract in the serum-free medium of one embodiment.
- Figure 24 shows the effect of increasing the adhesion and proliferation (culture) of umbilical cord-derived stem cells in a culture dish coated with umbilical cord-derived collagen according to one embodiment.
- 25 is a result of comparing the number of umbilical cord-derived stem cells proliferated 2 days after culturing the umbilical cord-derived stem cells in a culture dish coated with collagen concentration.
- Figure 26 is a diagram comparing the conventional umbilical cord-derived stem cell separation method and umbilical cord-derived stem cell separation method of one embodiment.
- FIG. 27 is a photograph showing the state of the cells in culture by separating by the six methods shown in FIG.
- Figure 28 shows the results of the total cell number recovered 15 days after umbilical cord-derived stem cell separation.
- FIG. 29 compares the results of immunomarker analysis of cells isolated and cultured by the six isolation and culture methods of FIG. 26.
- the x-axis is the intensity (intensity)
- the Y-axis is the number of cells (count). Changes in the x- and y-axes can identify the CD markers described above.
- FIG. 30 shows the results of performing cytokine arrray on a total of 507 human cytokines, chemokines, growth factors, and the like, which are included in extracts extracted from three different umbilical cords, and 10 cytokines most included in each umbilical cord. will be.
- FIG. 31 is a relative quantitative graph showing substances commonly included in extracts separated from three different umbilical cords. The most common are IGFBP-7 and lipocalin-1.
- FIG. 32 shows a total of 62 human cytokines identified in three different umbilical cords, starting from the highest amount.
- 33 illustrates a function performed in the human body for 10 well-known cytokines.
- stem cell capacity (stemnes) retention capacity of stem cells according to the continuous passage of the cells cultured using the umbilical cord extract and the cells cultured with medium added 10% FBS This shows a comparison of the stem cell division (doubling time) (Td) after the passage and the passage at least 10 times.
- U-MSCs stem cells isolated from umbilical cord tissue using umbilical cord extracts express embryonic stem cells (ESCs) specific markers.
- CD34 and CD45 show stem cells isolated from umbilical cord extracts obtained from tissues from three different donors from three different donors on CD29, CD73, CD90, CD105, and CD166, which are mesenchymal stem cell-specific cell surface markers. The results show that cells are commonly positive and negative for CD34 and CD45.
- the amount of protein eluted increased as the stirring time increased from the point of stirring start to 24 hours, but when stirring while replacing the PBS, eluting from about 60 hours after the start of stirring
- the amount of protein sharply decreased (FIG. 2).
- Umbilical cord was cut into 0.5-2.0 cm length (FIG. 3), washed twice with PBS (pH 7.0), and then placed in PBS about three times the weight of the umbilical cord and stirred at 4 ° C for 4 days. Supernatants collected by centrifugation at 4,500 rpm, 4 ° C. for 10 minutes were used as umbilical cord-derived extracts, and protein quantification was performed by Bradford assay. The change in protein elution amount according to the umbilical cord cutting size was not large, and the protein elution amount was drastically reduced when PBS was replaced during stirring (FIG. 4).
- the umbilical cord was cut into 0.5-2.0 cm lengths, and PBS (pH 2, pH 7 or pH 11) about three times the weight of the umbilical cord was added and stirred at 4 ° C for 24 hours.
- PBS pH 2, pH 7 or pH 11
- Supernatants collected by centrifugation at 4,500 rpm, 4 ° C. for 10 minutes were used as umbilical cord-derived extracts, and protein quantification was performed by Bradford assay.
- the method of stirring at 4 ° C. was a method capable of obtaining a large amount of protein while preventing protein denaturation and degradation of protein that may be caused by homogenization and 37 ° C. culture method.
- 73.78 mg (1.48 mg / ml) of protein was eluted, and as the amount of PBS increased, the amount of protein eluted also increased (FIG. 11).
- the amount of PBS increases, the amount of total protein increases, but the protein concentration of the final umbilical cord extract is lowered, so a separate enrichment process is required to produce a proper concentration, and the extracted protein is lost.
- the analysis was performed using the RayBio Human cytokine array kit, which is capable of analyzing 507 species for the types and relative quantitative analysis of growth factors, chemokines, and cytokines.
- the donor prepared 1 mg of each of the other three umbilical cord extracts, treated them with a membrane, and reacted. After that, the detected dots were analyzed by using the MultiGauge program.
- cytokine array 30 shows the results of performing a cytokine array on a total of 507 human cytokines, chemokines, growth factors, and the like, which are included in extracts from donors separated from three different umbilical cords, and most contained in extracts eluted from different donors. 10 cytokines are shown.
- FIG. 31 is a relative quantitative graph showing substances commonly included in extracts separated from three different umbilical cords. The most common ones are IGFBP-7 and lipocalin-1.
- FIG. 32 lists a total of 62 human cytokines identified in three different umbilical cords, starting with the highest amount.
- FIG. 33 is a table for explaining a function performed in the human body with 10 cytokines well-known in a large amount in the extract.
- Cytokine quantitative analysis showed an average of 1,400 pg / ml FGF-2, 1,480 pg / mlG-CSF, 860 pg / ml MCP-1, 900 pg / ml GRO, 700 pg / ml IL-1ra and 620 pg / ml IP-10 Among the extracted cytokines, it was confirmed as a main component (FIG. 12).
- Umbilical cord extracts were treated in serum-free medium at concentrations of 0, 0.1, 0.2, 0.5 or 1 mg / ml.
- Umbilical cord-derived stem cells (UC-MSC), bone marrow-derived stem cells (BM-MSC) and skin fibroblasts were cultured in medium containing 10% serum (Hyclone, SH30919.03) as a control group.
- % WST-1 assay (Daeillab, EZ-3000) was measured for 7 days at two-day intervals. WST-1 assay reagent was added to 10% of the medium and reacted for about 1 hour under the same conditions as the culture conditions, and the absorbance was measured at 450 nm.
- umbilical cord-derived stem cells, bone marrow-derived stem cells and dermal fibroblasts at 0.2 mg / ml were treated with umbilical cord extracts in serum-free medium at 10 mg FBS-mediated medium. It was confirmed that the umbilical cord extract was treated at a concentration of 0.5 mg / ml or more, it was confirmed that the cell proliferation is increased than cells in the medium condition of 10% FBS addition (Fig. 15-17).
- Bone marrow and umbilical cord stem cells were cultured in medium containing 10% FBS and 0.2 mg / ml umbilical cord extract, and then treated with markers of mesenchymal stem cells and FACS analysis was performed.
- stem cells cultured in serum-free medium containing umbilical cord extract did not change the characteristics of stem cells, such as differentiation, because mesenchymal stem cell-specific cell surface markers were maintained ( 19 and 20).
- stem cell cultivation stemness
- doubling time was compared with stem cells cultured in a medium added with 10% FBS while continually subcultured using umbilical cord extracts at three-day intervals. .
- FIG. 34 and FIG. 35 are experiments for comparing the stemness (stem cell capacity) maintenance capacity of stem cells in accordance with the continuous passage of stem cells cultured using the umbilical cord extract and cells cultured with medium added 10% FBS This is the result of comparing the Td time of the stem cells at the beginning of passage and after passage at least 10 times.
- the umbilical cord was cut to 1-2 cm in length and washed twice with DPBS.
- the cord was treated with 70% ethanol solution and reacted at 4 ° C. for 1 hour, and then washed twice with purified water to determine the weight of the cord.
- DPBS about three times the weight of the umbilical cord and after 24 hours at 4 °C
- umbilical cord extracts UAE was recovered.
- the recovered umbilical cord extracts were filtered through a final 0.22 ⁇ m filter and stored at 4 ° C.
- 3% H 2 O 2 solution was added to the remaining umbilical cord, and then treated at 4 ° C. for 12 to 24 hours. Then, washed twice with purified water until the bubbles disappear. After treatment with a 0.5 M acetic acid solution of about 10 times the weight of the umbilical cord, the tissue was ground using a blender (homenderizer) and a homogenizer (homogenizer). Umbilical cord weight was treated by 10% pepsin and reacted at 4 ° C for 24 hours. Centrifugation was performed at 10,000 rpm for 4 minutes at 4 ° C. After centrifugation, the pH of the collected supernatant was adjusted to 7 using NaOH to remove the pepsin enzyme activity.
- the recovered umbilical cord extract was quantified by Bradford assay, and the collagen prepared was quantified by hydroxyprolin assay.
- Collagen was dissolved in D.W. at a concentration of 50 ⁇ g / ml and treated in a culture dish and coated in the incubator for 1 hour. After coating, the collagen solution was removed, washed two more times with phosphate buffer, and completely dried at room temperature, followed by culturing the cells. Umbilical cord extracts were cultured by treating the serum-free medium at concentrations of 0, 0.1 and 0.2 mg / ml, and cultured with 10% FBS as a control. WST-1 assay was used to compare cell growth. (Daeillab, EZ-3000) was measured for 7 days at two-day intervals. After adding WST-1 assay reagent to 10% of the medium and reacting for about 1 hour in the same manner as in the culture conditions, the absorbance was measured at 450 nm (FIGS. 23 and 24).
- Blood outside the umbilical cord was removed with DPBS without Ca 2+ and Mg 2+ , and then the outer amnion was removed and two arteries were removed.
- Cells were cultured in a medium to which 10% FBS was added after collagenase treatment. Specifically, the blood-removed tissue was cut to a size of 1 mm 3 and treated with ⁇ -MEM containing 200 U / ml of collagenase type ⁇ for 5 hours to separate cells, and then 100 U / ml of Incubate in ⁇ -MEM containing penicillin, 0.1 ⁇ g / ml streptomycin, 10% FBS, and 2 ⁇ 10 3 cells per 1 cm 2 of the incubator in which 5% CO 2 is supplied at 37 ° C. It was.
- the cells were cultured in a medium containing 0.2 mg / ml UCE.
- the blood-removed tissue was cut into 1 mm 3 size and treated with ⁇ -MEM containing 200 U / ml of ⁇ collagenase for 5 hours to separate cells and then 100 U / ml penicillin, 0.1 ⁇ g / ml of streptomycin, 0.2 mg / ml umbilical cord extract the ⁇ -MEM containing the insert collagen-coated culture dish 1 cm 2 per 2 ⁇ 10 3 cells to put in the culture medium is from 37 °C temperature of 5% CO 2 is fed Incubated.
- Tissue culture was performed in the medium to which 10% FBS was added, followed by cell culture in the medium to which 10% FBS was added after collagenase treatment.
- the blood-removed tissue was cut to a size of 1 mm 3 and then placed in ⁇ -MEM containing 100 U / ml penicillin, 0.1 ⁇ g / ml streptomycin, 10% FBS, and cultured for 7 days.
- ⁇ -MEM containing 100 U / ml penicillin, 0.1 ⁇ g / ml streptomycin, 10% FBS, and cultured for 7 days.
- the cells were treated with ⁇ -MEM containing 200 U / ml of ll-type collagenase for 4 hours, and when the extracellular matrix was decomposed, the cells were separated by centrifugation and washing with PBS.
- Tissue culture was performed in a medium to which 0.2 mg / ml umbilical cord extract was added, followed by cell culture in a medium to which 0.2 mg / ml umbilical cord extract was added after collagenase treatment.
- the blood removed tissue was cut into 1 mm 3 size and placed in a collagen coated dish in ⁇ -MEM containing 100 U / ml penicillin, 0.1 ⁇ g / ml streptomycin and 0.2 mg / ml UCE. After culturing for a while, the cells attached to the bottom began to be seen and treated with ⁇ -MEM containing 200 U / ml of ⁇ -gal collagenase for 4 hours to separate the cells.
- Tissue cultures were performed in medium supplemented with 10% FBS. Specifically, the blood removed tissue was cut into 1 mm 3 size and placed in ⁇ -MEM containing 100 U / ml penicillin, 0.1 ⁇ g / ml streptomycin, 10% FBS, and 5% CO 2 at 37 ° C. The cells were cultured in the incubator supplied.
- Tissue cultures were performed in medium to which 0.2 mg / ml umbilical cord extract was added. Specifically, the blood-removed tissue was cut to a size of 1 mm 3 and placed in a collagen-coated culture dish with ⁇ -MEM containing 100 U / ml penicillin, 0.1 ⁇ g / ml streptomycin, and 0.2 mg / ml UCE. After the incubation was incubated in an incubator supplied with 5% CO 2 at 37 °C temperature.
- RNA samples were subjected to 2 ⁇ PCR Master mix solution kit (iNtRON Biotechnology) with 1 ⁇ Taq buffer, 0.25 U Taq polymerase, 10 pM sense and PCR was performed with 10 ⁇ L reaction solution mixed with antisense gene-specific primers. Amplification was performed for a total of 32 cycles. Each cycle consisted of 30 seconds of denaturation at 94 ° C, 30 seconds of annealing, and 30 seconds at 72 ° C. After completion of the reaction, the PCR product was loaded on a 2% agarose gel (agarose gel) and subjected to electrophoresis. After electrophoresis, stained with ethidium bromide and an image of DNA was obtained using UV light.
- Flow cytometry was performed to characterize the isolated cells.
- Cells are washed with PBS for flow cytometry and treated with trypsin-EDTA into a single cell population followed by washing with PBS containing 2% FBS.
- mice After the umbilical cord extract was mixed with the hyaluronic acid derivative and the hyaluronic acid derivative at a concentration of 500 ug / ml, the experiment was performed by treating mice (BALB / c-nuSlc, female, 5 weeks). 200 ul of each treatment group was subcutaneously injected and samples were taken one, four, eight and twelve weeks later. Mice were removed from all the tissues around the sample after the cervical spine, and each sample was weighed and fixed with 4% formalin (Neutral buffered formalin) for hematoxylin & eosin staining.
- formalin Neutral buffered formalin
- the umbilical cord extract of the present invention can be used as a serum replacement for culturing normal cells and stem cells derived from animals.
- the umbilical cord extract of the present invention can be used in tissue repair fillers, dressings and cosmetic compositions for improving skin conditions.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Dermatology (AREA)
- Reproductive Health (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Gynecology & Obstetrics (AREA)
- General Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Birds (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pregnancy & Childbirth (AREA)
- Hematology (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
유전자(gene) | 프라이머 서열(5'-3') | 온도(℃) | ||
OCT 4 | Sense | agaaggagtggtccgagtg | 서열번호 1 | 60 |
Antisense | agagtggtgacggagacagg | 서열번호 2 | ||
Nanog | Sense | atacctcagcctccagcaga | 서열번호 3 | 59 |
Antisense | cctgattgrrccaggattgg | 서열번호 4 | ||
KLF4 | Sense | accctgggtcttgaggaagt | 서열번호 5 | 59 |
Antisense | tgccttgagatgggaactct | 서열번호 6 | ||
Sox2 | Sense | gatgcacaactcggagatcag | 서열번호 7 | 60 |
Antisense | gccgttcatgtaggtctgcga | 서열번호 8 | ||
GAPDH | Sense | gaaggtgaaggtcggagtca | 서열번호 9 | 60 |
Antisense | ggaggcattgctgatgatct | 서열번호 10 |
Claims (23)
- 탯줄을 절단하는 단계;상기 절단된 탯줄을 완충액에 넣는 단계;상기 완충액에 함침된 탯줄을 교반하는 단계; 및교반하여 얻은 산물을 원심분리하여 탯줄 추출물로서 상층액을 수득하는 단계를 포함하는 포유동물 탯줄 추출물의 제조방법.
- 제1항에 의해 제조된 탯줄 추출물.
- 제2항에 있어서, 상기 탯줄 추출물은 IGFBP-7 (insulin-like growth factor binding protein-7), Lipocallin-1, CXCL14, Leptin R, IL-23, MIP-1a, Angiogenin, Thrombospondin-2, IL-29, IL-4R의 단백질을 성분으로 포함하는 탯줄 추출물.
- 탯줄 추출물을 포함하는 혈청 대체재.
- 제4항에 있어서, 상기 탯줄 추출물은 제1항의 방법에 의해 제조된 것인 혈청 대체재.
- 제4항의 혈청 대체재를 포함하는 세포 배양액.
- 제6항에 있어서, 상기 세포는 동물 세포인 것인 세포 배양액.
- 제7항에 있어서, 상기 동물 세포는 줄기 세포인 것인 세포 배양액.
- 제8항에 있어서, 상기 줄기 세포는 탯줄 유래 줄기세포인 것인 세포 배양액.
- 탯줄 추출물을 포함하는 조직 수복용 필러.
- 제10항에 있어서, 상기 탯줄 추출물은 제1항의 방법에 의해 제조된 것인 조직 수복용 필러.
- 세포배양용기에 포유류의 탯줄 추출물을 포함하는 배지 조성물과 세절된 탯줄 조직을 넣어 배양하는 단계;상기 탯줄 조직에 효소를 처리하는 단계; 및탯줄 조직에서 줄기세포를 분리하는 단계를 포함하는 포유류의 탯줄로부터 줄기세포 분리 방법.
- 제12항에 있어서, 상기 세포배양용기는 세포 부착 단백질(cell adhesion protein)로 코팅된 것인 줄기세포 분리 방법.
- 제12항에 있어서, 상기 세포 부착 단백질은 포유류 태반유래 콜라겐, 젤라틴, 피브로넥틴, 라미닌 및 폴리-D-라이신으로 이루어진 군으로부터 선택되는 것인 줄기세포 분리 방법.
- 줄기 세포 배양액에 탯줄 추출물을 첨가하는 단계;세포배양용기에서 상기 세포 배양액을 이용하여 줄기세포를 배양하는 단계를 포함하는 줄기세포 배양 방법.
- 제15항에 있어서, 상기 세포배양용기는 세포 부착 단백질(cell adhesion protein)로 코팅된 것인 줄기세포 배양 방법.
- 제15항에 있어서, 상기 줄기세포는 조직 유래 줄기세포인 것인 줄기세포 배양 방법.
- 제16항에 있어서, 상기 조직은 지방, 탯줄, 간 및 골막으로 이루어진 군에서 선택된 어느 하나인 것인 줄기세포 배양 방법.
- 제15항에 있어서, 상기 줄기 세포 배양액은 혈청을 포함하지 않는 것인 줄기세포 배양 방법.
- 제15항에 있어서, 상기 세포 부착 단백질은 포유류 태반유래 콜라겐, 젤라틴, 피브로넥틴, 라미닌 및 폴리-D-라이신으로 이루어진 군으로부터 선택되는 것인 줄기세포 배양 방법.
- 제15항에 있어서, 상기 줄기세포는 Oxt4, Sox2, KLF4 및 Nanog 유전자로 이루어진 그룹에서 적어도 어느 하나가 발현이 되는 세포인 것인 줄기세포 배양 방법.
- 제15항에 있어서, 상기 줄기세포는 CD29, CD73, CD90, CD105 및 CD166에 선택적으로 양성을 나타내는 세포이고 CD34 및 CD45에 선택적으로 음성을 나타내는 세포인 것인 줄기세포 배양 방법.
- 제15항에 있어서, 상기 줄기세포는 탯줄 추출물을 이용하여 배양하는 경우 배아줄기세포 특이적 유전자인 Oxt4, Sox2, KLF4 및 Nanog로 이루어진 그룹에서 적어도 어느 하나가 계대배양을 반복하여도 지속적으로 발현되는 세포인 것인 줄기세포 배양 방법.
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2014520127A JP2014520843A (ja) | 2011-07-11 | 2012-07-11 | 臍帯抽出物の製造方法及びその用途 |
US14/232,448 US20140295554A1 (en) | 2011-07-11 | 2012-07-11 | Method for manufacturing umbilical cord extract and usage of the same |
CN201280044164.3A CN103998048A (zh) | 2011-07-11 | 2012-07-11 | 脐带提取物的制备方法和使用方法 |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020110068261A KR20130007751A (ko) | 2011-07-11 | 2011-07-11 | 탯줄 추출물의 제조방법 및 그의 용도 |
KR10-2011-0068261 | 2011-07-11 | ||
KR1020110068761A KR20130008178A (ko) | 2011-07-12 | 2011-07-12 | 포유류의 탯줄유래 줄기세포 분리 또는 배양용 배지 조성물, 그리고 이를 이용한 포유류의 탯줄유래 줄기세포 분리 또는 배양 방법 |
KR10-2011-0068761 | 2011-07-12 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2013009100A2 true WO2013009100A2 (ko) | 2013-01-17 |
WO2013009100A3 WO2013009100A3 (ko) | 2013-06-13 |
Family
ID=47506715
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/KR2012/005514 WO2013009100A2 (ko) | 2011-07-11 | 2012-07-11 | 탯줄 추출물의 제조방법 및 그의 용도 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20140295554A1 (ko) |
JP (1) | JP2014520843A (ko) |
KR (1) | KR101462004B1 (ko) |
CN (1) | CN103998048A (ko) |
WO (1) | WO2013009100A2 (ko) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015137419A1 (ja) * | 2014-03-11 | 2015-09-17 | 北海道公立大学法人札幌医科大学 | 間葉系幹細胞の賦活化剤、賦活化された間葉系幹細胞およびその製造方法 |
CN106474155A (zh) * | 2016-10-19 | 2017-03-08 | 天津普瑞赛尔生物科技有限公司 | 含有人脐带间充质干细胞提取物的外用凝胶制剂及其制备方法和用途 |
EP3617306A4 (en) * | 2017-04-25 | 2021-01-20 | Sapporo Medical University | METHOD FOR PRODUCING MESENCHYMAL STEM CELLS, THERAPEUTIC EFFECT MARKERS FOR MESENCHYMAL STEM CELLS, METHOD FOR DETERMINING THERAPEUTIC EFFECTS OF MESENCHYMAL STEM CELLS AND CELL PREPARATIONS WITH MESENCHYMAL STEM CELLS |
CN113462641A (zh) * | 2021-07-15 | 2021-10-01 | 广州市宙斯生物科技有限责任公司 | 脐带间充质干细胞提取物、应用及检测方法 |
Families Citing this family (26)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10894066B2 (en) | 2014-03-06 | 2021-01-19 | Amnio Technology Llc | Amnion derived therapeutic compositions and methods of use |
US9132156B1 (en) | 2014-06-15 | 2015-09-15 | Amnio Technology Llc | Acellular amnion derived therapeutic compositions |
US10363278B2 (en) | 2014-06-15 | 2019-07-30 | Amnio Technology Llc | Frozen therapeutic dose and package |
CN105267243B (zh) * | 2014-07-23 | 2020-03-20 | 深圳培元生物科技有限公司 | 一种消除皮肤妊娠纹的干细胞提取物 |
TW201613621A (en) * | 2014-10-14 | 2016-04-16 | Bionet Corp | Composition for promoting growth of dermal papilla cells, pharmaceutical composition, and preparation method thereof |
CA2969690A1 (en) * | 2014-12-05 | 2016-06-09 | Janssen Biotech, Inc. | Treatment of ocular conditions using progenitor cells |
WO2017026838A1 (ko) * | 2015-08-12 | 2017-02-16 | 주식회사 차바이오텍 | 향상된 탯줄 유래 부착형 줄기세포, 그의 제조방법 및 용도 |
KR20170020273A (ko) * | 2015-08-12 | 2017-02-22 | (주)차바이오텍 | 향상된 탯줄 유래 부착형 줄기세포, 그의 제조방법 및 용도 |
US10517903B2 (en) | 2015-09-14 | 2019-12-31 | Amnio Technology Llc | Amnion derived therapeutic composition and process of making same |
US20200129563A1 (en) * | 2016-07-26 | 2020-04-30 | Equi-Stem Biotechnologies LLC | Composition derived from mammalian umbilical cord and whartons jelly for use in therapeutic and regenerative applications |
US20180028569A1 (en) * | 2016-07-26 | 2018-02-01 | Equi-Stem Biotechnologies LLC | Composition derived from mammalian umbilical cord and whartons jelly for use in therapeutic and regenerative applications |
US10987131B2 (en) | 2017-05-25 | 2021-04-27 | Coopersurgical, Inc. | Tissue containment systems and related methods |
KR102144593B1 (ko) * | 2017-06-07 | 2020-08-13 | 주식회사 엑소스템텍 | 인간줄기세포 유래 엑소좀을 포함하는 세포 배양용 무혈청 배지 조성물 |
KR102178778B1 (ko) * | 2017-12-26 | 2020-11-13 | (주) 차바이오에프앤씨 | 피부줄기세포 배양액을 포함하는 피부 노화 예방 또는 개선용 조성물 및 이의 용도 |
CN110179738B (zh) * | 2019-05-30 | 2022-03-15 | 科索瑞生物科技(天津)有限公司 | 活性精华素及其制备方法 |
CN110169946B (zh) * | 2019-05-30 | 2022-03-15 | 科索瑞生物科技(天津)有限公司 | 眼部精华乳及其制备方法 |
CN110179737B (zh) * | 2019-05-30 | 2022-03-15 | 科索瑞生物科技(天津)有限公司 | 护肤液及其制备方法 |
WO2021022377A1 (en) * | 2019-08-08 | 2021-02-11 | Tissue Regeneration Therapeutics Inc. | Perivascular lysates and uses thereof |
CN114732778A (zh) * | 2020-12-23 | 2022-07-12 | 天津拂瑞雅生物科技有限公司 | 修复面膜及其制备方法 |
CN114652665A (zh) * | 2020-12-23 | 2022-06-24 | 天津拂瑞雅生物科技有限公司 | 护肤精华液及其制备方法 |
CN114652664A (zh) * | 2020-12-23 | 2022-06-24 | 科索瑞生物科技(天津)有限公司 | 修复凝胶及其制备方法 |
CN114652620A (zh) * | 2020-12-23 | 2022-06-24 | 天津拂瑞雅生物科技有限公司 | 私密凝胶及其制备方法 |
CN114652663A (zh) * | 2020-12-23 | 2022-06-24 | 天津拂瑞雅生物科技有限公司 | 头发生长液及其制备方法 |
CN114652662A (zh) * | 2020-12-23 | 2022-06-24 | 天津拂瑞雅生物科技有限公司 | 修复霜及其制备方法 |
JP2023101072A (ja) * | 2022-01-07 | 2023-07-20 | 株式会社ホルス | 抽出物の製造方法、化粧品の製造方法、及び経口物の製造方法 |
ECSP22048957A (es) * | 2022-07-14 | 2022-11-30 | Univ San Francisco De Quito Usfq | Método para la preparación de un extracto de cordón umbilical |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20040034795A (ko) * | 2002-10-17 | 2004-04-29 | 박래옥 | 스테로이드 제제 및 태반 추출물을 함유한 염증성 피부병치료용 경피제 |
KR20100091762A (ko) * | 2009-02-11 | 2010-08-19 | 건국대학교 산학협력단 | 항산화 활성을 가지는 피부 보호용 조성물 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101525596A (zh) * | 2009-04-15 | 2009-09-09 | 大连理工大学 | 一种使用同一份脐带血的血清来收获干细胞的方法 |
-
2012
- 2012-07-11 US US14/232,448 patent/US20140295554A1/en not_active Abandoned
- 2012-07-11 WO PCT/KR2012/005514 patent/WO2013009100A2/ko active Application Filing
- 2012-07-11 CN CN201280044164.3A patent/CN103998048A/zh active Pending
- 2012-07-11 JP JP2014520127A patent/JP2014520843A/ja active Pending
- 2012-07-11 KR KR1020120075754A patent/KR101462004B1/ko active IP Right Grant
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20040034795A (ko) * | 2002-10-17 | 2004-04-29 | 박래옥 | 스테로이드 제제 및 태반 추출물을 함유한 염증성 피부병치료용 경피제 |
KR20100091762A (ko) * | 2009-02-11 | 2010-08-19 | 건국대학교 산학협력단 | 항산화 활성을 가지는 피부 보호용 조성물 |
Non-Patent Citations (3)
Title |
---|
JINYOUNG KIM ET AL.: 'Human Cord Serum as a Fetal Bovine Serum Substitute for the Culture of Human Amnion-Derived Stem Cells' DEV. REPROD. vol. 11, no. 2, 2007, pages 85 - 96 * |
LUCIANO OZZELLO ET AL.: 'Growth-promoting activity of acid mucopolysaccharides on a strain of human mammary carcinoma cell' CANCER RESEARCH vol. 20, 1960, pages 600 - 604 * |
YOUNG NAM LEE ET AL.: 'Comparison of Mycobacterial Growth in Dubos Medium, Hyaluronate Supplemented Medium and Umbilical Cord Extract Based Medium' YONSEI MEDICAL JOURNAL vol. 18, no. 2, 1977, pages 130 - 135 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015137419A1 (ja) * | 2014-03-11 | 2015-09-17 | 北海道公立大学法人札幌医科大学 | 間葉系幹細胞の賦活化剤、賦活化された間葉系幹細胞およびその製造方法 |
KR20160131112A (ko) | 2014-03-11 | 2016-11-15 | 훗카이도 코리츠 다이가쿠 호진 삿포르 이카 다이가쿠 | 간엽줄기세포 활성화제, 활성화된 간엽줄기세포 및 그 제조 방법 |
US10512660B2 (en) | 2014-03-11 | 2019-12-24 | Sapporo Medical University | Activator for mesenchymal stem cells, activated mesenchymal stem cells, and method for producing same |
CN106474155A (zh) * | 2016-10-19 | 2017-03-08 | 天津普瑞赛尔生物科技有限公司 | 含有人脐带间充质干细胞提取物的外用凝胶制剂及其制备方法和用途 |
EP3617306A4 (en) * | 2017-04-25 | 2021-01-20 | Sapporo Medical University | METHOD FOR PRODUCING MESENCHYMAL STEM CELLS, THERAPEUTIC EFFECT MARKERS FOR MESENCHYMAL STEM CELLS, METHOD FOR DETERMINING THERAPEUTIC EFFECTS OF MESENCHYMAL STEM CELLS AND CELL PREPARATIONS WITH MESENCHYMAL STEM CELLS |
CN113462641A (zh) * | 2021-07-15 | 2021-10-01 | 广州市宙斯生物科技有限责任公司 | 脐带间充质干细胞提取物、应用及检测方法 |
Also Published As
Publication number | Publication date |
---|---|
CN103998048A (zh) | 2014-08-20 |
KR101462004B1 (ko) | 2014-11-19 |
WO2013009100A3 (ko) | 2013-06-13 |
JP2014520843A (ja) | 2014-08-25 |
KR20140008749A (ko) | 2014-01-22 |
US20140295554A1 (en) | 2014-10-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2013009100A2 (ko) | 탯줄 추출물의 제조방법 및 그의 용도 | |
WO2016006885A1 (ko) | 자극된 줄기세포 배양액의 발모 촉진능 및 이의 용도 | |
Leyva-Leyva et al. | Characterization of mesenchymal stem cell subpopulations from human amniotic membrane with dissimilar osteoblastic potential | |
WO2011049414A2 (ko) | 지방조직 유래 성체 줄기세포 이동을 유도하는 방법 | |
US20110217385A1 (en) | Method for extracting mesenchymal stem cell from human or animal embryo and for extracting the secretion product thereof | |
AU2006304575A1 (en) | Methods for rejuvenating cells in vitro and in vivo | |
Little et al. | Procollagen and collagen produced by a teratocarcinoma-derived cell line, TSD4: evidence for a new molecular form of collagen | |
AU2021209278A1 (en) | Methods and compositions to modulate hair growth | |
WO2016006788A1 (ko) | 작은 크기 줄기세포의 발모 촉진능 및 이의 용도 | |
WO2018048220A1 (ko) | 염증 자극된 중간엽 줄기세포를 포함하는 면역질환 또는 염증 질환의 예방 또는 치료용 약학적 조성물 | |
WO2019209068A1 (ko) | Hla-g 단백질을 상시 분비 발현하는 세포주 및 이의 제조방법 | |
Martini et al. | Human placenta-derived mesenchymal stem cells acquire neural phenotype under the appropriate niche conditions | |
Hu et al. | Stem cells derived from human first-trimester umbilical cord have the potential to differentiate into oocyte-like cells in vitro | |
WO2012008733A2 (ko) | 1기 태반조직 유래 줄기세포 및 이를 함유하는 세포치료제 | |
WO2013165120A1 (ko) | 신경능선줄기세포의 배양방법 및 그 용도 | |
WO2019017691A9 (ko) | 인간 유래 만능줄기세포에서 모유두전구세포로의 분화방법 및 이의 용도 | |
WO2021118226A1 (ko) | 인간 만능줄기세포로부터 중간엽 줄기세포의 제조방법 및 이에 의해 제조된 중간엽 줄기세포 | |
WO2011102680A9 (ko) | Pi3k/akt/gsk3 경로를 통해 성체줄기세포의 증식, 다분화능 및 재프로그래밍을 촉진하는 cd49f | |
WO2021025533A1 (ko) | 골격근 줄기세포 유래 엑소좀을 유효성분으로 포함하는 피부상태 개선용 조성물 | |
WO2022004938A1 (ko) | 유사 중간엽 줄기세포의 제조방법 | |
KR20130007751A (ko) | 탯줄 추출물의 제조방법 및 그의 용도 | |
WO2019190246A1 (ko) | 인간 제대로부터 줄기세포를 분리하는 방법 | |
WO2022119418A1 (ko) | Gdf-3를 고발현하는 제대혈 줄기세포 분리 및 배양 방법 및 gdf-3의 용도 | |
US20050058628A1 (en) | Nuclear reprogramming of cells for therapeutic use | |
WO2022103129A1 (ko) | 노화가 감소되고 줄기세포능이 보존된 초기 중간엽 줄기세포, 및 그 배양방법 |
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: 12812015 Country of ref document: EP Kind code of ref document: A2 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 14232448 Country of ref document: US |
|
ENP | Entry into the national phase |
Ref document number: 2014520127 Country of ref document: JP Kind code of ref document: A |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 12812015 Country of ref document: EP Kind code of ref document: A2 |