WO2010040302A1 - 从人或动物胚胎提取间质性干细胞及提取其分泌物的方法 - Google Patents
从人或动物胚胎提取间质性干细胞及提取其分泌物的方法 Download PDFInfo
- Publication number
- WO2010040302A1 WO2010040302A1 PCT/CN2009/074031 CN2009074031W WO2010040302A1 WO 2010040302 A1 WO2010040302 A1 WO 2010040302A1 CN 2009074031 W CN2009074031 W CN 2009074031W WO 2010040302 A1 WO2010040302 A1 WO 2010040302A1
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- WIPO (PCT)
- Prior art keywords
- stem cells
- cells
- interstitial
- cell
- interstitial stem
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—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
-
- 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
-
- 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
- C12N5/0668—Mesenchymal stem cells from other natural sources
Definitions
- the non-adherent cells were aspirated by a pipette, washed with physiological saline and replaced with a new type of interstitial stem cell type A culture medium to continue the culture, and the trypsin was used when the adherent cells were expanded to the set volume of the culture flask. Digestion with EDTA to obtain a single cell suspension.
- the method further includes:
- the mixed enzyme digestion solution can be used as a digestive juice to obtain more single cells while minimizing cell damage;
- the mesenchymal stem cells screened by adherent purification and immunomagnetic beads were separated to improve the purity.
- interstitial stem cell type A culture medium is added to the isolated single cells, and the cells are inoculated and cultured, thereby promoting cell growth and proliferation.
- interstitial stem cell type B culture medium during the process of mesenchymal stem cell culture can stimulate the production of more secretory factors by interstitial stem cells. Therefore, the secretions from interstitial stem cells are more abundant. More cell secretion factors.
- Figure 9 is a flow chart showing a method for extracting interstitial stem cells from human or animal embryos according to still another embodiment of the present invention.
- 0.375 g of trypsin, 0.125 g of EDTA may be first dissolved in 500 ml of D-HBSS, and then 0.25 g of IV collagenase is dissolved in 500 ml of physiological saline, and then 500 ml of D- HBSS was mixed with 500 ml of physiological saline to prepare 1 L of the enzyme digest of the present invention.
- other methods, or buffers may be employed by those skilled in the art to configure the enzyme digests of the present invention.
- the enzyme digests of the invention may also be formulated in other ratios, buffers or methods.
- 0.2 g of trypsin, 0.05 g of EDTA, 4Na is dissolved in 500 ml of D-HBSS, while 0.5 g of IV collagenase is dissolved in 500 ml of physiological saline or PBS, and then 500 ml of D-HBSS and 500 ml of physiological saline are mixed to prepare 1 L of the enzyme digest of the present invention.
- Fig. 8 is a schematic view showing the cell adherent growth by cell seeding and culturing using a prior art culture solution. From the comparison of cell adherent growth shown in Fig. 5-8, it can be seen that when the interstitial stem cell type A medium was used for culture, the growth of the cells was significantly promoted.
- Adherent purification treatment including:
- the non-adherent cells were aspirated by a pipette, washed with physiological saline and replaced with a new type of interstitial stem cell type A culture medium, and cultured until the adherent cells were expanded to 70% of the volume of the culture flask. Digestion with EDTA (0.05%) yielded single cells.
- Magnetic bead sorting including:
- the interstitial stem cell type B culture medium comprises a matrix, a basic fibroblast growth factor, and a platelet-derived growth factor.
- the concentration of basic fibroblast growth factor in the matrix was 1 ng / ml, and the concentration of platelet-derived growth factor was 1 ng / ml.
- the matrix contains 98.8% by weight of DMEM medium, 1% by weight of penicillin-streptomycin-glutamine solution, 1% by weight of non-essential amino acids, 0.1% by weight of ⁇ - Mercaptoethanol and 1% by weight sodium pyruvate.
- the specific process of the above steps is: installing the diaphragm, the cup body, the stirring system according to the ultrafilter cup manual, adding the collecting liquid to the working volume, and then installing the cup cover and the protective cover. 2. Filter with nitrogen and collect the filtered concentrate.
- the specific process of the above step is: starting with nitrogen gas to start filtration, it should be noted that the pressure should be slowly pressurized at the beginning of the filtration, and then the magnetic stirrer is turned on after the pressure; the nitrogen pressure in the filtration cannot exceed 55PSI; should be slowly depressurized after filtration. Finally, when the concentrate in the ultrafiltration cup is concentrated to 25 times, the concentration is stopped, and the concentrate is collected in a clean bench and stored in a -80 °C refrigerator for use.
- the concentrated secretion contained a protein concentration of 0.2 mg/ml.
- the concentration of the protein in the concentrated secretion is 0.3 mg/ml ; when the interstitial stem cell type B medium of the second embodiment is used, the concentration is The reduced secretion contained a protein concentration of 1.5 mg/ml ; when the interstitial stem cell type B culture solution of the third embodiment described above was used, the concentrated secretion contained a protein concentration of 2 mg/ml.
- These gene products contain a large number of growth factor groups related to cell growth and metabolism, such as: BDNF, EGF, FGF, FGF17, FGF4, FGF6, FGF7, FGF9, GDNF, HGF, IGFBp, PDGFB, PIGF, TGFB1, TGFb2, TNFRSF11B, VEGF, IGF1 and LEP; and a large number of collagen molecules, such as: COLl lAl, COL12Al, COL16Al, C0L1A, C0L3A1, COL4Al, COL5A and COL6A.
- These protein molecular groups play an important role in promoting cell growth and metabolism. In the figure, it was found that the placental interstitial stem cell secretion contained a large amount of cell growth factor group and collagen molecule group, as indicated by the arrows in the figure.
- the interstitial stem cell secretion of the present invention has a significant protective effect on dermal fibroblasts.
- the addition of culture medium to placental interstitial stem cells (CM) can significantly reduce the cell death caused by hydrogen peroxide damage, thereby improving the survival of fiber young Cell survival rate.
- the addition of placental interstitial stem cell secretion to the culture medium can significantly reduce the cell death caused by hydrogen peroxide damage, and as the amount of CM increases, The survival rate of fibroblasts was also significantly increased (see Figure 15).
- the epidermal cell repairing solution of the invention can promote the fission and proliferation of fibroblasts and vascular endothelial cells and promote the synthesis and secretion of extracellular matrix, thereby promoting the growth of granulation tissue and lightening the sinus; promoting the division and reproduction of epithelial cells. Thereby promoting the skin of the wound.
- the reason why the skin is elastic is mainly because the collagen secreted by fibroblasts in the dermis of the skin forms a scaffold of the skin. This secretion increases the volume of collagen produced by the skin fibroblasts several times, and the fibroblasts. The volume increase is also more than 30%. Therefore, the continuous production of autologous collagen will increase the thickness and density of the dermis layer, fill in wrinkles, eliminate scars, and restore skin elasticity and luster.
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Developmental Biology & Embryology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- General Engineering & Computer Science (AREA)
- Gynecology & Obstetrics (AREA)
- Microbiology (AREA)
- Reproductive Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pregnancy & Childbirth (AREA)
- Dermatology (AREA)
- Rheumatology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Cosmetics (AREA)
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010532419A JP2010538681A (ja) | 2008-10-10 | 2009-09-18 | 人または動物胚から間葉系幹細胞を抽出及びその分泌物を抽出する方法 |
EP09818766A EP2368974A1 (en) | 2008-10-10 | 2009-09-18 | Methods for isolating mesenchymal stem cells from embryos of human or animals and extracting secretion substances thereof |
AU2009301517A AU2009301517A1 (en) | 2008-10-10 | 2009-09-18 | Methods for isolating mesenchymal stem cells from embryos of human or animals and extracting secretion substances thereof |
CN200980000501A CN101802177A (zh) | 2008-10-10 | 2009-09-18 | 从人或动物胚胎提取间质性干细胞及提取其分泌物的方法 |
CA2740092A CA2740092A1 (en) | 2008-10-10 | 2009-09-18 | Methods for isolating mesenchymal stem cells from embryo of human or animals and extracting secretion substances thereof |
US12/678,745 US20110217385A1 (en) | 2008-10-10 | 2009-09-18 | Method for extracting mesenchymal stem cell from human or animal embryo and for extracting the secretion product thereof |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/CN2008/072648 WO2010040262A1 (zh) | 2008-10-10 | 2008-10-10 | 分离动物胚胎间质性干细胞并提取其分泌物的方法 |
CNPCT/CN2008/072648 | 2008-10-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2010040302A1 true WO2010040302A1 (zh) | 2010-04-15 |
Family
ID=42100191
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2008/072648 WO2010040262A1 (zh) | 2008-10-10 | 2008-10-10 | 分离动物胚胎间质性干细胞并提取其分泌物的方法 |
PCT/CN2009/074031 WO2010040302A1 (zh) | 2008-10-10 | 2009-09-18 | 从人或动物胚胎提取间质性干细胞及提取其分泌物的方法 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2008/072648 WO2010040262A1 (zh) | 2008-10-10 | 2008-10-10 | 分离动物胚胎间质性干细胞并提取其分泌物的方法 |
Country Status (8)
Country | Link |
---|---|
US (1) | US20110217385A1 (zh) |
EP (1) | EP2368974A1 (zh) |
JP (1) | JP2010538681A (zh) |
KR (1) | KR20100065338A (zh) |
CN (1) | CN101802177A (zh) |
AU (1) | AU2009301517A1 (zh) |
CA (1) | CA2740092A1 (zh) |
WO (2) | WO2010040262A1 (zh) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2015077074A (ja) * | 2013-10-15 | 2015-04-23 | 佛教慈濟醫療財團法人 | 生体外でヒト間葉系幹細胞を増殖させる方法、およびそれに用いる補助剤、ならびに、生体外でヒト間葉系幹細胞の成長因子を取り出す方法およびその成長因子の用途 |
CN110079459A (zh) * | 2019-05-15 | 2019-08-02 | 刘宝全 | 一种干细胞培养容器内气体平衡的调节方法 |
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KR101583569B1 (ko) * | 2013-05-15 | 2016-01-08 | 라정찬 | 정맥 투여용 줄기세포 조성물 |
WO2015170347A2 (en) | 2014-05-09 | 2015-11-12 | Reelabs Private Limited | Foetal polymix of mesenchymal stem cells under hypoxic conditions for the treatment of clinical disorders |
CN108542877A (zh) * | 2017-03-23 | 2018-09-18 | 广州资生生物科技有限公司 | 一种皮米级细胞提取物的制备方法 |
WO2018186421A1 (ja) * | 2017-04-03 | 2018-10-11 | 株式会社カネカ | 間葉系幹細胞を含む細胞集団とその製造方法、及び医薬組成物 |
CN107142292B (zh) * | 2017-06-21 | 2019-11-12 | 广州润虹医药科技股份有限公司 | 一种诱导多能干细胞分泌素的制备方法及其应用 |
CN111565732A (zh) * | 2018-01-12 | 2020-08-21 | 国立大学法人大阪大学 | 复层扁平上皮细胞的正常分化-成熟促进剂、上皮疾病治疗剂以及复层扁平上皮细胞的正常分化-成熟促进方法 |
CN108815189A (zh) * | 2018-09-27 | 2018-11-16 | 天津欣普赛尔生物医药科技有限公司 | 一种含有自体子宫内膜间充质干细胞外泌体的制剂 |
CN110507597A (zh) * | 2019-08-20 | 2019-11-29 | 苏州诺普再生医学有限公司 | 一种组合物及其制备方法、应用 |
CN111150746A (zh) * | 2020-01-06 | 2020-05-15 | 湖南护宫福生物科技有限公司 | 一种用于阴道给药治疗妇科炎症的干细胞修复液 |
CA3177567A1 (en) * | 2020-03-31 | 2021-10-07 | Cell Exosome Therapeutics Inc. | Method of producing proliferated cells, method of producing cell product, mesenchymal stem cell population and method of producing same, culture supernatant of stem cells and method of producing same, and therapeutic agent |
CN111643390A (zh) * | 2020-07-16 | 2020-09-11 | 卡杜兰(广州)生命基因工程科技有限公司 | 一种将干细胞旁分泌因子浓缩、剔除无效杂质蛋白的制备方法 |
KR102435452B1 (ko) | 2020-11-11 | 2022-09-07 | 이엔셀 주식회사 | 노화가 감소되고 줄기세포능이 보존된 초기 중간엽 줄기세포, 및 그 배양방법 |
US20240066068A1 (en) * | 2020-12-22 | 2024-02-29 | Hans Pharma Co., Ltd. | Composition for skin improvement, containing culture liquid of umbilical-cord-derived mesenchymal stem cells as active ingredient |
CN113073077B (zh) * | 2021-04-07 | 2023-03-17 | 德泉生物医学技术(深圳)有限公司 | 一种使用封闭式系统培养临床级脐带血间充质干细胞的方法 |
CN113321709A (zh) * | 2021-05-18 | 2021-08-31 | 南方医科大学 | 自组装多肽、缓释外泌体多肽水凝胶及其制备方法和应用 |
CN113755445A (zh) * | 2021-07-26 | 2021-12-07 | 蓝海细胞(北京)生物科技有限公司 | 一种用于皮肤修复的细胞外泌体的制备方法 |
WO2023143522A1 (zh) * | 2022-01-28 | 2023-08-03 | 北京达尔文细胞生物科技有限公司 | 一种神经修复蛋白组合物及其制备方法和其应用 |
CN114717185A (zh) * | 2022-03-31 | 2022-07-08 | 秦岭大熊猫研究中心(陕西省珍稀野生动物救护基地) | 一种大熊猫胎盘间充质干细胞分离与培养方法 |
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WO2007027156A1 (en) * | 2005-09-02 | 2007-03-08 | Agency For Science, Technology And Research | Method of deriving mesenchymal stem cells |
CN101190174A (zh) * | 2006-12-01 | 2008-06-04 | 高宝化妆品(中国)有限公司 | 一种保湿抗皱护肤霜及其制作方法 |
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JP2004210713A (ja) * | 2002-12-27 | 2004-07-29 | Asahi Kasei Corp | 医療用胎盤由来細胞製剤 |
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2008
- 2008-10-10 WO PCT/CN2008/072648 patent/WO2010040262A1/zh active Application Filing
-
2009
- 2009-09-18 AU AU2009301517A patent/AU2009301517A1/en not_active Abandoned
- 2009-09-18 CA CA2740092A patent/CA2740092A1/en not_active Abandoned
- 2009-09-18 WO PCT/CN2009/074031 patent/WO2010040302A1/zh active Application Filing
- 2009-09-18 EP EP09818766A patent/EP2368974A1/en not_active Withdrawn
- 2009-09-18 CN CN200980000501A patent/CN101802177A/zh active Pending
- 2009-09-18 US US12/678,745 patent/US20110217385A1/en not_active Abandoned
- 2009-09-18 KR KR1020107006511A patent/KR20100065338A/ko active Search and Examination
- 2009-09-18 JP JP2010532419A patent/JP2010538681A/ja active Pending
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2015077074A (ja) * | 2013-10-15 | 2015-04-23 | 佛教慈濟醫療財團法人 | 生体外でヒト間葉系幹細胞を増殖させる方法、およびそれに用いる補助剤、ならびに、生体外でヒト間葉系幹細胞の成長因子を取り出す方法およびその成長因子の用途 |
CN110079459A (zh) * | 2019-05-15 | 2019-08-02 | 刘宝全 | 一种干细胞培养容器内气体平衡的调节方法 |
Also Published As
Publication number | Publication date |
---|---|
KR20100065338A (ko) | 2010-06-16 |
WO2010040262A1 (zh) | 2010-04-15 |
CA2740092A1 (en) | 2010-04-15 |
US20110217385A1 (en) | 2011-09-08 |
AU2009301517A1 (en) | 2010-04-15 |
JP2010538681A (ja) | 2010-12-16 |
CN101802177A (zh) | 2010-08-11 |
EP2368974A1 (en) | 2011-09-28 |
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