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1. (WO2017096607) METHOD FOR SEPARATING AND EXTRACTING HUC-MSC FROM OUTER LAYER OF AMNIOTIC MEMBRANE TISSUE OF UMBILICAL CORD
Latest bibliographic data on file with the International Bureau

Pub. No.: WO/2017/096607 International Application No.: PCT/CN2015/097112
Publication Date: 15.06.2017 International Filing Date: 11.12.2015
IPC:
C12N 5/0775 (2010.01)
C CHEMISTRY; METALLURGY
12
BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
N
MICRO-ORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICRO-ORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5
Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
07
Animal cells or tissues
071
Vertebrate cells or tissues, e.g. human cells or tissues
077
Mesenchymal cells, e.g. bone cells, cartilage cells, marrow stromal cells, fat cells or muscle cells
0775
Mesenchymal stem cells; Adipose-tissue derived stem cells
Applicants:
里程 LI, Cheng [CN/CN]; CN
郭镭 GUO, Lei [CN/CN]; CN
Inventors:
郭镭 GUO, Lei; CN
Agent:
北京瑞恒信达知识产权代理事务所(普通合伙) LEADING INTELLECTUAL PROPERTY FIRM; 中国北京市 海淀区大柳树路17号富海中心2号楼9层909房间 Room 909, 9th Floor, Tower B, Fortune Building, No.17, Daliushu Road, Haidian District Beijing 100081, CN
Priority Data:
Title (EN) METHOD FOR SEPARATING AND EXTRACTING HUC-MSC FROM OUTER LAYER OF AMNIOTIC MEMBRANE TISSUE OF UMBILICAL CORD
(FR) PROCÉDÉ DE SÉPARATION ET D’EXTRACTION DE HUC-MSC D’UNE COUCHE EXTERNE DE TISSU DE MEMBRANE AMNIOTIQUE DU CORDON OMBILICAL
(ZH) 一种从脐带外层羊膜组织中分离提取hUC-MSC的方法
Abstract:
(EN) Provided is a method for rapidly separating and extracting human umbilical cord mesenchymal stem cells (hUC-MSC). The method comprises the following steps: taking freshly collected healthy neonatal umbilical cord tissue, and after removing the blood vessels, bluntly dissecting the outer layer of amniotic membrane, mechanically pulverising same, and treating with erythrocyte lysate for 3 minutes; carrying out type IV collagenase digestion, and after sieving through a 100-200 mesh sieve, carrying out suspension culture in a serum-free culture medium, replacing the liquid every 3-5 days; taking the supernatant to detect cell contamination, and waiting for the plate adherence rate to reach 30-70%; carrying out trypsin digestion, and then collecting the cells by centrifugation for passage amplification, until the rate of confluence of the cells reaches over 90% confluence; collecting the cells for cryopreservation, and detecting the biological characteristics of the hUC-MSC.
(FR) La présente invention concerne un procédé de séparation et d’extraction rapide de cellules souches mésenchymateuses humaines de cordon ombilical (hUC-MSC). Le procédé comprend les étapes suivantes : prélèvement de tissu néonatal sain fraîchement recueilli d’un cordon ombilical, et après l’élimination des vaisseaux sanguins, la dissection franche de la couche externe de la membrane amniotique, sa pulvérisation mécanique, et son traitement avec un lysat d’érythrocytes durant 3 minutes ; la conduite d’une digestion collagénase de type IV, et après tamisage à travers un tamis de 100 à 200 mesh, la réalisation d’une culture en suspension dans un milieu de culture sans sérum, le remplacement du liquide tous les 3 à 5 jours ; le prélèvement du surnageant pour détecter la contamination des cellules, et l’attente que le taux d’adhérence à la plaque atteigne 30 à 70 % ; la réalisation d’une digestion à la trypsine, et ensuite le recueil des cellules par centrifugation pour l’amplification de passage, jusqu’à ce que le niveau de confluence des cellules atteigne plus de 90 % de confluence ; le recueil des cellules pour cryoconservation, et la détection des caractéristiques biologiques des hUC-MSC.
(ZH) 提供了一种快速分离提取人脐带间充质干细胞(human Umbilical Cord mesenchymal stem cells,hUC-MSC)的方法。该方法包括以下步骤:取新鲜采集的健康新生儿的脐带组织,剔除血管后钝性分离外层羊膜,机械粉碎,经红细胞裂解液处理3分钟,IV型胶原酶消化,过100-200目筛后以无血清培养基悬浮培养,每3-5天进行换液,取上清检测细胞污染,待平皿内贴壁率达30-70%,胰酶消化后,离心收集细胞进行传代扩增,至细胞汇合率达90%以上汇合,收集细胞冻存并检测hUC-MSC的生物学特性。
Designated States: AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW
African Regional Intellectual Property Organization (ARIPO) (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW)
Eurasian Patent Organization (AM, AZ, BY, KG, KZ, RU, TJ, TM)
European Patent Office (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR)
African Intellectual Property Organization (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG)
Publication Language: Chinese (ZH)
Filing Language: Chinese (ZH)