JP7015316B2 - ナノグを導入した羊水内胎児由来間葉系幹細胞から獲得したエクソソーム内に含まれた育毛促進用組成物の製造方法 - Google Patents
ナノグを導入した羊水内胎児由来間葉系幹細胞から獲得したエクソソーム内に含まれた育毛促進用組成物の製造方法 Download PDFInfo
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Description
(b)前記分離した胎児由来細胞をFBS(fetal bovine serum)および、bFGF(basic fibroblast growth factor)を含む培地で継代培養して、羊水内胎児由来間葉系幹細胞を収得する段階;
(c)前記収得した羊水内胎児由来間葉系幹細胞にナノグ(Nanog)を導入して、ナノグ(Nanog)が過発現した羊水内胎児由来間葉系幹細胞を収得する段階;
(d)前記収得したナノグ(Nanog)が過発現した羊水内胎児由来間葉系幹細胞を無血清培地で1~5日間培養して、コンディションド培地を製造する段階;および
(e)前記コンディションド培地からエクソソームを分離する段階を含む間葉系幹細胞由来エクソソームの製造方法を提供する。
実施例1:Nanogが導入された羊水由来間葉系幹細胞株の確立
本発明者らは、細胞拡張に容易な羊水由来間葉系幹細胞株を獲得するために、羊水由来間葉系幹細胞にレトロウイルスベクターシステム(Retroviral vector system)を利用してナノグ(Nanog)遺伝子を導入し、ナノグ(Nanog)の過発現を誘導し、これにより、ナノグ(Nanog)導入羊水由来間葉系幹細胞株を確立した。具体的な方法は、次の通りである。
RT-PCRを行うために、ナノグ(Nanog)導入羊水由来間葉系幹細胞をトリゾール(TRIzol)試薬(Invitrogen,USA)を利用して製造者の指示に応じてトータルRNAを分離した。分離したトータルRNA500ugをcDNA製造ミックス(Bioneer)を利用してcDNAに逆転写した。実験チューブを45℃で60分間静置した後、95℃で5分間静置し、以後、使用時まで-20℃で保管された。cDNAは、Taq合成酵素(Promega)とナノグ(Nanog)mRNAに特異的なプライマー(Exo-Nanog foward:5’-GCTTGGATACACGCCGC-3’(配列番号2);およびExo-Nanog reverse:5’-GATTGTTCCAGGATTGGGTG-3’(配列番号3))を通じて増幅された。RT-PCRは、94℃で20秒、60℃で30秒、および72℃で2分のサイクルを35回繰り返した後、最終合成のために72℃で10分間進めた。PCR結果物を1%アガロースゲルに電気泳動し、これを分析した。
実施例1および2で確立された羊水由来間葉系幹細胞株のうちそれぞれ一つずつの細胞部を選択し、コンディションド培地100mlを生産した後、エクソソームを分離した。
実施例3で分離したエクソソーム内部の物質を分析するために、最初にRT-PCRを通じてmRNAの含有を調査した。
獲得したエクソソームを様々な濃度に区別した後、毛乳頭細胞に処理して毛髪生成に決定的な役割をする二つ、すなわち、毛乳頭細胞の成長および毛髪生成マーカーの変化を確認した。濃度は、100Xから1Xまで分け、コンディションド培地とエクソソームを除いたコンディションド培地を共に比較分析した。
通常のヘアローションの製造方法によって、本発明のナノグ(Nanog)を導入した羊水由来間葉系幹細胞コンディションド培地内エクソソームを有効成分として含むヘアローションを表1のように製造した。
通常の親水性軟膏剤の製造方法によって、本発明のナノグ(Nanog)を導入した羊水由来間葉系幹細胞コンディションド培地内エクソソームを有効成分として含む親水性軟膏剤を表2のように製造した。
Claims (3)
- (a)妊婦から得た羊水から胎児由来細胞を分離する段階;
(b)前記分離した胎児由来細胞をFBS(fetal bovine serum)および、bFGF(basic fibroblast growth factor)を含む培地で継代培養して、羊水内胎児由来間葉系幹細胞を収得する段階;
(c)前記収得した羊水内胎児由来間葉系幹細胞にナノグ(Nanog)を導入して、ナノグ(Nanog)が過発現した羊水内胎児由来間葉系幹細胞を収得する段階;
(d)前記収得したナノグ(Nanog)が過発現した羊水内胎児由来間葉系幹細胞を無血清培地で1~5日間培養して、コンディションド培地を製造する段階;および
(e)前記コンディションド培地からエクソソームを分離する段階;を含み、
前記分離したエクソソームは、bFGF(basic fibroblast growth factor、塩基性線維芽細胞成長因子)、IGF(Insulin-like Growth Factor)、Wnt7a(Wingless-type MMTV integration site family member 7A)およびPDGF-AA(Platelet-derived Growth Factor)よりなる群から選ばれたいずれか一つ以上のヒト成長因子を含有することを特徴とする、間葉系幹細胞由来エクソソームを製造する方法。 - 前記段階(c)でナノグ(Nanog)は、レトロウイルスベクターを利用して導入するものである、請求項1に記載の間葉系幹細胞由来エクソソームを製造する方法。
- 前記段階(b)でセレニウム(selenium)およびアスコルビン酸(ascorbic acid)をさらに含む培地で継代培養する、請求項1に記載の間葉系幹細胞由来エクソソームを製造する方法。
Applications Claiming Priority (3)
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KR10-2017-0004242 | 2017-01-11 | ||
KR20170004242 | 2017-01-11 | ||
PCT/KR2018/000506 WO2018131900A2 (ko) | 2017-01-11 | 2018-01-11 | 나노그를 도입한 양수 내 태아 유래 중간엽 줄기세포로부터 획득한 엑소좀 내 포함된 육모 촉진용 조성물의 제조방법 |
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JP2020505058A JP2020505058A (ja) | 2020-02-20 |
JP7015316B2 true JP7015316B2 (ja) | 2022-02-02 |
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US (1) | US20190330594A1 (ja) |
EP (1) | EP3569239A4 (ja) |
JP (1) | JP7015316B2 (ja) |
KR (1) | KR102037038B1 (ja) |
CN (1) | CN110191714A (ja) |
WO (1) | WO2018131900A2 (ja) |
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JP7046421B2 (ja) * | 2018-11-07 | 2022-04-04 | 剛士 田邊 | 医薬品組成物及び化粧品組成物 |
KR102049505B1 (ko) | 2019-05-14 | 2019-11-29 | 이장호 | 젖산탈수소효소 b 및 페록시좀 증식인자-활성화 수용체 감마 활성화인자 1-알파가 함유된 면역관용화된 세포외소포의 제조방법 및 이를 이용한 항암용 조성물 |
KR102159915B1 (ko) * | 2019-09-18 | 2020-09-24 | 주식회사 엑소코바이오 | 엑소좀 및/또는 세포외 소포체의 생성 촉진 방법 |
CN110538197A (zh) * | 2019-09-27 | 2019-12-06 | 北京臻惠康生物科技有限公司 | 一种外泌体在治疗脱发的药物中的应用 |
CN110840812A (zh) * | 2019-11-27 | 2020-02-28 | 沣潮医药科技(上海)有限公司 | 胎体来源的外泌体在皮肤调节产品中的用途 |
WO2024015123A1 (en) * | 2022-07-14 | 2024-01-18 | Elevai, Inc. | Method of treating hair loss and formulation for treatment |
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- 2018-01-11 EP EP18739347.5A patent/EP3569239A4/en not_active Withdrawn
- 2018-01-11 CN CN201880006518.2A patent/CN110191714A/zh active Pending
- 2018-01-11 JP JP2019558322A patent/JP7015316B2/ja active Active
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WO2018131900A3 (ko) | 2018-12-06 |
KR20180082980A (ko) | 2018-07-19 |
CN110191714A (zh) | 2019-08-30 |
US20190330594A1 (en) | 2019-10-31 |
WO2018131900A2 (ko) | 2018-07-19 |
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